In vitro tests for aerosol deposition II: IVIVCs for different dry powder inhalers in normal adults.

Science.gov (United States)

Delvadia, Renishkumar; Hindle, Michael; Longest, P Worth; Byron, Peter R

2013-06-01

A new in vitro test method for dry powder inhalers (DPIs) was recently found to be predictive of the published in vivo results for Budelin Novolizer. The present study was intended to assess the method's robustness by evaluating correlations between average drug deposition in vitro and in vivo from five different DPIs. In vitro drug deposition from five marketed DPIs was assessed in a realistic physical airway model of a "medium" sized adult in an experimental setup that allowed deposition to be characterized regionally for carefully selected simulated air flow rate versus time profiles. The DPIs studied were Spiriva(®) HandiHaler(®), Relenza(®) Diskhaler(®), Salbutamol Easyhaler(®), Pulmicort(®) Turbuhaler(®), and Foradil(®) Aerolizer(®). In vitro regional deposition results were compared with those reported in the literature in order to create in vitro-in vivo correlations (IVIVCs) for each inhaler. Mean percent total lung deposition (TLD ± SD) in vitro for Spiriva HandiHaler, Relenza Diskhaler, Salbutamol Easyhaler, Pulmicort Turbuhaler, and Foradil Aerolizer were 17.3 ± 1.2, 22.6 ± 1.1, 29.0 ± 1.1, 28.0 ± 3.0, and 21.7 ± 1.2, respectively. These results showed excellent agreement with reported in vivo values, with absolute prediction errors in TLD of ≤ 2% for all DPIs except Relenza Diskhaler. Similarly, in vitro mouth-throat and device deposition results were stoichiometrically comparable to those reported in vivo for all DPIs except Relenza Diskhaler and Turbuhaler. Inspection of the scintigraphy studies for Relenza Diskhaler and Turbohaler revealed possible problems with powder labeling and result interpretation in their in vivo clinical assessments. A characteristic physical airway model representing a medium-sized adult, when coupled to carefully chosen characteristic inhalation maneuvers used in the clinic, produced results that correlated with regional drug deposition estimates from scintigraphy across a group of different DPIs.

Revealed preference tests for collective household behavior

NARCIS (Netherlands)

Cherchye, L.J.H.; de Rock, B.; Vermeulen, F.M.P.; Verriest, E.; Molina, J.A.

2011-01-01

This chapter contains a state of the art of revealed preference tests for consistency of observed household behavior with Pareto efficiency. These tests are entirely nonparametric, since they do not require any assumptions regarding the parametric form of individual preferences or the intrahousehold

Evaluation of the Ortho-Clinical Diagnostics Vitros ECi Anti-HCV test: comparison with three other methods.

Science.gov (United States)

Watterson, Jeannette M; Stallcup, Paulina; Escamilla, David; Chernay, Patrick; Reyes, Alfred; Trevino, Sylvia C

2007-01-01

After observing a high incidence of low positive hepatitis C virus (HCV) antibody screens by the Ortho-Clinical Vitros ECi test (Orthoclinical Diagnostics, Raritan, NJ), we compared results against those obtained using another chemiluminescent analyzer, as well as two U.S. Food and Drug Administration (FDA)-approved confirmatory methodologies. To ascertain the true anti-HCV status of samples deemed low-positive by the Ortho-Clinical Vitros ECi test, we tested samples using the ADVIA Centaur HCV screen test (Siemens Medical Solutions Diagnostics), the Chiron recombinant immunoblot assay (RIBA) test (Chiron Corp., Emeryville, CA), and the Roche COBAS Amplicor HCV qualitative test (Roche Diagnostics, Indianapolis, IN) in a series of studies. Of 94 specimens positive by Vitros ECi, 19% were observed to be negative by Centaur. A separate study of 91 samples with signal-to-cutoff (s/co) values less than 8.0 showed that all but one was negative for HCV ribonucleic acid (RNA). In comparison with RIBA, 100% (77) samples positive by the Vitros ECi test with s/co values less than 12.0 were negative or indeterminate by RIBA. A final study comparing all four methods side-by-side showed 63% disagreement by Centaur for Vitros ECi low-positive samples, 75% disagreement by RIBA, and 97% disagreement by polymerase chain reaction (PCR). In conclusion, the Ortho-Clinical Vitros ECi Anti-HCV test yields a high rate of false-positive results in the low s/co range in our patient population. (c) 2007 Wiley-Liss, Inc.

Implementation of in vitro replacement technologies in regulatory drug testing - An innovation systems perspective

NARCIS (Netherlands)

Kooijman, M.; Van Meer, P.J.K.; Moors, E.H.M.; Hekkert, M.P.; Schellekens, H.

2011-01-01

The replacement of in vivo methods by in vitro methods in regulatory drug testing is rare. The aim of this research is to identify barriers and drivers of the replacement of in vivo methods by in vitro methods in Europe. We studied two cases. The first case is the Draize eye test. Since 2009, the in

In vitro tests and ethnopharmacological investigations: wound healing as an example.

Science.gov (United States)

Houghton, P J; Hylands, P J; Mensah, A Y; Hensel, A; Deters, A M

2005-08-22

In vitro tests are now widely employed in ethnopharmacological research because of ethical reasons and their usefulness in bioactive-guided fractionation and determination of active compounds. For many disease conditions, a variety of in vitro tests can now be employed as the biochemical mechanisms underlying disease and healing processes are understood. Approaches to the in vitro investigations of wound healing processes are exemplified by studies on extracts of Buddleja species and three Ghanaian species Spathodea campanulata, Commelina diffusa and Secamone afzelii. Most studies have been carried out on Buddleja officinalis or Buddleja globosa. The extracts have been shown to have anti-inflammatory and antioxidant properties due to flavonoids, triterpenoids, diterpenoids and caffeic acid derivatives. There appears to a slight effect on proliferation of fibroblasts at lower concentrations, but this was not significant, and higher concentrations appeared to be cytotoxic. Novel findings are the ability of Buddleja globosa leaf extracts to induce differentiation in keratinocytes and to alter the profile of proteins produced by cultured fibroblasts. Extracts also had some effect on lattice contraction. The three Ghanaian species examined show a mixture of antimicrobial and antioxidant activities. The evolution over recent years of tests for wound healing, from in vivo tests to cell-based systems and chemical reactions and on to investigations into effects on secondary messengers and protein expression, is described.

A reproducible accelerated in vitro release testing method for PLGA microspheres.

Science.gov (United States)

Shen, Jie; Lee, Kyulim; Choi, Stephanie; Qu, Wen; Wang, Yan; Burgess, Diane J

2016-02-10

The objective of the present study was to develop a discriminatory and reproducible accelerated in vitro release method for long-acting PLGA microspheres with inner structure/porosity differences. Risperidone was chosen as a model drug. Qualitatively and quantitatively equivalent PLGA microspheres with different inner structure/porosity were obtained using different manufacturing processes. Physicochemical properties as well as degradation profiles of the prepared microspheres were investigated. Furthermore, in vitro release testing of the prepared risperidone microspheres was performed using the most common in vitro release methods (i.e., sample-and-separate and flow through) for this type of product. The obtained compositionally equivalent risperidone microspheres had similar drug loading but different inner structure/porosity. When microsphere particle size appeared similar, porous risperidone microspheres showed faster microsphere degradation and drug release compared with less porous microspheres. Both in vitro release methods investigated were able to differentiate risperidone microsphere formulations with differences in porosity under real-time (37 °C) and accelerated (45 °C) testing conditions. Notably, only the accelerated USP apparatus 4 method showed good reproducibility for highly porous risperidone microspheres. These results indicated that the accelerated USP apparatus 4 method is an appropriate fast quality control tool for long-acting PLGA microspheres (even with porous structures). Copyright © 2015 Elsevier B.V. All rights reserved.

The sensitivity testing of Wilms' tumors to cytostatic agents with an autoradiographic in vitro short-term test

International Nuclear Information System (INIS)

Willnow, U.

1984-01-01

Sensitivity of 15 Wilms' tumors in children was tested towards cytostatic agents in vitro by means of an autoradiographic short-term test. Sensitivity was measured as the magnitude of the inhibition of 3 H-thymidine or 3 H-uridine incorporation. The test was performed with Adriamycin, Actinomycin D, Daunomycin, Bleomycin, Cyclophosphamide, Ifosfamide, Trenimon, and Arabinosylcytosine. None of the tumors is resistant to all substances, they are responsive against 2 or more drugs. The most effective drugs tested are Adriamycin, Actinomycin D and Cyclophosphamide. The tumors show a marked individual sensitivity pattern. This behavior is explained mainly by the usually high proliferative activity of Wilms' tumors. The possibilities and limits of long-term and short-term methods for sensitivity testing are discussed critically. For the evaluation of the results of in vitro testing and in vivo effectiveness the close correlation should be considered between the type of cytostatic agent and proliferation kinetics of the tumor, cytostatic agent and effect on tumor metabolism as well as the effect of the cytostatics and the nucleic acid precursors used for the short-term test. Despite the methodological limitations preclinical testing should be preferred to unselected chemotherapy. (author)

In vitro chemical and cellular tests applied to uranium trioxide with different hydration states

International Nuclear Information System (INIS)

Ansoborlo, E.; Chalabreysse, J.; Henge-Napoli, M.H.; Pujol, E.

1992-01-01

A simple and rapid in vitro chemical solubility test applicable to industrial uranium trioxide (UO 3 ) was developed together with two in vitro cellular tests using rat alveolar macrophages maintained either in gas phase or in alginate beads at 37 degrees C. Industrial UO 3 was characterized by particle size, X-ray, and IR spectra, and chemical transformation (e.g., aging and hydration of the dust) was also studied. Solvents used for the in vitro chemical solubility study included carbonates, citrates, phosphates, water, Eagle's basal medium, and Gamble's solution (simulated lung fluid), alone, with oxygen, or with superoxide ions. Results, expressed in terms of the half-time of dissolution, according to International Commission on Radiological Protection (ICRP) classification (D,W,Y), varied for different hydration states of UO 3 , showing a lower solubility of hydrated UO 3 in solvents compared to basic UO 3 or UO 3 heated at 450 degrees C. Two in vitro cellular tests on cultured rat alveolar macrophages (cells maintained in gas phase and cells immobilized in alginate beads) were used on the same UO 3 samples and generally showed a lower solution transfer rate in the presence of macrophages than in the culture medium alone. The results of in vitro chemical and cellular tests were compared, with four main conclusions; a good reproducibility of the three tests in Eagle's basal medium of the effect of hydration state on solubility, the classification of UO 3 in terms of ICRP solubility criteria, and the ability of macrophoges to decrease uranium solubility in medium. 16 refs., 3 figs., 4 tabs

Validation of artificial skin equivalents as in vitro testing systems

Science.gov (United States)

Schmitt, Robert; Marx, Ulrich; Walles, Heike; Schober, Lena

2011-03-01

With the increasing complexity of the chemical composition of pharmaceuticals, cosmetics and everyday substances, the awareness of potential health issues and long term damages for humanoid organs is shifting into focus. Artificial in vitro testing systems play an important role in providing reliable test conditions and replacing precarious animal testing. Especially artificial skin equivalents ASEs are used for a broad spectrum of studies like penetration, irritation and corrosion of substances. One major challenge in tissue engineering is the qualification of each individual ASE as in vitro testing system. Due to biological fluctuations, the stratum corneum hornified layer of some ASEs may not fully develop or other defects might occur. For monitoring these effects we developed an fully automated Optical Coherence Tomography device. Here, we present different methods to characterize and evaluate the quality of the ASEs based on image and data processing of OCT B-scans. By analysing the surface structure, defects, like cuts or tears, are detectable. A further indicator for the quality of the ASE is the morphology of the tissue. This allows to determine if the skin model has reached the final growth state. We found, that OCT is a well suited technology for automatically characterizing artificial skin equivalents and validating the application as testing system.

Evaluation of seven in vitro alternatives for ocular safety testing.

Science.gov (United States)

Bruner, L H; Kain, D J; Roberts, D A; Parker, R D

1991-07-01

Seven in vitro assays were evaluated to determine if any were useful as screening procedures in ocular safety assessment. Seventeen test materials (chemicals, household cleaners, hand soaps, dishwashing liquids, shampoos, and liquid laundry detergents) were tested in each assay. In vivo ocular irritation scores for the materials were obtained from existing rabbit low volume eye test (LVET) data. The seven assays evaluated included the silicon microphysiometer (SM), luminescent bacteria toxicity test (LBT), neutral red assay (NR), total protein assay (TP), Tetrahymena thermophila motility assay (TTMA), bovine eye/chorioallantoic membrane assay (BE/CAM), and the EYTEX system (ETS). For the seventeen materials used in this study there was a significant correlation between the in vivo irritant potential and in vitro data for all the tests except the EYTEX System (SM, r = -0.87; LBT, r = -0.91; NR, r = -0.85; TTMA, r = 0.78; TP, r = -0.86; ETS, r = 0.29). The irritation classifications provided by the BE/CAM also did not correspond with the actual in vivo irritancy potential of the test materials. The result of this study suggested it may be possible to classify materials into broad irritancy categories with some of the assays. This would allow their use as screens prior to limited in vivo confirmation in the ocular safety assessment process.

The current limitations of in vitro genotoxicity testing and their relevance to the in vivo situation.

Science.gov (United States)

Nesslany, Fabrice

2017-08-01

The standard regulatory core battery of genotoxicity tests generally includes 2 or 3 validated tests with at least one in vitro test in bacteria and one in vitro test on cell cultures. However, limitations in in vitro genotoxicity testing may exist at many levels. The knowledge of the underlying mechanisms of genotoxicity is particularly useful to assess the level of relevance for the in vivo situation. In order to avoid wrong conclusions regarding the actual genotoxicity status of any test substance, it appears very important to be aware of the various origins of related bias leading to 'false positives and negatives' by using in vitro methods. Among these, mention may be made on the metabolic activation system, experimental (extreme) conditions, specificities of the test systems implemented, cell type used etc. The knowledge of the actual 'limits' of the in vitro test systems used is clearly an advantage and may contribute to avoid some pitfalls in order to better assess the level of relevance for the in vivo situation. Copyright © 2016. Published by Elsevier Ltd.

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

Science.gov (United States)

Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

2016-12-01

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

Science.gov (United States)

Thabet, Ahmed; Zhang, Runhui; Alnassan, Alaa-Aldin; Daugschies, Arwid; Bangoura, Berit

2017-01-15

Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC 95 , MIC 50/95 ) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%I SIA and%I RIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. Polyether ionophores applied in the RIA were highly effective at MIC 95 against Ref-1 and Ref-2 (%I RIA ≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%I RIA animal model (p89%) against all strains used in this study. However, adjusted GI (GI adj ) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

In-vitro orthodontic bond strength testing : A systematic review and meta-analysis

NARCIS (Netherlands)

Finnema, K.J.; Ozcan, M.; Post, W.J.; Ren, Y.J.; Dijkstra, P.U.

INTRODUCTION: The aims of this study were to systematically review the available literature regarding in-vitro orthodontic shear bond strength testing and to analyze the influence of test conditions on bond strength. METHODS: Our data sources were Embase and Medline. Relevant studies were selected

In vitro antifungal susceptibility testing of Scopulariopsis brevicaulis strains using agar diffusion method.

Science.gov (United States)

Skóra, Magdalena; Macura, Anna B

2011-01-01

The genus Scopulariopsis is a common soil saprotroph and has been isolated from air, organic waste and also from plant, animal and human tissues. Scopulariopsis has mainly been associated in humans with superficial mycoses, but it has also been described as the cause of subcutaneous and invasive infections. The most common aetiological agent of infections in humans is Scopulariopsis brevicaulis. This species has been reported to be resistant in vitro to broad-spectrum antifungal agents available today. The aim of the study was to establish in vitro antifungal susceptibility of 35 S. brevicaulis strains against amphotericin B (AMB), flucytosine (FC), caspofungin (CAS), terbinafine (TER), ciclopirox (CIC), voriconazole (VOR), clotrimazole (CTR), miconazole (MCZ), econazole (ECO), ketoconazole (KET), itraconazole (ITR), and fluconazole (FLU). Antifungal susceptibility tests were evaluated by an agar diffusion method (Neo-Sensitabs, Rosco, Denmark). AMB, FC, CAS, ITR and FLU showed no antifungal activity against S. brevicaulis. TER, CIC, CTR, KET, VOR, ECO, and MCZ revealed inhibitory activity for S. brevicaulis, but it varied for each of the drugs. The best antifungal effect was observed for TER and CIC. All isolates had large inhibition zones for TER and CIC. CTR was also inhibitory for all tested S. brevicaulis isolates, but the diameters of inhibition zones were smaller than for TER and CIC. Nearly 89% isolates showed inhibition zones for KET and the mean diameter of the inhibition zone was comparable to CTR. The least antifungal activity exhibited VQR, ECO and MCZ. Because of the multiresistance of S. brevicaulis, infections due to this species may not respond to particular antifungal treatment and other therapeutic approaches should be considered, e.g., combined therapy and/or surgery.

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability.

Science.gov (United States)

White, John; Mascarenhas, Anjali; Pereira, Ligia; Dash, Rashmi; Walke, Jayashri T; Gawas, Pooja; Sharma, Ambika; Manoharan, Suresh Kumar; Guler, Jennifer L; Maki, Jennifer N; Kumar, Ashwani; Mahanta, Jagadish; Valecha, Neena; Dubhashi, Nagesh; Vaz, Marina; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K

2016-01-22

Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. Culture adaptation of P. falciparum in a field setting is formally shown to be

Assuring safety without animal testing: the case for the human testis in vitro.

Science.gov (United States)

Chapin, Robert E; Boekelheide, Kim; Cortvrindt, Rita; van Duursen, Majorie B M; Gant, Tim; Jegou, Bernard; Marczylo, Emma; van Pelt, Ans M M; Post, Janine N; Roelofs, Maarke J E; Schlatt, Stefan; Teerds, Katja J; Toppari, Jorma; Piersma, Aldert H

2013-08-01

From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable. Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4

DEFF Research Database (Denmark)

Williams, Hywel D; Sassene, Philip; Kleberg, Karen

2014-01-01

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near...... a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF...... development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance...

Evaluation of genotoxicity of nitrile fragrance ingredients using in vitro and in vivo assays.

Science.gov (United States)

Bhatia, S P; Politano, V T; Api, A M

2013-09-01

Genotoxicity studies were conducted on a group of 8 fragrance ingredients that belong to the nitrile family. These nitriles are widely used in consumer products however there is very limited data in the literature regarding the genotoxicity of these nitriles. The 8 nitriles were assessed for genotoxicity using an Ames test, in vitro chromosome aberration test or in vitro micronucleus test. The positive results observed in the in vitro tests were further investigated using an in vivo micronucleus test. The results from these different tests were compared and these 8 nitriles are not considered to be genotoxic. Dodecanitrile and 2,2,3-trimethylcyclopent-3-enylacetonitrile were negative in the in vitro chromosome aberration test and in vitro micronucleus test, respectively. While citronellyl nitrile, 3-methyl-5-phenylpentanenitrile, cinnamyl nitrile, and 3-methyl-5-phenylpent-2-enenitrile revealed positive results in the in vitro tests, but confirmatory in vivo tests determined these nitriles to be negative in the in vivo micronucleus assay. The remaining two nitriles (benzonitrile and α-cyclohexylidene benzeneacetonitrile) were negative in the in vivo micronucleus test. This study aims to evaluate the genotoxicity potential of these nitriles as well as enrich the literature with genotoxicity data on fragrance ingredients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of an in vitro Endotoxin Test for Monoolein–Water ...

African Journals Online (AJOL)

Purpose: Drugs that are administered by parenteral route must be apyrogenic. The aim of this study was to develop an in vitro endotoxin test for liquid crystalline gels for use as implants, using a monoolein–water liquid crystalline gel as a model. Methods: The gel-clot technique was used. The gel was dissolved first in ...

An in vitro human skin test for assessing sensitization potential.

Science.gov (United States)

Ahmed, S S; Wang, X N; Fielding, M; Kerry, A; Dickinson, I; Munuswamy, R; Kimber, I; Dickinson, A M

2016-05-01

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.

In vivo and in vitro testing for selenium and selenium compounds bioavailability assessment in foodstuff.

Science.gov (United States)

Moreda-Piñeiro, Jorge; Moreda-Piñeiro, Antonio; Bermejo-Barrera, Pilar

2017-03-04

The assessment of selenium and selenium species bioavailability in foodstuff is of special concern on the context of human nutrition. In vivo (human and animal), and in vitro tests are important approaches for estimating the bioavailability of toxic and essential compounds to humans. An overview on in vivo and in vitro bioavailability assays for releasing selenium and selenium species in foodstuffs is summarized. Se and Se species content in a foodstuff critically influence Se bioavailability and bioactivity to humans and animals. Se bioavailability is affected by foodstuff-matrix major composition and minor components. Foodstuffs processing and/or treatments could enhancement or decrease Se bioavailability. Experimental conditions such as the selection of healthy status of examined people (in in vivo humans approaches), the selection of animal model (in vivo animals approaches), or the selection of GI conditions (in in vitro tests) could determines the results. Thus, international standardized protocol for in vivo and in vitro approaches assessment is mandatory.

THE ANTIGEN-SPECIFIC CELL IN VITRO TESTS FOR POST-VACCINATION ANTIPLAGUE IMMUNITY FORMATION

Directory of Open Access Journals (Sweden)

A. N. Kulichenko

2017-01-01

Full Text Available The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+ and late (CD45+CD3+HLA-DR+ lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37% and at 3 (47.41% months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days and late (6 months periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.

Selective localization of different types of opiate receptors in hippocampus as revealed by in vitro autoradiography

International Nuclear Information System (INIS)

Duka, T.; Wuester, M.; Schubert, P.; Stoiber, R.; Herz, A.

1981-01-01

The visualization of opiate binding sites within the hippocampus of the rat has been achieved by means of an in vitro autoradiography. In line with the concept of multiple opiate receptors, different opioid agonists revealed a particular distribution pattern. Whereas the selective delta-receptor agonist [ 3 H]D-Ala 2 , D-Leu 5 -enkephalin specifically labelled binding sites in the CA 2 area, [ 3 H]etorphine grains displayed a uniform dense distribution throughout the pyramidal cell layers from CA 1 to CA 4 . (Auth.)

Possibilities for in-vitro testing of controlled-release parenterals.

OpenAIRE

Kožák, Jan

2014-01-01

The objective of this study was to start with the development of a gel-based medium simulating intramuscular environment for in-vitro drug release testing as an alternative to currently used water-based media. A pork meat was selected as a model of the muscle tissue. At the first part of the study, diffusion rate was proposed as the most determining factor of drug release in gel-based medium. Consequently, the diffusion rate of sodium fluorescein in pork meat was compared with the diffusion r...

Etest cannot be recommended for in vitro susceptibility testing of mucorales.

Science.gov (United States)

Caramalho, Rita; Maurer, Elisabeth; Binder, Ulrike; Araújo, Ricardo; Dolatabadi, Somayeh; Lass-Flörl, Cornelia; Lackner, Michaela

2015-01-01

Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales species in vitro, while the activities of posaconazole were more restricted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

In Vitro Screening of Environmental Chemicals for Targeted Testing Prioritization: The ToxCast Project

OpenAIRE

Judson, Richard S.; Houck, Keith A.; Kavlock, Robert J.; Knudsen, Thomas B.; Martin, Matthew T.; Mortensen, Holly M.; Reif, David M.; Rotroff, Daniel M.; Shah, Imran; Richard, Ann M.; Dix, David J.

2009-01-01

Background Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use, and the thousands of environmental chemicals lacking toxicity data. The U.S. Environmental Protection Agency?s ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives This project aims to evaluate the use of in vitro assays for understanding the ty...

In vitro screening of environmental chemicals for targeted testing prioritization: the ToxCast project.

Science.gov (United States)

Judson, Richard S; Houck, Keith A; Kavlock, Robert J; Knudsen, Thomas B; Martin, Matthew T; Mortensen, Holly M; Reif, David M; Rotroff, Daniel M; Shah, Imran; Richard, Ann M; Dix, David J

2010-04-01

Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use, and the thousands of environmental chemicals lacking toxicity data. The U.S. Environmental Protection Agency's ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. We tested 309 mostly pesticide active chemicals in 467 assays across nine technologies, including high-throughput cell-free assays and cell-based assays, in multiple human primary cells and cell lines plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Chemicals displayed a broad spectrum of activity at the molecular and pathway levels. We saw many expected interactions, including endocrine and xenobiotic metabolism enzyme activity. Chemicals ranged in promiscuity across pathways, from no activity to affecting dozens of pathways. We found a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also found associations between a small set of in vitro assays and rodent liver lesion formation. This approach promises to provide meaningful data on the thousands of untested environmental chemicals and to guide targeted testing of environmental contaminants.

RNA biology in a test tube--an overview of in vitro systems/assays.

Science.gov (United States)

Roca, Xavier; Karginov, Fedor V

2012-01-01

In vitro systems have provided a wealth of information in the field of RNA biology, as they constitute a superior and sometimes the unique approach to address many important questions. Such cell-free methods can be sorted by the degree of complexity of the preparation of enzymatic and/or regulatory activity. Progress in the study of pre-mRNA processing has largely relied on traditional in vitro methods, as these reactions have been recapitulated in cell-free systems. The pre-mRNA capping, editing, and cleavage/polyadenylation reactions have even been reconstituted using purified components, and the enzymes responsible for catalysis have been characterized by such techniques. In vitro splicing using nuclear or cytoplasmic extracts has yielded clues on spliceosome assembly, kinetics, and mechanisms of splicing and has been essential to elucidate the function of splicing factors. Coupled systems have been important to functionally connect distinct processes, like transcription and splicing. Extract preparation has also been adapted to cells from a variety of tissues and species, revealing general versus species-specific mechanisms. Cell-free assays have also been applied to newly discovered pathways such as those involving small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs). The first two pathways have been well characterized largely by in vitro methods, which need to be developed for piRNAs. Finally, new techniques, such as single-molecule studies, are continuously being established, providing new and important insights into the field. Thus, in vitro approaches have been, are, and will continue being at the forefront of RNA research. Copyright © 2012 John Wiley & Sons, Ltd.

10 CFR 31.11 - General license for use of byproduct material for certain in vitro clinical or laboratory testing.

Science.gov (United States)

2010-01-01

... therefrom, to human beings or animals. (2) Iodine-131, in units not exceeding 10 microcuries each for use in... microcuries each for use in in vitro clinical or laboratory tests not involving internal or external... (tritium), in units not exceeding 50 microcuries each for use in in vitro clinical or laboratory tests not...

Recommendations for In Vitro and In Vivo Testing of Magnetic Nanoparticle Hyperthermia Combined with Radiation Therapy

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Spiridon V. Spirou

2018-05-01

Full Text Available Magnetic nanoparticle (MNP-mediated hyperthermia (MH coupled with radiation therapy (RT is a novel approach that has the potential to overcome various practical difficulties encountered in cancer treatment. In this work, we present recommendations for the in vitro and in vivo testing and application of the two treatment techniques. These recommendations were developed by the members of Working Group 3 of COST Action TD 1402: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy (“Radiomag”. The purpose of the recommendations is not to provide definitive answers and directions but, rather, to outline those tests and considerations that a researcher must address in order to perform in vitro and in vivo studies. The recommendations are divided into 5 parts: (a in vitro evaluation of MNPs; (b in vitro evaluation of MNP-cell interactions; (c in vivo evaluation of the MNPs; (d MH combined with RT; and (e pharmacokinetic studies of MNPs. Synthesis and characterization of the MNPs, as well as RT protocols, are beyond the scope of this work.

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

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Albrekt Ann-Sofie

2011-08-01

Full Text Available Abstract Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

Science.gov (United States)

2011-01-01

Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests. PMID:21824406

Magnesium alloys: predicting in vivo corrosion with in vitro immersion testing.

Science.gov (United States)

Walker, Jemimah; Shadanbaz, Shaylin; Kirkland, Nicholas T; Stace, Edward; Woodfield, Tim; Staiger, Mark P; Dias, George J

2012-05-01

Magnesium (Mg) and its alloys have been proposed as degradable replacements to commonly used orthopedic biomaterials such as titanium alloys and stainless steel. However, the corrosion of Mg in a physiological environment remains a difficult characteristic to accurately assess with in vitro methods. The aim of this study was to identify a simple in vitro immersion test that could provide corrosion rates similar to those observed in vivo. Pure Mg and five alloys (AZ31, Mg-0.8Ca, Mg-1Zn, Mg-1Mn, Mg-1.34Ca-3Zn) were immersed in either Earle's balanced salt solution (EBSS), minimum essential medium (MEM), or MEM-containing 40 g/L bovine serum albumin (MEMp) for 7, 14, or 21 days before removal and assessment of corrosion by weight loss. This in vitro data was compared to in vivo corrosion rates of the same materials implanted in a subcutaneous environment in Lewis rats for equivalent time points. The results suggested that, for the alloys investigated, the EBSS buffered with sodium bicarbonate provides a rate of degradation comparable to those observed in vivo. In contrast, the addition of components such as (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), vitamins, amino acids, and albumin significantly increased corrosion rates. Based on these findings, it is proposed that with this in vitro protocol, immersion of Mg alloys in EBSS can be used as a predictor of in vivo corrosion. Copyright © 2012 Wiley Periodicals, Inc.

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

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Ingo Ahrens

Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

In vitro antitumour activity, safety testing and subcellular distribution of two poly[oxyethylene(aminophosphonate-co-H-phosphonate]s in Ehrlich ascites carcinoma and BALB/c 3T3 cell culture systems

Directory of Open Access Journals (Sweden)

Ani Georgieva

2016-01-01

Full Text Available Two polyphosphoesters containing anthracene-derived aminophosphonate and hydrophilic H-phosphonate repeating units, poly[oxyethylene(aminophosphonate-co-H-phosphonate]s (1 and 2, were tested for the in vitro antitumour activity on cell cultures derived from ascitic form of Ehrlich mammary adenocarcinoma by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT-dye reduction assay. The in vitro safety testing of the copolymers was performed by BALB/c 3T3 neutral red uptake assay. A study on their uptake and subcellular distribution in non-tumourigenic and tumour cells was performed by means of fluorescence microscopy. Both copolymers showed significant antitumour activity towards Ehrlich ascites carcinoma (EAC cells. However, the in vitro safety testing revealed significant toxicity of polymer 2 to BALB/c 3T3 mouse embryo cells. In contrast, polymer 1 showed complete absence of cytotoxicity to BALB/c 3T3 cells. The fluorescent studies showed that the substances were diffusely distributed in the cytoplasm in both cell culture systems. As opposed to BALB/c 3T3 cells, in EAC cells, intense fluorescent signal was observed in the nuclei and in the perinuclear region. The tested polyphosphoesters are expected to act under physiological conditions as prodrugs of aminophosphonates.

ID-check: Online concealed information test reveals true identity

NARCIS (Netherlands)

Verschuere, B.; Kleinberg, B.

2016-01-01

The Internet has already changed people's lives considerably and is likely to drastically change forensic research. We developed a web-based test to reveal concealed autobiographical information. Initial studies identified a number of conditions that affect diagnostic efficiency. By combining these

In vitro and in vivo tests of PLA/d-HAp nanocomposite

Science.gov (United States)

Thom Nguyen, Thi; Hoang, Thai; Mao Can, Van; Son Ho, Anh; Hai Nguyen, Song; Thu Trang Nguyen, Thi; Pham, Thi Nam; Phuong Nguyen, Thu; Le Hien Nguyen, Thi; Thanh Dinh Thi, Mai

2017-12-01

The bioactivity of the PLA/d-HAp nanocomposite with 30 wt.% d-HAp was evaluated by in vitro tests and indicated that after 7 immersion days in SBF solution, PLA amorphous part was hydrolyzed and PLA crystal part was remained. The formation of apatite on the surface of the material was observed. The in vivo test results of PLA/d-HAp nanocomposite (70/30 wt/wt) on femur of dogs displayed that 3 months after grafting, the materials did not induce any osteitis, osteomyelitis or structural abnormalities. The histological and x-ray image demonstrated a growth of the bone into the material area, while osteitis and osteomyelitis were not observed.

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

Science.gov (United States)

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

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

Science.gov (United States)

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

Chapter 31: Common in vitro tests for allergy and immunology.

Science.gov (United States)

Makhija, Melanie; O'Gorman, Maurice R G

2012-01-01

Allergen-specific IgE antibody is the most commonly ordered in vitro test in the practice of allergy and is used to diagnose type I hypersensitivity reactions to foods or reactivity to aeroallergens in patients with relative contraindications to skin-prick testing such as dermatographism. The Phadebas radioallergosorbent test (RAST; Pharmacia, Uppsala, Sweden) was the first assay reported for the detection of the allergen-specific IgE antibody. In a RAST, antigen (allergen) is bound to a solid phase, such as a paper disk, and then incubated with human serum. A buffer wash removes unbound serum proteins, and radiolabeled anti-human IgE is added to detect bound IgE, if present. The results are reported in arbitrary units of IgE per milliliter of serum. The term RAST was originally a brand name but it is now often used colloquially (and incorrectly) to describe any in vitro assay for allergen-specific IgE. Total serum IgE can be measured and is helpful in determining atopic presentations such as in allergic bronchopulmonary aspergillosis or in patients with persistent asthma who are candidates for monoclonal anti-IgE antibody therapy with, omalizumab. In patients with recurrent bacterial infections of the sinopulmonary tract, the basic humoral immune system testing includes measuring quantitative immunoglobulins (IgG, IgA, and IgM) and comparing them to age-matched normal ranges. Most clinical laboratories use nephelometry to measure immunoglobulin levels quantitatively. Nephelometry detects either the rate or the end point of soluble immune complex formation (the IgG in sera complexes with an anti-IgG antibody forming a classic immunoprecipitation reaction) by monitoring the scatter of transmitted light. The most common method for the screening of cellular immunodeficiency involved the measurement of the absolute and relative representation of the major lymphocyte subsets, T-cells, T-helper cells, T-cytotoxic cells, B-cells and NK-cells.

Radiosensitivity of lymphocytes in vitro

International Nuclear Information System (INIS)

Albrecht, S.

1979-01-01

The radiation-induced impairment of human T-lymphocytes was studied after in vitro exposure to 25.8 - 825.6 mC/kg (100 - 3200 R) of 60 Co γ-radiation by ascertaining the change in lymphocyte response to phytohaemagglutin stimulation. Following methods were used: (1) measurement of 3 H-thymidine uptake, (2) E-rosette test, and (3) morphological examination of transformed T-cells. The results revealed a dose-dependent decline in T-cell number which was still somewhat more marked with lymphocytes purified over Ficoll-Isopaque prior to irradiation. (author)

Accelerated in vitro release testing method for naltrexone loaded PLGA microspheres.

Science.gov (United States)

Andhariya, Janki V; Choi, Stephanie; Wang, Yan; Zou, Yuan; Burgess, Diane J; Shen, Jie

2017-03-30

The objective of the present study was to develop a discriminatory and reproducible accelerated release testing method for naltrexone loaded parenteral polymeric microspheres. The commercially available naltrexone microsphere product (Vivitrol ® ) was used as the testing formulation in the in vitro release method development, and both sample-and-separate and USP apparatus 4 methods were investigated. Following an in vitro drug stability study, frequent media replacement and addition of anti-oxidant in the release medium were used to prevent degradation of naltrexone during release testing at "real-time" (37°C) and "accelerated" (45°C), respectively. The USP apparatus 4 method was more reproducible than the sample-and-separate method. In addition, the accelerated release profile obtained using USP apparatus 4 had a shortened release duration (within seven days), and good correlation with the "real-time" release profile. Lastly, the discriminatory ability of the developed accelerated release method was assessed using compositionally equivalent naltrexone microspheres with different release characteristics. The developed accelerated USP apparatus 4 release method was able to detect differences in the release characteristics of the prepared naltrexone microspheres. Moreover, a linear correlation was observed between the "real-time" and accelerated release profiles of all the formulations investigated, suggesting that the release mechanism(s) may be similar under both conditions. These results indicate that the developed accelerated USP apparatus 4 method has the potential to be an appropriate fast quality control tool for long-acting naltrexone PLGA microspheres. Copyright © 2017 Elsevier B.V. All rights reserved.

ACETURATO DE DIMINAZENO LIPOSSOMAL NO TRATAMENTO DA INFECÇÃO POR Trypanosoma evansi: TESTES in vitro E in vivo

OpenAIRE

Camila Belmonte Oliveira

2014-01-01

Este estudo teve como objetivo desenvolver e testar lipossomas de aceturato de diminazeno em testes in vitro e in vivo visando o controle de Trypanosoma evansi. O teste in vitro foi realizado em meio de cultura nas concentrações de 0,25, 0,5, 1, 2 e 3 μg/mL de aceturato de diminazeno convencional (C-DMZ) e lipossomal (L-DMZ). Para os testes in vivo foram utilizados 114 ratos (Rattus norvegicus) divididos em seis grupos (A, B, C, D, E e F) em dois experimentos, um para aval...

In vitro and in vivo antitrypanosomal activity of Xanthium strumarium leaves.

Science.gov (United States)

Talakal, T S; Dwivedi, S K; Sharma, S R

1995-12-15

Antitrypanosomal activity of crude 50% ethanolic extract of Xanthium strumarium leaves was studied in vitro and in vivo. The extract exhibited trypanocidal activity at all four concentrations tested i.e. 5, 50, 500 and 1000 micrograms/ml, in vitro. In vivo trial revealed that the extract exerted antitrypanosomal effect at dosage of 100, 300 and 1000 mg/kg, intraperitoneally. At 100 and 300 mg/kg doses the survival period of the Trypanosoma evansi infected mice was significantly prolonged. However, the extract was found to be toxic to the animals at 1000 mg/kg dose.

Correlation of In Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study.

Science.gov (United States)

Soeteman-Hernández, Lya G; Fellows, Mick D; Johnson, George E; Slob, Wout

2015-12-01

In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement). © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

NARCIS (Netherlands)

Park, M.V.; Annema, W.; Salvati, A.; Lesniak, A.; Elsaesser, A.; Barnes, C.; McKerr, G.; Howard, C.; Lynch, I.; Dawson, K.; Piersma, A.H.; de Jong, W.H.

2009-01-01

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining

An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine.

Science.gov (United States)

Asciolla, James J; Miele, Matthew M; Hendrickson, Ronald C; Resh, Marilyn D

2017-08-18

Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC 50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O -acyl transferase family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Validation of Alternative In Vitro Methods to Animal Testing: Concepts, Challenges, Processes and Tools.

Science.gov (United States)

Griesinger, Claudius; Desprez, Bertrand; Coecke, Sandra; Casey, Warren; Zuang, Valérie

This chapter explores the concepts, processes, tools and challenges relating to the validation of alternative methods for toxicity and safety testing. In general terms, validation is the process of assessing the appropriateness and usefulness of a tool for its intended purpose. Validation is routinely used in various contexts in science, technology, the manufacturing and services sectors. It serves to assess the fitness-for-purpose of devices, systems, software up to entire methodologies. In the area of toxicity testing, validation plays an indispensable role: "alternative approaches" are increasingly replacing animal models as predictive tools and it needs to be demonstrated that these novel methods are fit for purpose. Alternative approaches include in vitro test methods, non-testing approaches such as predictive computer models up to entire testing and assessment strategies composed of method suites, data sources and decision-aiding tools. Data generated with alternative approaches are ultimately used for decision-making on public health and the protection of the environment. It is therefore essential that the underlying methods and methodologies are thoroughly characterised, assessed and transparently documented through validation studies involving impartial actors. Importantly, validation serves as a filter to ensure that only test methods able to produce data that help to address legislative requirements (e.g. EU's REACH legislation) are accepted as official testing tools and, owing to the globalisation of markets, recognised on international level (e.g. through inclusion in OECD test guidelines). Since validation creates a credible and transparent evidence base on test methods, it provides a quality stamp, supporting companies developing and marketing alternative methods and creating considerable business opportunities. Validation of alternative methods is conducted through scientific studies assessing two key hypotheses, reliability and relevance of the

The limits of testing particle-mediated oxidative stress in vitro in predicting diverse pathologies; relevance for testing of nanoparticles

Directory of Open Access Journals (Sweden)

Gulumian Mary

2009-04-01

Full Text Available Abstract In vitro studies with particles are a major staple of particle toxicology, generally used to investigate mechanisms and better understand the molecular events underlying cellular effects. However, there is ethical and financial pressure in nanotoxicology, the new sub-specialty of particle toxicology, to avoid using animals. Therefore an increasing amount of studies are being published using in vitro approaches and such studies require careful interpretation. We point out here that 3 different conventional pathogenic particle types, PM10, asbestos and quartz, which cause diverse pathological effects, have been reported to cause very similar oxidative stress effects in cells in culture. We discuss the likely explanation and implications of this apparent paradox, and its relevance for testing in nanotoxicology.

Potency testing of veterinary vaccines: the way from in vivo to in vitro.

Science.gov (United States)

Romberg, Judith; Lang, Stefan; Balks, Elisabeth; Kamphuis, Elisabeth; Duchow, Karin; Loos, Daniela; Rau, Henriette; Motitschke, Andreas; Jungbäck, Carmen

2012-01-01

Current quality control of inactivated animal vaccines still focuses on the potency of final products in a batch-wise manner. Animal welfare concerns as well as scientific considerations have led to the '3Rs-concept' that comprises the refinement of animal procedures, the reduction of animal numbers, and the replacement of animal models. Although the 3Rs-concept has been widely accepted as a fundamental principle, the number of approved alternatives for in vivo tests is still limited. To promote further progress, the international scientific workshop 'Potency Testing of Veterinary Vaccines: The Way from in vivo to in vitro' was held at the Paul-Ehrlich-Institut in Langen, Germany, on 01-03 December 2010. More than 130 participants from industry, academia and regulatory authorities discussed the current state of the 3Rs-concept, examples of its successful implementation as well as still existing hurdles. Special emphasis was laid on the 'consistency approach' that aims to ensure relevant quality attributes of vaccine batches by in vitro analyses during production rather than by in vivo potency tests on the final product. This report provides an overview of the insights gained, including the recommendations produced at the end of the workshop. Copyright © 2011. Published by Elsevier Ltd.. All rights reserved.

In vitro mechanical integrity of hydroxyapatite coated magnesium alloy

International Nuclear Information System (INIS)

Kannan, M Bobby; Orr, Lynnley

2011-01-01

The mechanical integrity of resorbable implants during service, especially in load bearing orthopaedic applications, is critical. The high degradation rate of resorbable magnesium and magnesium-based implants in body fluid may potentially cause premature in-service failure. In this study, a magnesium alloy (AZ91) was potentiostatically coated with hydroxyapatite at different cathodic voltages in an attempt to enhance the mechanical integrity. The mechanical integrity of the uncoated and hydroxyapatite coated alloys was evaluated after in vitro testing of the coated samples in simulated body fluid (SBF). The uncoated alloy showed 40% loss in the mechanical strength after five days exposure to SBF. However, the hydroxyapatite coated alloy exposed to SBF showed 20% improvement in the mechanical strength as compared to that of the uncoated alloy. The alloy coated potentiostatically at -2 V performed better than the -3 V coated alloy. The cross-sectional analysis of the coatings revealed relatively uniform coating thickness for the -2 V coated alloy, whereas the -3 V coated alloy exhibited areas of uneven coating. This can be attributed to the increase in hydrogen evolution on the alloy during -3 V coating as compared to -2 V coating. The scanning electron micrographs of the in vitro tested alloy revealed that hydroxyapatite coating significantly reduced the localized corrosion of the alloy, which is critical for better in-service mechanical integrity. Thus, the study suggests that the in vitro mechanical integrity of resorbable magnesium-based alloy can be improved by potentiostatic hydroxyapatite coating.

In vitro mechanical integrity of hydroxyapatite coated magnesium alloy

Energy Technology Data Exchange (ETDEWEB)

Kannan, M Bobby; Orr, Lynnley, E-mail: bobby.mathan@jcu.edu.au [Discipline of Chemical Engineering, School of Engineering and Physical Sciences, James Cook University, Townsville, Queensland 4811 (Australia)

2011-08-15

The mechanical integrity of resorbable implants during service, especially in load bearing orthopaedic applications, is critical. The high degradation rate of resorbable magnesium and magnesium-based implants in body fluid may potentially cause premature in-service failure. In this study, a magnesium alloy (AZ91) was potentiostatically coated with hydroxyapatite at different cathodic voltages in an attempt to enhance the mechanical integrity. The mechanical integrity of the uncoated and hydroxyapatite coated alloys was evaluated after in vitro testing of the coated samples in simulated body fluid (SBF). The uncoated alloy showed 40% loss in the mechanical strength after five days exposure to SBF. However, the hydroxyapatite coated alloy exposed to SBF showed 20% improvement in the mechanical strength as compared to that of the uncoated alloy. The alloy coated potentiostatically at -2 V performed better than the -3 V coated alloy. The cross-sectional analysis of the coatings revealed relatively uniform coating thickness for the -2 V coated alloy, whereas the -3 V coated alloy exhibited areas of uneven coating. This can be attributed to the increase in hydrogen evolution on the alloy during -3 V coating as compared to -2 V coating. The scanning electron micrographs of the in vitro tested alloy revealed that hydroxyapatite coating significantly reduced the localized corrosion of the alloy, which is critical for better in-service mechanical integrity. Thus, the study suggests that the in vitro mechanical integrity of resorbable magnesium-based alloy can be improved by potentiostatic hydroxyapatite coating.

In vitro antioxidant, hypoglycemic and oral glucose tolerance test of banana peels

Directory of Open Access Journals (Sweden)

V.V. Navghare

2017-08-01

Full Text Available Banana fruit is claimed to have antidiabetic effects despite its high calorie content, and its peels also contain vital phytoconstituents including gallocatechin. Previously banana pulp has been studied for antihyperglycemic effects, and in the present investigation antihyperglycemic effect of ethanolic extract of inner peels of Musa sapientum (EMS, Musa paradisiaca (EMP, Musa cavendish (EMC and Musa acuminata (EMA fruit was evaluated using oral glucose tolerance test in normoglycemic rats. In vitro antioxidant study was conducted using DPPH, H2O2 radical scavenging assay and ferric reducing power assay. Wistar rats were divided into fourteen groups and twelve groups received different doses of aforementioned extracts, while control group received gum acacia solution and remaining group received standard drug, glimepiride. All the rats received glucose load at a dose of 2 g/kg body weight. Groups treated with EMC and EMA showed significant decrease in glucose level (p < 0.01 at 150 min as compared to control group. In hypoglycemic study, only EMP 500 mg/kg, p.o. treated group revealed a significant decrease (p < 0.05 in glucose level at 120 min, while other groups did not show any sign of hypoglycemia. In glucose tolerance test, animals treated with EMC and EMA depicted dose dependent antihyperglycemic effect at 150 min while EMS and EMP showed significant reduction in plasma glucose at higher doses. In a similar fashion, EMA i.e. M. acuminata demonstrated highest antioxidant activity followed by EMC against DPPH radical. In ferric reducing power and H2O2 scavenging assay, EMA demonstrated maximal antioxidant activity when compared with other extracts.

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

Science.gov (United States)

Shivhare, Devendra; Mueller-Cajar, Oliver

2017-07-01

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice ( Oryza sativa ) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. © 2017 American Society of Plant Biologists. All Rights Reserved.

Toxicity assessment of industrial chemicals and airborne contaminants: transition from in vivo to in vitro test methods: a review.

Science.gov (United States)

Bakand, S; Winder, C; Khalil, C; Hayes, A

2005-12-01

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

In vitro metabolism and bioavailability tests for endocrine active substances: What is needed next for regulatory purposes?

Science.gov (United States)

Legistation and prospective legislative proposals internationally (may) require that chemicals be tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive in vitro are considered to be endocrine active substances (EAS) and may be puta...

Influence of HEPES buffer on the local pH and formation of surface layer during in vitro degradation tests of magnesium in DMEM

Directory of Open Access Journals (Sweden)

S. Naddaf Dezfuli

2014-10-01

Full Text Available The human body is a buffered environment where pH is effectively maintained. HEPES is a biological buffer often used to mimic the buffering activity of the body in in vitro studies on the degradation behavior of magnesium. However, the influence of HEPES on the degradation behavior of magnesium in the DMEM pseudo-physiological solution has not yet been determined. The research aimed at elucidating the degradation mechanisms of magnesium in DMEM with and without HEPES. The morphologies and compositions of surface layers formed during in vitro degradation tests for 15–3600 s were characterized. The effect of HEPES on the electrochemical behavior and corrosion tendency was determined by performing electrochemical tests. HEPES indeed retained the local pH, leading to intense intergranular/interparticle corrosion of magnesium made from powder and an increased degradation rate. This was attributed to an interconnected network of cracks formed at the original powder particle boundaries and grain boundaries in the surface layer, which provided pathways for the corrosive medium to interact continuously with the internal surfaces and promoted further dissolution. Surface analysis revealed significantly reduced amounts of precipitated calcium phosphates due to the buffering activity of HEPES so that magnesium became less well protected in the buffered environment.

In vitro cell culture, platelet adhesion tests and in vivo implant tests of plasma-polymerized para-xylene films

Energy Technology Data Exchange (ETDEWEB)

Chou, Chia-Man [Department of Surgery, Taichung Veterans General Hospital, Taiwan, ROC (China); National Yang-Ming University, Taipei, Taiwan, ROC (China); Yeh, Chou-Ming, E-mail: cmchou4301@gmail.com [Taichung Hospital, Department of Health, Executive Yuan, Taiwan, ROC (China); Chung, Chi-Jen [Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taiwan, ROC (China); He, Ju-Liang [Department of Materials Science and Engineering, Feng Chia University, Taiwan, ROC (China)

2013-09-01

Plasma-polymerized para-xylene (PPX) was developed in a previous study by adjusting the process parameters: pulse frequency of the power supply (ω{sub p}) and para-xylene monomer flow rate (f{sub p}). All the obtained PPX films exhibit an amorphous structure and present hydrophobicity (water contact angle ranging from 98.5° to 121.1°), higher film growth rate and good fibroblast cell proliferation. In this study, in vitro tests (fibroblast cell compatibility and platelet adhesion) and an in vivo animal study were performed by using PPX deposited industrial-grade silicone sheets (IGS) and compared with medical-grade silicone ones (MS), which were commonly manufactured into catheters or drainage tubes in clinical use. The results reveal that PPX deposited at high ω{sub p} or high f{sub p}, in comparison with MS, exhibit better cell proliferation and clearly shows less cell adhesion regardless of ω{sub p} and f{sub p}. PPX also exhibit a comparatively lower level of platelet adhesion than MS. In the animal study, PPX-coated IGS result in similar local tissue responses at 3, 7 and 28 days (short-term) and 84 days (long-term) after subcutaneous implantation the abdominal wall of rodents compared with respective responses to MS. These results suggest that PPX-coated industrial-grade silicone is one alternative to high cost medical-grade silicone.

In vitro cell culture, platelet adhesion tests and in vivo implant tests of plasma-polymerized para-xylene films

International Nuclear Information System (INIS)

Chou, Chia-Man; Yeh, Chou-Ming; Chung, Chi-Jen; He, Ju-Liang

2013-01-01

Plasma-polymerized para-xylene (PPX) was developed in a previous study by adjusting the process parameters: pulse frequency of the power supply (ω p ) and para-xylene monomer flow rate (f p ). All the obtained PPX films exhibit an amorphous structure and present hydrophobicity (water contact angle ranging from 98.5° to 121.1°), higher film growth rate and good fibroblast cell proliferation. In this study, in vitro tests (fibroblast cell compatibility and platelet adhesion) and an in vivo animal study were performed by using PPX deposited industrial-grade silicone sheets (IGS) and compared with medical-grade silicone ones (MS), which were commonly manufactured into catheters or drainage tubes in clinical use. The results reveal that PPX deposited at high ω p or high f p , in comparison with MS, exhibit better cell proliferation and clearly shows less cell adhesion regardless of ω p and f p . PPX also exhibit a comparatively lower level of platelet adhesion than MS. In the animal study, PPX-coated IGS result in similar local tissue responses at 3, 7 and 28 days (short-term) and 84 days (long-term) after subcutaneous implantation the abdominal wall of rodents compared with respective responses to MS. These results suggest that PPX-coated industrial-grade silicone is one alternative to high cost medical-grade silicone.

Immunochemical faecal occult blood tests have superior stability and analytical performance characteristics over guaiac-based tests in a controlled in vitro study.

LENUS (Irish Health Repository)

Lee, Chun Seng

2011-06-01

The aims of this study were (1) to determine the measurement accuracy of a widely used guaiac faecal occult blood test (gFOBT) compared with an immunochemical faecal occult blood test (iFOBT) during in vitro studies, including their analytical stability over time at ambient temperature and at 4°C; and (2) to compare analytical imprecision and other characteristics between two commercially available iFOBT methods.

Statistical comparative study on a combined radioiodine test and extended protirelin test and correlation with the common in vitro parameters of hyroid function

International Nuclear Information System (INIS)

Kraemer, H.A.

1982-01-01

Using the data of 339 patients, the following parameters of thyroid function were statistically evaluated. The in vitro parameters ET 3 U, TT 4 (D), FT 4 -index and PB 127 I and the radioiodine test with determination of PB 131 I before i.v. injection of 400 μg protirelin (DHP) and 120 minutes after the injection. There was no correlation between the percentage Change of the PB 121 I level 120 min after protirelin (DHP) administration and the percentage change of the TSH level 30 min after protirelin (DTP1) administration. The accuracies of the in vitro parameters ET 3 U, TT 4 (D) and FT 4 -index on the one hand and the extended protirelin test on the other hand were compared. (orig./MG) [de

Surface modification of nano-silica on the ligament advanced reinforcement system for accelerated bone formation: primary human osteoblasts testing in vitro and animal testing in vivo.

Science.gov (United States)

Li, Mengmeng; Wang, Shiwen; Jiang, Jia; Sun, Jiashu; Li, Yuzhuo; Huang, Deyong; Long, Yun-Ze; Zheng, Wenfu; Chen, Shiyi; Jiang, Xingyu

2015-05-07

The Ligament Advanced Reinforcement System (LARS) has been considered as a promising graft for ligament reconstruction. To improve its biocompatibility and effectiveness on new bone formation, we modified the surface of a polyethylene terephthalate (PET) ligament with nanoscale silica using atom transfer radical polymerization (ATRP) and silica polymerization. The modified ligament is tested by both in vitro and in vivo experiments. Human osteoblast testing in vitro exhibits an ∼21% higher value in cell viability for silica-modified grafts compared with original grafts. Animal testing in vivo shows that there is new formed bone in the case of a nanoscale silica-coated ligament. These results demonstrate that our approach for nanoscale silica surface modification on LARS could be potentially applied for ligament reconstruction.

Cost effectiveness of ovarian reserve testing in in vitro fertilization : a Markov decision-analytic model

NARCIS (Netherlands)

Moolenaar, Lobke M.; Broekmans, Frank J. M.; van Disseldorp, Jeroen; Fauser, Bart C. J. M.; Eijkemans, Marinus J. C.; Hompes, Peter G. A.; van der Veen, Fulco; Mol, Ben Willem J.

2011-01-01

Objective: To compare the cost effectiveness of ovarian reserve testing in in vitro fertilization (IVF). Design: A Markov decision model based on data from the literature and original patient data. Setting: Decision analytic framework. Patient(s): Computer-simulated cohort of subfertile women aged

In vitro mechanical integrity of hydroxyapatite coated magnesium alloy.

Science.gov (United States)

Kannan, M Bobby; Orr, Lynnley

2011-08-01

The mechanical integrity of resorbable implants during service, especially in load bearing orthopaedic applications, is critical. The high degradation rate of resorbable magnesium and magnesium-based implants in body fluid may potentially cause premature in-service failure. In this study, a magnesium alloy (AZ91) was potentiostatically coated with hydroxyapatite at different cathodic voltages in an attempt to enhance the mechanical integrity. The mechanical integrity of the uncoated and hydroxyapatite coated alloys was evaluated after in vitro testing of the coated samples in simulated body fluid (SBF). The uncoated alloy showed 40% loss in the mechanical strength after five days exposure to SBF. However, the hydroxyapatite coated alloy exposed to SBF showed 20% improvement in the mechanical strength as compared to that of the uncoated alloy. The alloy coated potentiostatically at -2 V performed better than the -3 V coated alloy. The cross-sectional analysis of the coatings revealed relatively uniform coating thickness for the -2 V coated alloy, whereas the -3 V coated alloy exhibited areas of uneven coating. This can be attributed to the increase in hydrogen evolution on the alloy during -3 V coating as compared to -2 V coating. The scanning electron micrographs of the in vitro tested alloy revealed that hydroxyapatite coating significantly reduced the localized corrosion of the alloy, which is critical for better in-service mechanical integrity. Thus, the study suggests that the in vitro mechanical integrity of resorbable magnesium-based alloy can be improved by potentiostatic hydroxyapatite coating. © 2011 IOP Publishing Ltd

Recommended procedures for performance testing of radiobioassay laboratories: Volume 2, In vitro samples

International Nuclear Information System (INIS)

Fenrick, H.W.; MacLellan, J.A.

1988-11-01

Draft American National Standards Institute (ANSI) Standard N13.30 (Performance Criteria for Radiobioassay) was developed for the US Department of Energy and the US Nuclear Regulatory Commission to help ensure that bioassay laboratories provide accurate and consistent results. The draft standard specifies the criteria for defining the procedures necessary to establish a bioassay performance-testing laboratory and program. The bioassay testing laboratory will conduct tests to evaluate the performance of service laboratories. Pacific Northwest Laboratory helped develop testing procedures as part of an effort to evaluate the performance criteria by testing the existing measurement capabilities of various bioassay laboratories. This report recommends guidelines for the preparation, handling, storage, distribution, shipping, and documentation of in vitro test samples (artificial urine and fecal matter) for indirect bioassay. The data base and recommended records system for documenting radiobioassay performance at the service laboratories are also presented. 8 refs., 3 tabs

Particular Characterisation of an In-Vitro-DTH Test to Monitor Cellular Immunity - Applications for Patient Care and Space Flight

Science.gov (United States)

Feurecker, M.; Mayer, W.; Gruber, M.; Muckenthaler, F.; Draenert, R.; Bogner, J.; Kaufmann, I.; Crucian, B.; Rykova, M.; Morukov, B.;

Gas transport during in vitro and in vivo preclinical testing of inert gas therapies

Directory of Open Access Journals (Sweden)

Ira Katz

2016-01-01

Full Text Available New gas therapies using inert gases such as xenon and argon are being studied, which require in vitro and in vivo preclinical experiments. Examples of the kinetics of gas transport during such experiments are analyzed in this paper. Using analytical and numerical models, we analyze an in vitro experiment for gas transport to a 96 cell well plate and an in vivo delivery to a small animal chamber, where the key processes considered are the wash-in of test gas into an apparatus dead volume, the diffusion of test gas through the liquid media in a well of a cell test plate, and the pharmacokinetics in a rat. In the case of small animals in a chamber, the key variable controlling the kinetics is the chamber wash-in time constant that is a function of the chamber volume and the gas flow rate. For cells covered by a liquid media the diffusion of gas through the liquid media is the dominant mechanism, such that liquid depth and the gas diffusion constant are the key parameters. The key message from these analyses is that the transport of gas during preclinical experiments can be important in determining the true dose as experienced at the site of action in an animal or to a cell.

Good cell culture practices &in vitro toxicology.

Science.gov (United States)

Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

2017-12-01

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cost effectiveness of ovarian reserve testing in in vitro fertilization: a Markov decision-analytic model

NARCIS (Netherlands)

Moolenaar, Lobke M.; Broekmans, Frank J. M.; van Disseldorp, Jeroen; Fauser, Bart C. J. M.; Eijkemans, Marinus J. C.; Hompes, Peter G. A.; van der Veen, Fulco; Mol, Ben Willem J.

2011-01-01

To compare the cost effectiveness of ovarian reserve testing in in vitro fertilization (IVF). A Markov decision model based on data from the literature and original patient data. Decision analytic framework. Computer-simulated cohort of subfertile women aged 20 to 45 years who are eligible for IVF.

Summary, the 16th quality control survey for radioisotope in vitro tests in Japan, 1994

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-11-01

The results of the 16th quality control survey for radioisotope in vitro tests in Japan (1994) are summarized. Of 399 medical facilities conducting radioisotope in vitro tests, 201 were enrolled in this study. Forty items including ACTH and {alpha}-fetoprotein were selected as the subjects. Freeze-drying samples were sent to the facilities. The quality of assay tubes, duration between fusion of the samples and assay, and the condition of preservation were examined, and those influence on the assay values were studied. Radioimmunoassay, immunoradiometric assay, and other procedures using enzymes, fluorescence, and chemiluminescense were conducted. The assay values of some of the items were significantly influenced by repeated freezing and fusion of the samples. Data were collected from individual items and kits used, and analyzed. The significant difference of values between different facilities and kits used were considered due to difference of assay principle, antibodies used, and standard items. The concentration of the samples needs to be improved. (S.Y.).

The 14th quality control survey for radioisotope in vitro tests in Japan, 1992

Energy Technology Data Exchange (ETDEWEB)

1993-11-01

This report presents the results of the 14th quality control nationwide survey. Of 490 facilities performing RI in vitro tests as of December 1992, 261 (53.3%) participated in the present 1992 survey. Free testosterone and renin were added to the following conventional 37 test items: adrenocorticotropic hormone (ACTH), growth hormone (GH), somatomedin C, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, thyroid stimulating hormone (TSH), triiodothyronine (T[sub 3]), free T[sub 3], thyroxine (T[sub 4]), free T[sub 4], T[sub 3] uptake, thyroglobulin, T[sub 3] binding globulin (TBG), parathyroid hormone (PTH), calcitonin, insulin, C-peptide, glucagon, gastrin, testosterone, estradiol, progesterone, 17[alpha]-hydroxyprogesterone, aldosterone, cortisol, immunoglobulin E (IgE), digoxin, [alpha]-fetoprotein, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), CA125, CA19-9, CA15-3, prostatic acid phosphatase (PAP), [beta][sub 2]-microglobulin, and ferritin. Measurement data for each kit were analyzed by a mean value of measurements, standard deviation, and coefficient of variation (CV). Both 'within kit variation' between facilities and 'between kit variation' showed a CV of 20% or less for GH, somatomedin C, TSH, T[sub 3], T[sub 4], T[sub 3] uptake, TBG, cortisol, IgE, CA125, PAP, and [beta][sub 2]-microglobulin, revealing satisfactory results. There was a great 'within kit variation' between facilities in ACTH, free T[sub 4], and calcitonin; and there was a great 'between kit variation' in ACTH, LH, free T[sub 4], thyroglobulin, PTH, calcitonin, and [alpha]-fetoprotein. (N.K.).

In vitro biocompatibility tests of two commercial types of mineral trioxide aggregate Testes de biocompatibilidade in vitro de duas formas comerciais do agregado de trióxido mineral

Directory of Open Access Journals (Sweden)

Daniel Araki Ribeiro

2005-09-01

Full Text Available Recently, regular and white mineral trioxide aggregate (MTA are being used in Dentistry as retrofilling materials. Genotoxicity and cytotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. Thus, the goal of this study was to examine the genotoxicity and cytotoxicity of regular and white MTA in vitro by the single cell gel (comet assay and trypan blue exclusion test, respectively. Mouse lymphoma cells were exposed to two presentation forms of MTA at final concentrations ranging from 1 to 1,000 µg/mL for 3 h at 37°C. The results showed that both compounds tested did not produce genotoxic effects at all concentrations evaluated. Likewise, no statistically significant differences (p > 0.05 were observed in cytotoxicity. Taken together, our results suggest that regular and white MTA are not genotoxins and are not able to interfere in cellular viability as assessed by single cell gel (comet assay and trypan blue assay, respectively.Recentemente, o agregado de trióxido mineral (MTA regular e branco estão sendo utilizados na Odontologia como materiais para obturação retrógrada de canais radiculares. Testes de genotoxicidade e citotoxicidade formam uma importante parte da pesquisa do câncer e da avaliação de risco de carcinógenos potenciais. Assim, o objetivo deste estudo foi examinar a genotoxicidade e citotoxicidade do MTA branco e regular in vitro pelo teste do cometa e teste de exclusão pelo azul de tripan, respectivamente. Células do linfoma murino foram expostas às duas formas de apresentação do MTA nas concentrações finais de 1 a 1.000 µg/mL por 3 horas a 37°C. Os resultados mostraram que ambos os compostos testados não produziram efeito genotóxico em todas as concentrações testadas. Da mesma forma, nenhuma diferença estatisticamente significativa (p > 0,05 foi observada na citotoxicidade. Em suma, nossos resultados sugerem que o MTA regular e branco não são genotoxinas e

Development of diagnostic test instruments to reveal level student conception in kinematic and dynamics

Science.gov (United States)

Handhika, J.; Cari, C.; Suparmi, A.; Sunarno, W.; Purwandari, P.

2018-03-01

The purpose of this research was to develop a diagnostic test instrument to reveal students' conceptions in kinematics and dynamics. The diagnostic test was developed based on the content indicator the concept of (1) displacement and distance, (2) instantaneous and average velocity, (3) zero and constant acceleration, (4) gravitational acceleration (5) Newton's first Law, (6) and Newton's third Law. The diagnostic test development model includes: Diagnostic test requirement analysis, formulating test-making objectives, developing tests, checking the validity of the content and the performance of reliability, and application of tests. The Content Validation Index (CVI) results in the category are highly relevant, with a value of 0.85. Three questions get negative Content Validation Ratio CVR) (-0.6), after revised distractors and clarify visual presentation; the CVR become 1 (highly relevant). This test was applied, obtained 16 valid test items, with Cronbach Alpha value of 0.80. It can conclude that diagnostic test can be used to reveal the level of students conception in kinematics and dynamics.

Etoposide; colchicine; mitomycin C and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster lung (CHL) cells at Covance laboratories; Harrogate UK in support of OECD draft Test Guideline 487.

Science.gov (United States)

Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

2010-10-29

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the Chinese hamster lung cell line CHL. Etoposide (a topoisomerase inhibitor), colchicine (an aneugen), mitomycin C (a DNA cross linking agent) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

Summary, the 20th quality control survey for radioisotopes in vitro tests in Japan, 1998

Energy Technology Data Exchange (ETDEWEB)

NONE

1999-11-01

For advancement of radioisotope in vitro tests such as radioimmunoassay and immunoradiometric assay, the Subcommittee for Radioisotope in vitro Test in Medical and Pharmaceutical Committee of Japan Radioisotope Association has conducted the yearly quality control survey for the test facilities in Japan since 1978. This is the summary of the 20th survey in 1998 where non-radioisotope tests like enzyme-immunoassay were involved as well. The survey was done for 143 facilities: 20 national and public university hospitals, 18 private university hospitals, 8 national hospitals, 13 public hospitals, 21 private hospitals, 41 hygienic laboratories and 22 manufacturers of reagents. Facilities examined intra- and between day-reproducibility, freeze-thaw effect and time change of the measured values on the same samples. Assays were for: growth hormone (h), somatomedin C, follicle stimulating h, luteinizing h, prolactin, thyroid stimulating h, triiodothyronines, thyroxines, thyroxine binding protein, calcitonin, insulin, C-peptide, glucagons, gastrin, testosterones, estradiol, progesterone, gonadotropin, 17{alpha}-hydroxyprogesterone, aldosterone, cortisol, dehydroepiandorosterone sulfate, renin, IgE, digoxin, {alpha}-fetoprotein, carcinoembryonic antigen, tissue polypeptide antigen, CA (125, 19-9 and 15-3), prostatic acid phosphatase, prostate specific antigen, {beta}2-microglobulin, ferritin, and neuron specific enolase. There was no great difference between this and last survey results although tendency of improvement was recognized. There were problems to be solved from the standpoint of clinical practice. (K.H.)

In vitro performance investigation of bioresorbable scaffolds - Standard tests for vascular stents and beyond.

Science.gov (United States)

Schmidt, Wolfram; Behrens, Peter; Brandt-Wunderlich, Christoph; Siewert, Stefan; Grabow, Niels; Schmitz, Klaus-Peter

2016-09-01

Biodegradable polymers are the main materials for coronary scaffolds. Magnesium has been investigated as a potential alternative and was successfully tested in human clinical trials. However, it is still challenging to achieve mechanical parameters comparative to permanent bare metal (BMS) and drug-eluting stents (DES). As such, in vitro tests are required to assess mechanical parameters correlated to the safety and efficacy of the device. In vitro bench tests evaluate scaffold profiles, length, deliverability, expansion behavior including acute elastic and time-dependent recoil, bending stiffness and radial strength. The Absorb GT1 (Abbott Vascular, Temecula, CA), DESolve (Elixir Medical Corporation, Sunnyvale, CA) and the Magmaris (BIOTRONIK AG, Bülach, Switzerland) that was previously tested in the BIOSOLVE II study, were tested. Crimped profiles were 1.38±0.01mm (Absorb GT1), 1.39±0.01mm (DESolve) and 1.44±0.00mm (Magmaris) enabling 6F compatibility. Trackability was measured depending on stiffness and force transmission (pushability). Acute elastic recoil was measured at free expansion and within a mock vessel, respectively, yielding results of 5.86±0.76 and 5.22±0.38% (Absorb), 7.85±3.45 and 9.42±0.21% (DESolve) and 5.57±0.72 and 4.94±0.31% (Magmaris). Time-dependent recoil (after 1h) was observed for the Absorb and DESolve scaffolds but not for the Magmaris. The self-correcting wall apposition behavior of the DESolve did not prevent time-dependent recoil under vessel loading. The results of the suggested test methods allow assessment of technical feasibility based on objective mechanical data and highlight the main differences between polymeric and metallic bioresorbable scaffolds. Copyright © 2016 Elsevier Inc. All rights reserved.

Preclinical Testing of an Oncolytic Parvovirus in Ewing Sarcoma: Protoparvovirus H-1 Induces Apoptosis and Lytic Infection In Vitro but Fails to Improve Survival In Vivo.

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Lacroix, Jeannine; Kis, Zoltán; Josupeit, Rafael; Schlund, Franziska; Stroh-Dege, Alexandra; Frank-Stöhr, Monika; Leuchs, Barbara; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane

2018-06-03

About 70% of all Ewing sarcoma (EWS) patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV) to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU)/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.

Preclinical Testing of an Oncolytic Parvovirus in Ewing Sarcoma: Protoparvovirus H-1 Induces Apoptosis and Lytic Infection In Vitro but Fails to Improve Survival In Vivo

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Jeannine Lacroix

2018-06-01

Full Text Available About 70% of all Ewing sarcoma (EWS patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.

Revealing Students' Cognitive Structure about Physical and Chemical Change: Use of a Word Association Test

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Yildirir, Hasene Esra; Demirkol, Hatice

2018-01-01

The current study aimed at examining the utility of a word association test in revealing students' cognitive structure in a specific chemistry topic through a word association test. The participants were 153 6th graders in a western Turkish city. The results revealed that the word association test serves a useful purpose in exploring the students'…

Investigation of thyroid parameters in farm animal by means of 125I in vitro tests

International Nuclear Information System (INIS)

Reinecke, P.; Leuthold, G.

1988-01-01

125 I in vitro tests especially thyroid hormone radioimmunoassays rendered it possible to study thyroidal activity of domestic animals even in large random tests. Parameters of thyroidal activity, such as effective T 4 quotient, T 3 value and total T 3 content, were investigated as to their connection to growth and environmental influence. The estimation of the hereditability yielded only low h 2 coefficients except in the T 3 value. All parameters studied depended to a great extent on farm conditions

In Vitro Drug Sensitivity Tests to Predict Molecular Target Drug Responses in Surgically Resected Lung Cancer.

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Ryohei Miyazaki

Full Text Available Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs and anaplastic lymphoma kinase (ALK inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI test and collagen gel droplet embedded culture drug sensitivity test (CD-DST are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs.The growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status.HCC827 (Exon19: E746-A750 del and H3122 (EML4-ALK cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003. The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026 in CD-DST.In vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically.

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions

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Nora Freyer

2018-03-01

Full Text Available The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p < 0.05 and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p < 0.0001, indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p < 0.05, suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions.

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Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Gerstmann, Florian; Storch, Lisa; Damm, Georg; Seehofer, Daniel; Foster Harris, Jennifer; Iyer, Rashi; Schubert, Frank; Zeilinger, Katrin

2018-03-15

The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP ( p < 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP ( p < 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP ( p < 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.

Testing strategies for embryo-fetal toxicity of human pharmaceuticals. Animal models vs. in vitro approaches: a workshop report.

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van der Laan, Jan Willem; Chapin, Robert E; Haenen, Bert; Jacobs, Abigail C; Piersma, Aldert

2012-06-01

Reproductive toxicity testing is characterized by high animal use. For registration of pharmaceutical compounds, developmental toxicity studies are usually conducted in both rat and rabbits. Efforts have been underway for a long time to design alternatives to animal use. Implementation has lagged, partly because of uncertainties about the applicability domain of the alternatives. The reproductive cycle is complex and not all mechanisms of development can be mimicked in vitro. Therefore, efforts are underway to characterize the available alternative tests with regard to the mechanism of action they include. One alternative test is the mouse embryonic stem cell test (EST), which has been studied since the late 1990s. It is a genuine 3R "alternative" assay as it is essentially animal-free. A meeting was held to review the state-of-the-art of various in vitro models for prediction of developmental toxicity. Although the predictivity of individual assays is improving, a battery of several assays is likely to have even higher predictivity, which is necessary for regulatory acceptance. The workshop concluded that an important first step is a thorough survey of the existing rat and rabbit studies, to fully characterize the frequency of responses and the types of effects seen. At the same time, it is important to continue the optimization of in vitro assays. As more experience accumulates, the optimal conditions, assay structure, and applicability of the alternative assays are expected to emerge. Copyright © 2012 Elsevier Inc. All rights reserved.

Diagnostic efficacy of in vitro methods vs. skin testing in patients with inhalant allergies

International Nuclear Information System (INIS)

Corey, J.P.; Liudahl, J.J.; Young, S.A.; Rodman, S.M.

1991-01-01

The purpose of our study was to investigate the diagnostic efficacy of two selected methods of in vitro allergy testing. Specifically, the PRIST/modified RAST I125 isotope systems and the Quantizyme/modified EAST alkaline phosphatase method were compared. The time, expense, convenience, and diagnostic efficacy of the two procedures are discussed. Special attention is given to the practicality of each method for the practicing physician

In vitro susceptibility testing of Aspergillus spp. against voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin.

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Shi, Jun-yan; Xu, Ying-chun; Shi, Yi; Lü, Huo-xiang; Liu, Yong; Zhao, Wang-sheng; Chen, Dong-mei; Xi, Li-yan; Zhou, Xin; Wang, He; Guo, Li-na

2010-10-01

During recent years, the incidence of serious infections caused by opportunistic fungi has increased dramatically due to alterations of the immune status of patients with hematological diseases, malignant tumors, transplantations and so forth. Unfortunately, the wide use of triazole antifungal agents to treat these infections has lead to the emergence of Aspergillus spp. resistant to triazoles. The present study was to assess the in vitro activities of five antifungal agents (voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin) against different kinds of Aspergillus spp. that are commonly encountered in the clinical setting. The agar-based Etest MIC method was employed. One hundred and seven strains of Aspergillus spp. (5 species) were collected and prepared according to Etest Technique Manuel. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 hours at 35°C. MIC(50), MIC(90) and MIC range were acquired by Whonet 5.4 software. The MIC(90) of caspofungin against A. fumigatus, A. flavus and A. nidulans was 0.094 µg/ml whereas the MIC(90) against A. niger was 0.19 µg/ml. For these four species, the MIC(90) of caspofungin was the lowest among the five antifungal agents. For A. terrus, the MIC(90) of posaconazole was the lowest. For A. fumigatus and A. flavus, the MIC(90) in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, and amphotericin B. The MIC of amphotericin B against A. terrus was higher than 32 µg/ml in all 7 strains tested. The in vitro antifungal susceptibility test shows the new drug caspofungin, which is a kind of echinocandins, has good activity against the five species of Aspergillus spp. and all the triazoles tested have better in vitro activity than traditional amphotericin B.

Surface enhanced Raman spectroscopy (SERS for in vitro diagnostic testing at the point of care

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Marks Haley

2017-06-01

Full Text Available Point-of-care (POC device development is a growing field that aims to develop low-cost, rapid, sensitive in-vitro diagnostic testing platforms that are portable, self-contained, and can be used anywhere – from modern clinics to remote and low resource areas. In this review, surface enhanced Raman spectroscopy (SERS is discussed as a solution to facilitating the translation of bioanalytical sensing to the POC. The potential for SERS to meet the widely accepted “ASSURED” (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable criterion provided by the World Health Organization is discussed based on recent advances in SERS in vitro assay development. As SERS provides attractive characteristics for multiplexed sensing at low concentration limits with a high degree of specificity, it holds great promise for enhancing current efforts in rapid diagnostic testing. In outlining the progression of SERS techniques over the past years combined with recent developments in smart nanomaterials, high-throughput microfluidics, and low-cost paper diagnostics, an extensive number of new possibilities show potential for translating SERS biosensors to the POC.

Surface enhanced Raman spectroscopy (SERS) for in vitro diagnostic testing at the point of care

Science.gov (United States)

Marks, Haley; Schechinger, Monika; Garza, Javier; Locke, Andrea; Coté, Gerard

2017-06-01

Point-of-care (POC) device development is a growing field that aims to develop low-cost, rapid, sensitive in-vitro diagnostic testing platforms that are portable, self-contained, and can be used anywhere - from modern clinics to remote and low resource areas. In this review, surface enhanced Raman spectroscopy (SERS) is discussed as a solution to facilitating the translation of bioanalytical sensing to the POC. The potential for SERS to meet the widely accepted "ASSURED" (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable) criterion provided by the World Health Organization is discussed based on recent advances in SERS in vitro assay development. As SERS provides attractive characteristics for multiplexed sensing at low concentration limits with a high degree of specificity, it holds great promise for enhancing current efforts in rapid diagnostic testing. In outlining the progression of SERS techniques over the past years combined with recent developments in smart nanomaterials, high-throughput microfluidics, and low-cost paper diagnostics, an extensive number of new possibilities show potential for translating SERS biosensors to the POC.

In vitro adaptation of Plasmodium falciparum reveal variations in cultivability

OpenAIRE

White, John; Mascarenhas, Anjali; Pereira, Ligia; Dash, Rashmi; Walke, Jayashri T.; Gawas, Pooja; Sharma, Ambika; Manoharan, Suresh Kumar; Guler, Jennifer L.; Maki, Jennifer N.; Kumar, Ashwani; Mahanta, Jagadish; Valecha, Neena; Dubhashi, Nagesh; Vaz, Marina

2016-01-01

Background Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. Methods Venous blood was collected f...

In Vitro Antimicrobial Susceptibility Testing of Animal Nocardia Isolated from Field Cases of Skin Diseases

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M. A. Oyekunle

2001-03-01

Full Text Available In vitro antimicrobial tests were carried out on strains of Nocardia isolated from field cases of cutaneous nocardiosis in farm animals. Results with the disc diffusion test showed the multiresistant nature of the isolates, but 23.81 and 21.43% were sensitive to ciprofloxacin and gentamycin, respectively. The MIC mode and range for oxytetracycline were 12.5 and 3.12–25 μg/ml, respectively, while those of erythromycin were 3.12 and 0.78–6.25 μg/ml, respectively.

Novel Antiseptic Urinary Catheters for Prevention of Urinary Tract Infections: Correlation of In Vivo and In Vitro Test Results▿

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Hachem, Ray; Reitzel, Ruth; Borne, Agatha; Jiang, Ying; Tinkey, Peggy; Uthamanthil, Rajesh; Chandra, Jyotsna; Ghannoum, Mahmoud; Raad, Issam

2009-01-01

Urinary catheters are widely used for hospitalized patients and are often associated with high rates of urinary tract infection. We evaluated in vitro the antiadherence activity of a novel antiseptic Gendine-coated urinary catheter against several multidrug-resistant bacteria. Gendine-coated urinary catheters were compared to silver hydrogel-coated Foley catheters and uncoated catheters. Bacterial biofilm formation was assessed by quantitative culture and scanning electron microscopy. These data were further correlated to an in vivo rabbit model. We challenged 31 rabbits daily for 4 days by inoculating the urethral meatus with 1.0 × 109 CFU streptomycin-resistant Escherichia coli per day. In vitro, Gendine-coated urinary catheters reduced the CFU of all organisms tested for biofilm adherence compared with uncoated and silver hydrogel-coated catheters (P < 0.004). Scanning electron microscopy analysis showed that a thick biofilm overlaid the control catheter and the silver hydrogel-coated catheters but not the Gendine-coated urinary catheter. Similar results were found with the rabbit model. Bacteriuria was present in 60% of rabbits with uncoated catheters and 71% of those with silver hydrogel-coated catheters (P < 0.01) but not in those with Gendine-coated urinary catheters. No rabbits with Gendine-coated urinary catheters had invasive bladder infections. Histopathologic assessment revealed no differences in toxicity or staining. Gendine-coated urinary catheters were more efficacious in preventing catheter-associated colonization and urinary tract infections than were silver hydrogel-coated Foley catheters and uncoated catheters. PMID:19805562

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop1[OPEN

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Shivhare, Devendra

2017-01-01

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. PMID:28546437

Accelerated in vitro release testing of implantable PLGA microsphere/PVA hydrogel composite coatings.

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Shen, Jie; Burgess, Diane J

2012-01-17

Dexamethasone loaded poly(lactic-co-glycolic acid) (PLGA) microsphere/PVA hydrogel composites have been investigated as an outer drug-eluting coating for implantable devices such as glucose sensors to counter negative tissue responses to implants. The objective of this study was to develop a discriminatory, accelerated in vitro release testing method for this drug-eluting coating using United States Pharmacopeia (USP) apparatus 4. Polymer degradation and drug release kinetics were investigated under "real-time" and accelerated conditions (i.e. extreme pH, hydro-alcoholic solutions and elevated temperatures). Compared to "real-time" conditions, the initial burst and lag phases were similar using hydro-alcoholic solutions and extreme pH conditions, while the secondary apparent zero-order release phase was slightly accelerated. Elevated temperatures resulted in a significant acceleration of dexamethasone release. The accelerated release data were able to predict "real-time" release when applying the Arrhenius equation. Microsphere batches with faster and slower release profiles were investigated under "real-time" and elevated temperature (60°C) conditions to determine the discriminatory ability of the method. The results demonstrated both the feasibility and the discriminatory ability of this USP apparatus 4 method for in vitro release testing of drug loaded PLGA microsphere/PVA hydrogel composites. This method may be appropriate for similar drug/device combination products and drug delivery systems. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro cytotoxicity testing of Ubiquicidin 29-41-99mTc

International Nuclear Information System (INIS)

Ocampo, Ivette Z.; Okazaki, Kayo; Dias, Luis Alberto Pereira; Higa, Olga Z.; Silva, Fabiana M. da; Vieira, Daniel P.; Passos, Priscila; Esteves-Pedro, Natalia M.

2015-01-01

The work carried out cytotoxicity tests using a radiopharmaceutical compound produced at IPEN/CNEN-SP to certify its safety through in vitro cytotoxicity tests. Since 2009, the Brazilian regulatory agency (ANVISA) requires that such tests have to be carried out following good laboratory practices (GLP) and in according to the OECD (Organisation for Economic Co-operation and Development) guidelines in order to certify its safety for medical use. Those guidelines comprises series of technical recommendations performed to assure quality of experiments. The study chose Ubiquicidin 29-41, an antimicrobial peptide used to discriminate bacterial infection foci from inflammatory sites. Amounts of UBI 29-41 were conjugated or not to 99m Tc and diluted to equivalent concentrations of 10, 100 and 1000% of the maximum dose (or activity) administered in adults. Possible cytotoxic effects were evaluated in comparison to untreated controls as well as positive and negative damage controls. Both full (radioactive) radiopharmaceuticals, as their precursors (only molecules without conjugation to isotopes) showed no significant cytotoxic effect (cytotoxicity ≤ 10%). The study was conducted for the first time in the country comprising preclinical testing of this radiopharmaceutical in accordance with internationally accepted quality parameters, ensuring the safety of its use and enabling inclusion in the pharmaceutical regulatory agenda. (author)

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

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Claudia Patricia Cordero Camacho

2002-01-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

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Claudia Patricia Cordero Camacho

2011-12-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence Factors.

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Vítor Borges

Full Text Available Research on the obligate intracellular bacterium Chlamydia trachomatis demands culture in cell-lines, but the adaptive process behind the in vivo to in vitro transition is not understood. We assessed the genomic and transcriptomic dynamics underlying C. trachomatis in vitro adaptation of strains representing the three disease groups (ocular, epithelial-genital and lymphogranuloma venereum propagated in epithelial cells over multiple passages. We found genetic features potentially underlying phase variation mechanisms mediating the regulation of a lipid A biosynthesis enzyme (CT533/LpxC, and the functionality of the cytotoxin (CT166 through an ON/OFF mechanism. We detected inactivating mutations in CT713/porB, a scenario suggesting metabolic adaptation to the available carbon source. CT135 was inactivated in a tropism-specific manner, with CT135-negative clones emerging for all epithelial-genital populations (but not for LGV and ocular populations and rapidly increasing in frequency (~23% mutants per 10 passages. RNA-sequencing analyses revealed that a deletion event involving CT135 impacted the expression of multiple virulence factors, namely effectors known to play a role in the C. trachomatis host-cell invasion or subversion (e.g., CT456/Tarp, CT694, CT875/TepP and CT868/ChlaDub1. This reflects a scenario of attenuation of C. trachomatis virulence in vitro, which may take place independently or in a cumulative fashion with the also observed down-regulation of plasmid-related virulence factors. This issue may be relevant on behalf of the recent advances in Chlamydia mutagenesis and transformation where culture propagation for selecting mutants/transformants is mandatory. Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time. In general, these data shed light on the adaptive process underlying the C. trachomatis in vivo to in vitro transition, and indicates that it would be prudent to

RNA-Seq Revealed Differences in Transcriptomes between 3ADON and 15ADON Populations of Fusarium graminearum In Vitro and In Planta.

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Puri, Krishna D; Yan, Changhui; Leng, Yueqiang; Zhong, Shaobin

2016-01-01

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) in barley and wheat in North America. The fungus not only causes yield loss of the crops but also produces harmful trichothecene mycotoxins [Deoxynivalenol (DON) and its derivatives-3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV)] that contaminate grains. Previous studies showed a dramatic increase of 3ADON-producing isolates with higher aggressiveness and DON production than the 15ADON-producing isolates in North America. However, the genetic and molecular basis of differences between the two types of isolates is unclear. In this study, we compared transcriptomes of the 3ADON and 15ADON isolates in vitro (in culture media) and in planta (during infection on the susceptible wheat cultivar 'Briggs') using RNA-sequencing. The in vitro gene expression comparison identified 479 up-regulated and 801 down-regulated genes in the 3ADON isolates; the up-regulated genes were mainly involved in C-compound and carbohydrate metabolism (18.6%), polysaccharide metabolism (7.7%) or were of unknown functions (57.6%). The in planta gene expression analysis revealed that 185, 89, and 62 genes were up-regulated in the 3ADON population at 48, 96, and 144 hours after inoculation (HAI), respectively. The up-regulated genes were significantly enriched in functions for cellular import, C-compound and carbohydrate metabolism, allantoin and allantoate transport at 48 HAI, for detoxification and virulence at 96 HAI, and for metabolism of acetic acid derivatives, detoxification, and cellular import at 144 HAI. Comparative analyses of in planta versus in vitro gene expression further revealed 2,159, 1,981 and 2,095 genes up-regulated in the 3ADON isolates, and 2,415, 2,059 and 1,777 genes up-regulated in the 15ADON isolates at the three time points after inoculation. Collectively, our data provides a foundation for further understanding of molecular mechanisms involved in

RNA-Seq Revealed Differences in Transcriptomes between 3ADON and 15ADON Populations of Fusarium graminearum In Vitro and In Planta.

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Krishna D Puri

Full Text Available Fusarium graminearum is the major causal agent of Fusarium head blight (FHB in barley and wheat in North America. The fungus not only causes yield loss of the crops but also produces harmful trichothecene mycotoxins [Deoxynivalenol (DON and its derivatives-3-acetyldeoxynivalenol (3ADON and 15-acetyldeoxynivalenol (15ADON, and nivalenol (NIV] that contaminate grains. Previous studies showed a dramatic increase of 3ADON-producing isolates with higher aggressiveness and DON production than the 15ADON-producing isolates in North America. However, the genetic and molecular basis of differences between the two types of isolates is unclear. In this study, we compared transcriptomes of the 3ADON and 15ADON isolates in vitro (in culture media and in planta (during infection on the susceptible wheat cultivar 'Briggs' using RNA-sequencing. The in vitro gene expression comparison identified 479 up-regulated and 801 down-regulated genes in the 3ADON isolates; the up-regulated genes were mainly involved in C-compound and carbohydrate metabolism (18.6%, polysaccharide metabolism (7.7% or were of unknown functions (57.6%. The in planta gene expression analysis revealed that 185, 89, and 62 genes were up-regulated in the 3ADON population at 48, 96, and 144 hours after inoculation (HAI, respectively. The up-regulated genes were significantly enriched in functions for cellular import, C-compound and carbohydrate metabolism, allantoin and allantoate transport at 48 HAI, for detoxification and virulence at 96 HAI, and for metabolism of acetic acid derivatives, detoxification, and cellular import at 144 HAI. Comparative analyses of in planta versus in vitro gene expression further revealed 2,159, 1,981 and 2,095 genes up-regulated in the 3ADON isolates, and 2,415, 2,059 and 1,777 genes up-regulated in the 15ADON isolates at the three time points after inoculation. Collectively, our data provides a foundation for further understanding of molecular mechanisms involved

In vitro testing of daptomycin plus rifampin againstmethicillin-resistant Staphylococcus aureus resistant to rifampin

International Nuclear Information System (INIS)

Khaswneh, Faisal A.; Ashcraft, Deborah S.; Pankey, George A.

2008-01-01

Objective was to test for synergy between daptomycin (DAP) and rifampin(RIF) against RIF-resistant methicillin-resistant Staphylococcus aureus(MRSA) isolates. Synergy testing using time-kill assay (TKA) was performed on6 clinically and genetically unique RIF-resistant MRSA isolates. The isolateswere identified out of 489 (1.2%) samples collected during April 2003 toAugust 2006, from patients at the Ochsner Medical Center in New Orleans,Louisiana, United States of America. Synergy testing of DAP plus RIF by TKAshowed that 5 isolates were different, but one isolate was antagonistic. Ourin-vitro study failed to demonstrate synergy between DAP plus RIF, againstour RIF-resistant MRSA isolates. Clinical failure of this combination shouldprompt the clinician to consider antagonism as one of the potential causes.(author)

The development of in vitro mutagenicity testing systems using T-lymphocytes

International Nuclear Information System (INIS)

Albertini, R.J.

1992-05-01

This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs

In vitro anti-proliferative effect of interferon alpha in solid tumors: A potential predicative test

International Nuclear Information System (INIS)

Fuchsberger, N.; Kubes, M.; Kontsek, P.; Borecky, L.; Hornak, M.; Silvanova; Godal, A.; Svec, J.

1993-01-01

An in vitro test for the anti-proliferative effect of human leukocyte interferon (IFN-alpha) was performed in primary cultures of tumor cells obtained from 32 patients with either malignant melanoma (13), renal carcinoma (4) or bladder carcinoma (15). Our results demonstrated activity of IFN in all three groups of solid tumors. However, appreciable differences in sensitivity to anti-proliferative effect of IFN between individual tumors of the same type were found. The potential of this anti-proliferative test for prediction of treatment response in IFN-therapy is discussed. (author)

Diagnosis of stinging insect allergy: utility of cellular in-vitro tests.

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Scherer, Kathrin; Bircher, Andreas J; Heijnen, Ingmar Afm

2009-08-01

Diagnosis of stinging insect allergy is based on a detailed history, venom skin tests, and detection of venom-specific IgE. As an additional diagnostic tool, basophil responsiveness to venom allergens has been shown to be helpful in selected patients. This review summarizes the current diagnostic procedures for stinging insect allergy and discusses the latest developments in cellular in-vitro tests. Cellular assays have been evaluated in patients with Hymenoptera venom allergy. The diagnostic performance of the cellular mediator release test is similar to that of the flow cytometric basophil activation test (BAT), but the BAT has been the most intensively studied. BAT offers the possibility to assess basophil reactivity to allergens in their natural environment and to simultaneously analyze surface marker expression and intracellular signaling. It has been demonstrated that BAT represents a valuable additional diagnostic tool in selected patients when used in combination with other well established tests. A major limitation is the current lack of unified, standardized protocols. Flow cytometry offers huge possibilities to enhance knowledge of basophil functions. The BAT may be used as an additional test to confirm the diagnosis of stinging insect allergy in selected patients, provided that it is performed by an experienced laboratory using a validated assay. Test results have to be interpreted by clinicians familiar with the methodological aspects. The utility of the BAT to confirm allergy diagnosis and to predict the risk of subsequent systemic reactions may be improved by combined analysis of multiple surface markers and intracellular signaling pathways.

Labelled T{sub 3}, T{sub 4} and TBP for In Vitro Testing of Thyroid Function in Man and Animals

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Czerniak, P.; Boruchowski, Sabina; Shomron, I. [Dept. of Radiotherapy and Isotopes, Tel-Hashomer Hospital, Tel-Aviv University Medical School, Faculty of Continuing Medical Education, Tel-Aviv (Israel)

1970-02-15

Iodothyronines are bound to determined electrophoretic fractions of serum proteins - TBP (TBG, TBA, TBPA). Radioiodine labelled T{sub 3} and T{sub 4} complex the free TBP fractions until saturation. The excess of added in vitro thyronines is then absorbed by the RBC. The changes described can be detected and quantitatively determined by radioisotope tests: radioelectrophoresis - T{sub 3/4} BP test, and RBC - {sup 125}I T{sub 3} test (Hamolsky test). The in vitro tests of the thyroid function can be clinically reliable if the protein fractions are normal, and they may be altered without thyroid pathology if the TBG fractions are abnormal. The electrophoretic fractions vary quantitatively and qualitatively in animals of different classes and orders. We chose these features to study the correlation between iodothyronines, plasma proteins and the above-mentioned thyroid tests. Twenty-two animal species (arranged according to the increasing percentage of the Hamolsky test) were examined: goat (8.6%, cow, lamb, calf, man, camel, goose, hamster, rat, turkey, marmot, duck, horse, donkey, hen, dog, pigeon, rabbit, guinea-pig, mouse, fish and frog (91.5%). The following additional parameters were examined: PBI, serum quantitative electrophoresis, T{sub 3} BP studies. All the tests were performed under identical technical conditions. Results and conclusions: (1) The Hamolsky test in the examined animals ranges from 9% to 92%. It is highest in the poicolothermics, in which much prealbumin and few glubulins are found on electrophoresis. The T{sub 3} BP is low, and about a half of the added {sup 125}I T{sub 3} remains unbound. (2) T{sub 3} and T{sub 4} are complexed with T{sub 3} BP and T{sub 4} BP fractions, which correspond to prealbumin, albumin, alpha 1-2, beta and exceptionally gamma globulin. The fractions are variable but characteristic for each animal species. T{sub 3} BP does not correlate exactly with T{sub 4} BP, and seems to be distributed over more fractions

Validity of in vitro tests on aqueous spray pumps as surrogates for nasal deposition, absorption, and biologic response.

Science.gov (United States)

Suman, Julie D; Laube, Beth L; Dalby, Richard

2006-01-01

This research investigated the impact of the full range of in vitro spray characterization tests described in the FDA Draft Bioequivalence Guidance on nasal deposition pattern, pharmacokinetics, and biological response to nicotine administered by two aqueous nasal spray pumps in human volunteers. Nicotine was selected as a model drug (even though it is not locally acting) based on its ability to alter cardiac function and available plasma assay. Significant differences in pump performance-including mean volume diameters, spray angle, spray width, and ovality ratios-were observed between the two pumps. There were no significant differences in deposition pattern, or pharmacokinetic or pharmacodynamic response to the nasally administered nicotine. Although there were statistical differences in the in vitro tests between the two pumps, these differences did not result in significant alterations in the site of droplet deposition within the nose, the rate and extent of nicotine absorption, or the physiologic response it induced. These results suggest that current measures of in vitro performance, particularly spray angle and spray pattern (ovality), may not be clinically relevant. Additional research is needed to define what spray pump characteristics are likely to produce differences in deposition pattern and drug response.

Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.

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Biswas, Nidhan K; Chandra, Vikas; Sarkar-Roy, Neeta; Das, Tapojyoti; Bhattacharya, Rabindra N; Tripathy, Laxmi N; Basu, Sunandan K; Kumar, Shantanu; Das, Subrata; Chatterjee, Ankita; Mukherjee, Ankur; Basu, Pryiadarshi; Maitra, Arindam; Chattopadhyay, Ansuman; Basu, Analabha; Dhara, Surajit

2015-01-21

Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

In vitro and ex vivo testing of tenofovir shows it is effective as an HIV-1 microbicide.

Directory of Open Access Journals (Sweden)

Lisa C Rohan

2010-02-01

Full Text Available Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective

In vitro performance investigation of bioresorbable scaffolds – Standard tests for vascular stents and beyond

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Schmidt, Wolfram, E-mail: wolfram.schmidt@uni-rostock.de [Institute for Biomedical Engineering, University Medicine Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany); Behrens, Peter, E-mail: peter.behrens@uni-rostock.de [Institute for Biomedical Engineering, University Medicine Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany); Brandt-Wunderlich, Christoph, E-mail: christoph.brandt@uni-rostock.de [Institute for ImplantTechnology and Biomaterials – IIB e.V., Associated Institute of the University of Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany); Siewert, Stefan, E-mail: stefan.siewert@uni-rostock.de [Institute for ImplantTechnology and Biomaterials – IIB e.V., Associated Institute of the University of Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany); Grabow, Niels, E-mail: niels.grabow@uni-rostock.de [Institute for Biomedical Engineering, University Medicine Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany); Schmitz, Klaus-Peter, E-mail: klaus-peter.schmitz@uni-rostock.de [Institute for ImplantTechnology and Biomaterials – IIB e.V., Associated Institute of the University of Rostock, Friedrich-Barnewitz-Strasse 4, D-18119 Rostock-Warnemünde (Germany)

2016-09-15

Background/Purpose: Biodegradable polymers are the main materials for coronary scaffolds. Magnesium has been investigated as a potential alternative and was successfully tested in human clinical trials. However, it is still challenging to achieve mechanical parameters comparative to permanent bare metal (BMS) and drug-eluting stents (DES). As such, in vitro tests are required to assess mechanical parameters correlated to the safety and efficacy of the device. Methods/Materials: In vitro bench tests evaluate scaffold profiles, length, deliverability, expansion behavior including acute elastic and time-dependent recoil, bending stiffness and radial strength. The Absorb GT1 (Abbott Vascular, Temecula, CA), DESolve (Elixir Medical Corporation, Sunnyvale, CA) and the Magmaris (BIOTRONIK AG, Bülach, Switzerland) that was previously tested in the BIOSOLVE II study, were tested. Results: Crimped profiles were 1.38 ± 0.01 mm (Absorb GT1), 1.39 ± 0.01 mm (DESolve) and 1.44 ± 0.00 mm (Magmaris) enabling 6F compatibility. Trackability was measured depending on stiffness and force transmission (pushability). Acute elastic recoil was measured at free expansion and within a mock vessel, respectively, yielding results of 5.86 ± 0.76 and 5.22 ± 0.38% (Absorb), 7.85 ± 3.45 and 9.42 ± 0.21% (DESolve) and 5.57 ± 0.72 and 4.94 ± 0.31% (Magmaris). Time-dependent recoil (after 1 h) was observed for the Absorb and DESolve scaffolds but not for the Magmaris. The self-correcting wall apposition behavior of the DESolve did not prevent time-dependent recoil under vessel loading. Conclusions: The results of the suggested test methods allow assessment of technical feasibility based on objective mechanical data and highlight the main differences between polymeric and metallic bioresorbable scaffolds.

In vitro performance investigation of bioresorbable scaffolds – Standard tests for vascular stents and beyond

International Nuclear Information System (INIS)

Schmidt, Wolfram; Behrens, Peter; Brandt-Wunderlich, Christoph; Siewert, Stefan; Grabow, Niels; Schmitz, Klaus-Peter

2016-01-01

Background/Purpose: Biodegradable polymers are the main materials for coronary scaffolds. Magnesium has been investigated as a potential alternative and was successfully tested in human clinical trials. However, it is still challenging to achieve mechanical parameters comparative to permanent bare metal (BMS) and drug-eluting stents (DES). As such, in vitro tests are required to assess mechanical parameters correlated to the safety and efficacy of the device. Methods/Materials: In vitro bench tests evaluate scaffold profiles, length, deliverability, expansion behavior including acute elastic and time-dependent recoil, bending stiffness and radial strength. The Absorb GT1 (Abbott Vascular, Temecula, CA), DESolve (Elixir Medical Corporation, Sunnyvale, CA) and the Magmaris (BIOTRONIK AG, Bülach, Switzerland) that was previously tested in the BIOSOLVE II study, were tested. Results: Crimped profiles were 1.38 ± 0.01 mm (Absorb GT1), 1.39 ± 0.01 mm (DESolve) and 1.44 ± 0.00 mm (Magmaris) enabling 6F compatibility. Trackability was measured depending on stiffness and force transmission (pushability). Acute elastic recoil was measured at free expansion and within a mock vessel, respectively, yielding results of 5.86 ± 0.76 and 5.22 ± 0.38% (Absorb), 7.85 ± 3.45 and 9.42 ± 0.21% (DESolve) and 5.57 ± 0.72 and 4.94 ± 0.31% (Magmaris). Time-dependent recoil (after 1 h) was observed for the Absorb and DESolve scaffolds but not for the Magmaris. The self-correcting wall apposition behavior of the DESolve did not prevent time-dependent recoil under vessel loading. Conclusions: The results of the suggested test methods allow assessment of technical feasibility based on objective mechanical data and highlight the main differences between polymeric and metallic bioresorbable scaffolds.

Cadmium chloride, benzo[a]pyrene and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Covance laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

Science.gov (United States)

Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

2010-10-29

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

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Stacey, Glyn N; Coecke, Sandra; Price, Anna-Bal; Healy, Lyn; Jennings, Paul; Wilmes, Anja; Pinset, Christian; Ingelman-Sundberg, Magnus; Louisse, Jochem; Haupt, Simone; Kidd, Darren; Robitski, Andrea; Jahnke, Heinz-Georg; Lemaitre, Gilles; Myatt, Glenn

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

Characterisation, in vitro release study, and antibacterial activity of montmorillonite-gentamicin complex material

International Nuclear Information System (INIS)

Rapacz-Kmita, A.; Bućko, M.M.; Stodolak-Zych, E.; Mikołajczyk, M.; Dudek, P.; nd Department of Surgery, Kopernika 21, 31-501 Krakow (Poland))" data-affiliation=" (Jagiellonian University, Medical College, 2nd Department of Surgery, Kopernika 21, 31-501 Krakow (Poland))" >Trybus, M.

2017-01-01

The present paper concerns the potential use of montmorillonite as a drug carrier and focusses on the intercalation of the studied clay with gentamicin (an aminoglycoside antibiotic) at various temperatures (20, 50 and 80 °C). The experiments were performed to identify the temperature required for the optimum intercalation of gentamicin into the interlayer of montmorillonite. The structural and microstructural properties of gentamicin and the potential for introducing it between smectite clay layers were investigated by means of X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopic techniques, and SEM with EDS analysis. Additionally, the in vitro drug release behaviour of the montmorillonite-gentamicin complex and its antibacterial activity against Escherichia coli (E. coli) bacteria was investigated. Based on these studies, the impact of temperature on the intercalation of the drug between layers of smectite was evaluated. It was found that an intercalation temperature of 50 °C resulted in the highest shift in the position of principle peak d (001) as measured by XRD, suggesting, that the greatest amount of gentamicin had been introduced into the interlayer space of montmorillonite at this temperature. Subsequently, the montmorillonite-gentamicin complex material obtained at 50 °C revealed the greatest capacity for killing E. coli bacteria during an in vitro test. - Highlights: • A novel montmorillonite-gentamicin hybrid materials was prepared as potential drug carrier. • Optimal conditions for the intercalation of gentamicin into the interlayer space of montmorillonite were tested. • The MMT-G complex material obtained at 50 °C revealed the greatest capacity for killing E. coli during the inhibitory zone test. • Modulating drug delivery was monitored and confirmed in in vitro drug release study.

Characterisation, in vitro release study, and antibacterial activity of montmorillonite-gentamicin complex material

Energy Technology Data Exchange (ETDEWEB)

Rapacz-Kmita, A., E-mail: kmita@agh.edu.pl [AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Mickiewicza 30, 30-059 Krakow (Poland); Bućko, M.M.; Stodolak-Zych, E. [AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Mickiewicza 30, 30-059 Krakow (Poland); Mikołajczyk, M. [The University Hospital in Krakow, Division of Microbiology, Kopernika 19, 31-501 Krakow (Poland); Dudek, P. [AGH University of Science and Technology, Faculty of Mechanical Engineering and Robotics, Mickiewicza 30, 30-059 Krakow (Poland); Trybus, M. [Jagiellonian University, Medical College, 2" n" d Department of Surgery, Kopernika 21, 31-501 Krakow (Poland)

2017-01-01

The present paper concerns the potential use of montmorillonite as a drug carrier and focusses on the intercalation of the studied clay with gentamicin (an aminoglycoside antibiotic) at various temperatures (20, 50 and 80 °C). The experiments were performed to identify the temperature required for the optimum intercalation of gentamicin into the interlayer of montmorillonite. The structural and microstructural properties of gentamicin and the potential for introducing it between smectite clay layers were investigated by means of X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopic techniques, and SEM with EDS analysis. Additionally, the in vitro drug release behaviour of the montmorillonite-gentamicin complex and its antibacterial activity against Escherichia coli (E. coli) bacteria was investigated. Based on these studies, the impact of temperature on the intercalation of the drug between layers of smectite was evaluated. It was found that an intercalation temperature of 50 °C resulted in the highest shift in the position of principle peak d{sub (001)} as measured by XRD, suggesting, that the greatest amount of gentamicin had been introduced into the interlayer space of montmorillonite at this temperature. Subsequently, the montmorillonite-gentamicin complex material obtained at 50 °C revealed the greatest capacity for killing E. coli bacteria during an in vitro test. - Highlights: • A novel montmorillonite-gentamicin hybrid materials was prepared as potential drug carrier. • Optimal conditions for the intercalation of gentamicin into the interlayer space of montmorillonite were tested. • The MMT-G complex material obtained at 50 °C revealed the greatest capacity for killing E. coli during the inhibitory zone test. • Modulating drug delivery was monitored and confirmed in in vitro drug release study.

In vitro cross-linking of bovine lens proteins photosensitized by promazines

International Nuclear Information System (INIS)

Merville, M.P.; Decuyper, J.; Piette, J.; Calberg-Bacq, C.M.; Van de Vorst, A.

1984-01-01

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O 2 , as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [TFPZ], methoxypromazine [MTPZ], and acepromazine [ACPZ]), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo

Rapid prototyping compliant arterial phantoms for in-vitro studies and device testing

Directory of Open Access Journals (Sweden)

Biglino Giovanni

2013-01-01

Full Text Available Abstract 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 Tango

Rapid prototyping compliant arterial phantoms for in-vitro studies and device testing.

Science.gov (United States)

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

In vitro testing of thiolated poly(aspartic acid) from ophthalmic formulation aspects.

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Budai-Szű Cs, Mária; Horvát, Gabriella; Gyarmati, Benjámin; Szilágyi, Barnabás Áron; Szilágyi, András; Csihi, Tímea; Berkó, Szilvia; Szabó-Révész, Piroska; Mori, Michela; Sandri, Giuseppina; Bonferoni, Maria Cristina; Caramella, Carla; Csányi, Erzsébet

2016-08-01

Ocular drug delivery formulations must meet anatomical, biopharmaceutical, patient-driven and regulatory requirements. Mucoadhesive polymers can serve as a better alternative to currently available ophthalmic formulations by providing improved bioavailability. If all requirements are addressed, a polymeric formulation resembling the tear film of the eye might be the best solution. The optimum formulation must not have high osmotic activity, should provide appropriate surface tension, pH and refractive index, must be non-toxic and should be transparent and mucoadhesive. We would like to highlight the importance of in vitro polymer testing from a pharmaceutical aspect. We, therefore, carried out physical-chemical investigations to verify the suitability of certain systems for ophthalmic formulations. In this work, in situ gelling, mucoadhesive thiolated poly(aspartic acid)s were tested from ophthalmic formulation aspects. The results of preformulation measurements indicate that these polymers can be used as potential carriers in ophthalmic drug delivery.

In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

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Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

2016-12-06

The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing

NARCIS (Netherlands)

Nicolas, J.A.Y.; Hendriksen, P.J.M.; Haan, de L.H.J.; Koning, R.; Rietjens, I.M.C.M.; Bovee, T.F.H.

2015-01-01

The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on

40 CFR 799.9537 - TSCA in vitro mammalian chromosome aberration test.

Science.gov (United States)

2010-07-01

... effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples... if applicable. (H) Modal number of chromosomes. (iv) Test conditions. (A) Identity of metaphase... Mutation Assay. Environmental Mutagenesis 5, 795-801 (1983). (17) Locke-Huhle, C. Endoreduplication in...

Modular synthesis and in vitro and in vivo antimalarial assessment of C-10 pyrrole mannich base derivatives of artemisinin.

Science.gov (United States)

Pacorel, Bénédicte; Leung, Suet C; Stachulski, Andrew V; Davies, Jill; Vivas, Livia; Lander, Hollie; Ward, Stephen A; Kaiser, Marcel; Brun, Reto; O'Neill, Paul M

2010-01-28

In two steps from dihydroartemisinin, a small array of 16 semisynthetic C-10 pyrrole Mannich artemisinin derivatives (7a-p) have been prepared in moderate to excellent yield. In vitro analysis against both chloroquine sensitive and resistant strains has demonstrated that these analogues have nanomolar antimalarial activity, with several compounds being more than 3 times more potent than the natural product artemisinin. In addition to a potent antimalarial profile, these molecules also have very high in vitro therapeutic indices. Analysis of the optimal Mannich side chain substitution for in vitro and in vivo activity reveals that the morpholine and N-methylpiperazine Mannich side chains provide analogues with the best activity profiles, both in vitro and in vivo in the Peter's 4 day test.

In vitro binding of estrogens by dietary fiber and the in vivo apparent digestibility tested in pigs

NARCIS (Netherlands)

Arts, C.J.M.; Govers, C.A.R.L.; Berg, H. van den; Wolters, M.G.E.; Leeuwen, P. van; Thijsen, J.H.

1991-01-01

Within the framework of experiments related to the association between dietary fiber and breast cancer an in vitro test system was used to study the binding of estrogens to various fibers (e.g. cholestyramin, lignin and cellulose) and fiber sources (e.g. wheat bran, cereals, seeds and legumes).

Evaluation of the medical devices benchmark materials in the controlled human patch testing and in the RhE in vitro skin irritation protocol.

NARCIS (Netherlands)

Kandárová, Helena; Bendova, Hana; Letasiova, Silvia; Coleman, Kelly P; De Jong, Wim H; Jírova, Dagmar

2018-01-01

Several irritants were used in the in vitro irritation medical device round robin. The objective of this study was to verify their irritation potential using the human patch test (HPT), an in vitro assay, and in vivo data. The irritants were lactic acid (LA), heptanoic acid (HA), sodium dodecyl

Screening of wheat endophytes as biological control agents against Fusarium head blight using two different in vitro tests.

Science.gov (United States)

Comby, Morgane; Gacoin, Marie; Robineau, Mathilde; Rabenoelina, Fanja; Ptas, Sébastien; Dupont, Joëlle; Profizi, Camille; Baillieul, Fabienne

2017-09-01

In order to find biological control agents (BCAs) for the management of Fusarium head blight (FHB), a major disease on wheat crops worldwide, 86 microorganisms isolated from inner tissues of wheat plants were discriminated for their ability to inhibit the growth of Fusarium graminearum and Fusarium culmorum by in vitro dual culture assays. A group of 22 strains appeared very effective to inhibit F. graminearum (inhibition of 30-51%) and they were also globally effective in controlling F. culmorum (inhibition of 15-53%). Further evaluation of a subselection of strains by screening on detached spikelets in vitro confirmed three species, namely Phoma glomerata, Aureobasidium proteae and Sarocladium kiliense, that have not yet been reported for their efficacy against Fusarium spp., indicating that looking for BCAs toward FHB among wheat endophytes proved to be promising. The efficacy of some strains turned out different between both in vitro screening approaches, raising the importance of finding the most appropriate screening approach for the search of BCAs. This study pointed out the interest of the test on detached wheat spikelets that provided information about a potential pathogenicity, the growth capacity and efficacy of the endophyte strains on the targeted plant, before testing them on whole plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

In Vitro Susceptibility Test of Different Clinical Isolates against Ceftriaxone

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Syed Hakim Masood

2010-06-01

Full Text Available Objectives: Because of the prevailing penicillin resistance in microorganisms, broad spectrum cephalosporins are used empirically specially in developing countries. The aim of this study is to determine the susceptibility pattern of different gram positive and gram negative pathogens against third generation cephalosporin-ceftriaxone to explore the existing effectiveness of this antibiotic.Methods: 180 clinical isolates of different gram positive and gram negative pathogens including P.mirabilis, S. typhi P.aeruginosa, E. coli, S. aureus and Klebsiella were collected from blood and urine samples of in-patients. 30 isolates of all species were tested against each of six brands of ceftriaxone using in vitro sensitivity tests by disc diffusion method (NCCLS criteria. The susceptibility limit was ≥21 mm zone of inhibition, while moderately susceptible was considered at 20-14 mm, and those isolates which showed >13 mm or no zone of inhibition were resistant to this antibacterial drug.Results: Ceftriaxone was found most effective against S. aureus. While 96.1% of the isolates showed susceptibility towards ceftriaxone, followed by E. coli (95%, P. aeruginosa (92.7%, K. pneumonia (89.4% and S. typhi (87.2%. P. mirabilis showed lowest susceptibility amongst all the test organisms (83.8%.Conclusion: Ceftriaxone can be used as a drug of choice in infections caused by S. aureus, E. coli, P. aurigenosa, K. pneumonia and S. typhi. However, it should be used with other antimicrobial agents in order to increase its effectiveness against P. mirabilis.

Experimental manipulation reveals a trade-off between weapons and testes.

Science.gov (United States)

Somjee, U; Miller, C W; Tatarnic, N J; Simmons, L W

2018-01-01

Theory predicts a trade-off between sexually selected weapons used to secure mates and post-copulatory traits used to maximize fertilization success. However, individuals that have a greater capacity to acquire resources from the environment may invest more in both pre- and post-copulatory traits, and trade-offs may not be readily apparent. Here, we manipulate the phenotype of developing individuals to examine allocation trade-offs between weapons and testes in Mictis profana (Hemiptera: Coreidae), a species where the hind legs are sexually selected weapons used in contests over access to females. We experimentally prevented males from developing weapons by inducing them to autotomize their hind legs before the final moult to adulthood. We compared trait expression in this group to males where autotomy was induced in the mid-legs, which are presumably not under sexual selection to the same extent. We found males without weapons invested proportionally more in testes mass than those with their mid-legs removed. Males that developed to adulthood without weapons did not differ from the mid-leg removal group in other traits potentially under precopulatory sexual selection, other post-copulatory traits or naturally selected traits. In addition, a sample of adult males from the same population in the wild revealed a positive correlation between investment in testes and weapons. Our study presents a critical contribution to a growing body of literature suggesting the allocation of resources to pre- and post-copulatory sexual traits is influenced by a resource allocation trade-off and that this trade-off may only be revealed with experimental manipulation. © 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.

ID-Check: Online Concealed Information Test Reveals True Identity.

Science.gov (United States)

Verschuere, Bruno; Kleinberg, Bennett

2016-01-01

The Internet has already changed people's lives considerably and is likely to drastically change forensic research. We developed a web-based test to reveal concealed autobiographical information. Initial studies identified a number of conditions that affect diagnostic efficiency. By combining these moderators, this study investigated the full potential of the online ID-check. Participants (n = 101) tried to hide their identity and claimed a false identity in a reaction time-based Concealed Information Test. Half of the participants were presented with personal details (e.g., first name, last name, birthday), whereas the others only saw irrelevant details. Results showed that participants' true identity could be detected with high accuracy (AUC = 0.98; overall accuracy: 86-94%). Online memory detection can reliably and validly detect whether someone is hiding their true identity. This suggests that online memory detection might become a valuable tool for forensic applications. © 2015 American Academy of Forensic Sciences.

Implementation challenges for designing Integrated In Vitro Testing Strategies (ITS) aiming at reducing and replacing animal experimentation

NARCIS (Netherlands)

Wever, B.de; Fuchs, H.W.; Gaca, M.; Krul, C.A.M.; Mikulowski, S.; Poth, A.; Roggen, E.L.; Vilà, M.R.

2012-01-01

At the IVTIP (In Vitro Testing Industrial Platform) meeting of November 26th 2009 entitled 'Toxicology in the 21st century ('21C') - working our way towards a visionary reality' all delegates endorsed the emerging concept of the '21C' vision as the way forward to enable a thorough, reliable and

In vitro cytotoxicity testing of Ubiquicidin 29-41-{sup 99m}Tc

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Ocampo, Ivette Z.; Okazaki, Kayo; Dias, Luis Alberto Pereira; Higa, Olga Z.; Silva, Fabiana M. da; Vieira, Daniel P., E-mail: dpvieira@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Passos, Priscila; Esteves-Pedro, Natalia M., E-mail: fabiana@biosintesis.com.br [Laboratorio Biosintesis Ltda, Sao Paulo, SP (Brazil)

2015-07-01

The work carried out cytotoxicity tests using a radiopharmaceutical compound produced at IPEN/CNEN-SP to certify its safety through in vitro cytotoxicity tests. Since 2009, the Brazilian regulatory agency (ANVISA) requires that such tests have to be carried out following good laboratory practices (GLP) and in according to the OECD (Organisation for Economic Co-operation and Development) guidelines in order to certify its safety for medical use. Those guidelines comprises series of technical recommendations performed to assure quality of experiments. The study chose Ubiquicidin 29-41, an antimicrobial peptide used to discriminate bacterial infection foci from inflammatory sites. Amounts of UBI{sub 29-41} were conjugated or not to {sup 99m}Tc and diluted to equivalent concentrations of 10, 100 and 1000% of the maximum dose (or activity) administered in adults. Possible cytotoxic effects were evaluated in comparison to untreated controls as well as positive and negative damage controls. Both full (radioactive) radiopharmaceuticals, as their precursors (only molecules without conjugation to isotopes) showed no significant cytotoxic effect (cytotoxicity ≤ 10%). The study was conducted for the first time in the country comprising preclinical testing of this radiopharmaceutical in accordance with internationally accepted quality parameters, ensuring the safety of its use and enabling inclusion in the pharmaceutical regulatory agenda. (author)

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Science.gov (United States)

Sakaguchi, H; Ashikaga, T; Miyazawa, M; Yoshida, Y; Ito, Y; Yoneyama, K; Hirota, M; Itagaki, H; Toyoda, H; Suzuki, H

2006-08-01

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen

Revealing barriers and facilitators to use a new genetic test: comparison of three user involvement methods.

Science.gov (United States)

Rhebergen, Martijn D F; Visser, Maaike J; Verberk, Maarten M; Lenderink, Annet F; van Dijk, Frank J H; Kezic, Sanja; Hulshof, Carel T J

2012-10-01

We compared three common user involvement methods in revealing barriers and facilitators from intended users that might influence their use of a new genetic test. The study was part of the development of a new genetic test on the susceptibility to hand eczema for nurses. Eighty student nurses participated in five focus groups (n = 33), 15 interviews (n = 15) or questionnaires (n = 32). For each method, data were collected until saturation. We compared the mean number of items and relevant remarks that could influence the use of the genetic test obtained per method, divided by the number of participants in that method. Thematic content analysis was performed using MAXQDA software. The focus groups revealed 30 unique items compared to 29 in the interviews and 21 in the questionnaires. The interviews produced more items and relevant remarks per participant (1.9 and 8.4 pp) than focus groups (0.9 and 4.8 pp) or questionnaires (0.7 and 2.3 pp). All three involvement methods revealed relevant barriers and facilitators to use a new genetic test. Focus groups and interviews revealed substantially more items than questionnaires. Furthermore, this study suggests a preference for the use of interviews because the number of items per participant was higher than for focus groups and questionnaires. This conclusion may be valid for other genetic tests as well.

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

Directory of Open Access Journals (Sweden)

Donohue-Rolfe Arthur

2011-06-01

Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

Influence of different test parameters on in vitro drug release from topical diclofenac formulations in a vertical diffusion cell setup.

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Klein, S

2013-07-01

In the past decades, the vertical diffusion cell has emerged as a useful device for testing drug release of topical dosage forms. However, to date neither a general USP method nor formulation-related monographs have been published in international pharmacopoeia. The purpose of the present work was to examine the influence of different test parameters in a vertical diffusion cell setup on in vitro drug release from semi-solid preparations for cutaneous application. Diclofenac was selected as the model compound. Release experiments were performed in a 7 ml Microett vertical diffusion cell system. Various test parameters, including the media composition and pH, degassing, membrane material and pore size, stirring speed and stirrer type, were varied. Results obtained with different test parameter settings clearly indicate that both drug properties and instrumental details can have a huge impact on the outcome of in vitro diffusion/drug release studies with the vertical diffusion cell. Thus, the selection of adequate test parameters is crucial for the success of the release experiments and, as shown in the present study, optimal test parameters/conditions need to be established and validated on a case by case study.

A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA).

Science.gov (United States)

Ashikaga, Takao; Sakaguchi, Hitoshi; Sono, Sakiko; Kosaka, Nanae; Ishikawa, Makie; Nukada, Yuko; Miyazawa, Masaaki; Ito, Yuichi; Nishiyama, Naohiro; Itagaki, Hiroshi

2010-08-01

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential. 2010 FRAME.

[Studies on preparation and dissolution test in vitro of sustained-release dropping pills of curcumin].

Science.gov (United States)

Fang, Yu; Xiang, Bai; Pan, Zhen-Hua; Cao, De-Ying

2010-01-01

To study the prescription and technique of sustained-release dropping pills of curcumin and inspect their release property in vitro. The orthogonal test was used to screen the prescription and technique which were definited with the colligation evaluation of release and formation of dropping pills. The optimization of prescription and technique were as follows: stearic acid 70 mg, glycery monostearate 25 mg, solutol 6 mg, viscosity of cooling liquid was 100 mm2/s; the temperature of material liquid was 80 degrees C; the cooling temperature was 30 - 0 degrees C; the dropping speed was (21 +/- 2) dripping/min. The release behavior of sustained-release dropping pills of curcumin coincidented with Higuchi equation well and the character of sustained-release was transparent. The sustained-release dropping pills of curcumin have good property of sustained-release in vitro and their release behavior in vivo need to be inspected.

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

Science.gov (United States)

Honkalampi-Hämäläinen, U; Bradley, E L; Castle, L; Severin, I; Dahbi, L; Dahlman, O; Lhuguenot, J-C; Andersson, M A; Hakulinen, P; Hoornstra, D; Mäki-Paakkanen, J; Salkinoja-Salonen, M; Turco, L; Stammati, A; Zucco, F; Weber, A; von Wright, A

2010-03-01

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Preparation, characterization and in vitro cytotoxicity of BSA-based nanospheres containing nanosized magnetic particles and/or photosensitizer

International Nuclear Information System (INIS)

Rodrigues, Marcilene M.A.; Simioni, Andreza R.; Primo, Fernando L.; Siqueira-Moura, Marigilson P.; Morais, Paulo C.; Tedesco, Antonio C.

2009-01-01

This study reports on the preparation, characterization and in vitro toxicity test of a new nano-drug delivery system (NDDS) based on bovine serum albumin (BSA) nanospheres which incorporates surface-functionalized magnetic nanoparticles (MNP) and/or the silicon(IV) phthalocyanine (NzPc). The new NDDS was engineered for use in photodynamic therapy (PDT) combined with hyperthermia (HPT) to address cancer treatment. The BSA-based nanospheres, hosting NzPc, MNP or both (NzPc and MNP), present spherical shape with hydrodynamic average diameter values ranging from 170 to 450 nm and zeta potential of around -23 mV. No difference on the fluorescence spectrum of the encapsulated NzPc was found regardless of the presence of MNP. Time-dependent fluorescence measurements of the encapsulated NzPc revealed a bi-exponential decay for samples incorporating only NzPc and NzPc plus MNP, in the time window ranging from 1.70 to 5.20 ns. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic NDDS.

Free Fatty Acid Concentration and Carboxy methyl cellulase Activity of Some Formulas of Protected Fat-proteins Tested In Vitro

Directory of Open Access Journals (Sweden)

Lilis Hartati

2015-05-01

Full Text Available The aim of this study was to determine the levels of free fatty acids and carboxymethylcellulase activity (cmc-ase activity of some protected fat-proteins base on in vitro Tilley and Terry method. Two sources of fat, i.e. crude palm oil and fish oil and three sources of protein i.e. skim milk, soybean flour and soybean meal were used in the formulation of protected fat-protein, and thus there were six treatment combinations. The filtrate from the in vitro test was analyzed for the levels of free fatty acids and  cmcase activity. The result of this research indicates that different combinations of feed materials and fat give different content of free fatty acid in first stage and second stage in vitro, with the best results in the combination treatment of skim milk and palm oil that give the lowest result of  free fatty acid concentration in fisrt stage in vitro (0.168% and the highest result free fatty acid concentration in second stage in vitro ( 4.312% . The activity of CMC-ase was not influenced by different  sources of fat and protein. It can be concluded was that the protection of the combination between skim milk and CPO gives the highest protection results.

In-vitro diagnostics of Hymenoptera venom allergy

NARCIS (Netherlands)

Rueff, F.; Vos, B.; Przybilla, B.

In-vitro diagnostics of Hymenoptera venom allergy Patients with a history of anaphylactic sting reactions require an allergological work-up (history, in-vitro tests, and skin tests) to clarify indications on venom immunotherapy and on the type of venom to be used. To demonstrate a venom

Rapid lymphocyte immunoreactivity test utilizing [3H]uridine in vitro

International Nuclear Information System (INIS)

Pienkowski, M.M.; Lyerly, M.M.; Miller, H.C.

1978-01-01

A microculture assay utilizing [ 3 H]uridine incorporation was developed to test murine spleen lymphocyte immunoreactivity in vitro. Parameters of the culture technique which included cell density, doses of LPS, Con A, PHA, [ 3 H]uridine levels, and length of culture time were investigated. Responses were detectable at 4 h for all 3 mitogens, with labelling ranging up to 180% of the control value. By 8 h there was a 200-350% increase in mitogen-induced incorporation of radioactivity. Similar increases were observed in a serum-free system. The responses were the result of increased incorporation of label by stimulated cultures rather than decreased labeling of non-mitogen treated cultures over time. The [ 3 H]uridine incorporation was demonstrated to be the selective response of T or B cell populations when stimulated with appropriate lectins. This assay detects early RNA synthesis, as supported by experimental observations in which accumulation of radioactivity in stimulated lymphocytes was TCA precipitable, resistant to SDS treatment, and inhibited by actinomycin D. (Auth.)

Biological evaluation of dental materials, in vitro and in vivo

International Nuclear Information System (INIS)

Kawahara, H.

1982-01-01

In this paper, the correlation between the user of tissue culture for in vitro tests and the tissue irritability and pupal response observed in in vitro tests, will be discussed. It would produce confusion if dental materials were standardised with the unreliable parameter of the living system in dynamic balance. Biological tests, both in vitro and in vivo, should be used for pre-standards testing, without any political control to establish physicochemical standards. As a first step, corrosion tests and the dissolution dosje of toxic components from the material in the tissue culture medium and/or artificial salvia should be standardised under conditions simulating the oral environment. The CNC method and photo-pattern analysis are used for the interpretation of cytotoxicity. The need for biological testing, both in vitro and in vivo, definitely exists in order to obtain physicochemical standards, with a biological simulation depending upon the feedback obtained from the results of in vitro and in vivo tests

5-Fluorouracil, colchicine, benzo[a]pyrene and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster V79 cells at Covance Laboratories, Harrogate, UK in support of OECD draft Test Guideline 487.

Science.gov (United States)

Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, Debbie; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

2010-10-29

The reference genotoxic agents 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster V79 cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In Vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

In Vitro Susceptibility Testing of Tedizolid against Isolates of Nocardia.

Science.gov (United States)

Brown-Elliott, Barbara A; Wallace, Richard J

2017-12-01

There is a paucity of efficacious antimicrobials (especially oral) against clinically relevant species of Nocardia To date, all species of Nocardia have been susceptible to linezolid, the first commercially available oxazolidinone. Tedizolid is a new oxazolidinone with previously reported improved in vitro and in vivo (intracellular) potency against multidrug-resistant strains of Mycobacterium sp. and Nocardia brasiliensis Using the current Clinical and Laboratory Standards Institute-recommended broth microdilution method, 101 isolates of Nocardia spp., including 29 Nocardia cyriacigeorgica , 17 Nocardia farcinica , 13 Nocardia nova complex, 21 Nocardia brasiliensis , 5 Nocardia pseudobrasiliensis , and 5 Nocardia wallacei isolates and 11 isolates of less common species, were tested for susceptibility to tedizolid and linezolid. For the most common clinically significant species of Nocardia , tedizolid MIC 50 values were 0.25 μg/ml for N. nova complex, N. brasiliensis , N. pseudobrasiliensis , and N. wallacei , compared to linezolid MIC 50 values of 1, 2, 0.5, and 1 μg/ml, respectively. Tedizolid and linezolid MIC 90 values were 2 μg/ml for N. nova complex and N. brasiliensis Tedizolid MIC 50 and MIC 90 values for both N. cyriacigeorgica and N. farcinica were 0.5 μg/ml and 1 μg/ml, respectively, compared to linezolid MIC 50 and MIC 90 values of 2 and 4 μg/ml, respectively. Based on MIC 90 values, this study showed that tedizolid was 2- to 3-fold more active than linezolid in vitro against most common species of Nocardia , with the exception of the N. nova complex and N. brasiliensis , for which values were the same. These results may warrant evaluation of tedizolid as a potential treatment option for Nocardia infections. Copyright © 2017 American Society for Microbiology.

Short-term incubation in vitro with precursors of nucleic acids on human primary tumors and metastases of carcinoma of the breast

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, M; Kubli, F; Volm, M; Fournier, D V; Reus, W [Heidelberg Univ. (Germany, F.R.). Frauenklinik; Deutsches Krebsforschungszentrum, Heidelberg (Germany, F.R.). Inst. fuer Experimentelle Pathologie)

1978-04-01

A technique of short-term tests in vitro by means of the incubation of tumor cell suspensions is utilized as a radioactive-biochemical method for pretherapeutic determination of the resistance in human cancers of the breast. Cell suspensions from primary tumors and metastases reveal individually different responses to cytostatics in vitro. It is possible, therewith, to differentiate two tumor collectives related to in vivo resistant or in vivo sensitive tumors. The responses of the primary lesion and the axillary lymphatic metastasis of the same carcinoma may in single cases also differ in vitro, according to clinical experience with the therapy of breast cancer. A distinct relation can be shown between the histological type of a carcinoma and its in vitro capacity of resistance.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

International Nuclear Information System (INIS)

Vieira, J.M.B.D.; Seabra, S.H.; Vallim, D.C.; Americo, M.A.; Fracallanza, S.E.L.; Vommaro, R.C.; Domingues, R.M.C.P.

2009-01-01

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Energy Technology Data Exchange (ETDEWEB)

Vieira, J.M.B.D., E-mail: jmanya@terra.com.br [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil); Seabra, S.H. [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Vallim, D.C. [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil); Americo, M.A.; Fracallanza, S.E.L. [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil); Vommaro, R.C. [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil); Domingues, R.M.C.P. [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)

2009-10-02

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Investigation of moxifloxacin loaded chitosan–dextran nanoparticles for topical instillation into eye: In-vitro and ex-vivo evaluation

Science.gov (United States)

Kaskoos, Raad A.

2014-01-01

Introduction: Management of ocular surface disease by conventional formulation is limited by poor residence of drug at cul-de-sac of eye. To overcome this limitation, prolonged released mucoadhesive chitosan (CS)–dextran sulfate (DS) nanoparticles (NPs) were investigated for the prolonged topical ophthalmic delivery of moxifloxacin (Mox). Methods: Formulation was optimized by 3-factors (CS, DS, and Mox concentration), 3-levels (−1, 0, +1) Box-Behnken design. Optimized formulation was characterized for various in-vitro attributes, including particles size, zeta potential, shape and morphology, in-vitro release profile, corneal permeation, corneal retention, ocular tolerance test as well as antimicrobial activity. Results: Average hydrodynamic particle size of statistically optimized formulation was found to be 279.18 ± 15.63 nm with good polydispersity index, 0.367 ± 0.016 and positive zeta potential, +31.23 ± 1.32. NPs showed entrapment efficiency, 72.82 ± 3.6% and transmission electron microscopic analysis revealed a spherical shape of particles. Formulation exhibited biphasic release profile with an initial fast release (≈25% in 1st h) followed by sustained release (≈95% in next 24 h) following Korsmeyer–Peppas model with a nonFickian diffusion process. Mox loaded CS-DS NPs exhibited a significantly higher (P < 0.01), approximately 1.8-fold transcorneal permeation as well as significantly higher corneal retention (P < 0.01), around 4-5-fold when compared to free solution. Developed formulation exhibited safety profile comparable to normal saline, which was revealed by ocular tolerance test (Hen's egg test-chorioallantoic membrane). Mox-CS-DS NPs exhibited significantly high (P < 0.01) antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Conclusion: In-vitro and ex-vivo studies revealed that developed formulation could be a potential substitute for prolonged topical ocular delivery. PMID:25426437

Implementation challenges for designing integrated in vitro testing strategies (ITS) aiming at reducing and replacing animal experimentation.

Science.gov (United States)

De Wever, Bart; Fuchs, Horst W; Gaca, Marianna; Krul, Cyrille; Mikulowski, Stan; Poth, Albrecht; Roggen, Erwin L; Vilà, Maya R

2012-04-01

At the IVTIP (in vitro testing industrial platform) meeting of November 26th 2009 entitled 'Toxicology in the 21st century ('21C')--working our way towards a visionary reality' all delegates endorsed the emerging concept of the '21C' vision as the way forward to enable a thorough, reliable and systematic approach to future toxicity testing without the use of animals. One of the emerging concepts focused on integrating a defined number of tests modelling in vivo-relevant and well-characterised toxicity pathways representing mechanistic endpoints. At this meeting the importance of Integrated Testing Strategies (ITS) as tools towards reduction and eventually replacement of the animals currently used for hazard identification and risk assessment was recognised. A follow-up IVTIP Spring 2010 meeting entitled 'Integrated In Vitro Testing Strategies (ITS)--Implementation Challenges' was organised to address pending questions about ITS. This report is not a review of the ITS literature, but a summary of the discussions triggered by presented examples on how to develop and implement ITS. Contrasts between pharmaceutical and chemical industry, as well as a list of general but practical aspects to be considered while developing an ITS emerged from the discussions. In addition, current recommendations on the validation of ITS were discussed. In conclusion, the outcome of this workshop improved the understanding of the participants of some important factors that may impact the design of an ITS in function of its purpose (e.g., screening, or early decision making versus regulatory), the context in which they need to be applied (e.g., ICH guidelines, REACH) and the status and quality of the available tools. A set of recommendations of best practices was established and the importance of the applicability of the individual tests as well as the testing strategy itself was highlighted. Copyright © 2012 Elsevier Ltd. All rights reserved.

A realistic in vitro exposure revealed seasonal differences in (pro-)inflammatory effects from ambient air in Fribourg, Switzerland.

Science.gov (United States)

Bisig, Christoph; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2018-01-01

Ambient air pollutant levels vary widely in space and time, therefore thorough local evaluation of possible effects is needed. In vitro approaches using lung cell cultures grown at the air-liquid interface and directly exposed to ambient air can offer a reliable addition to animal experimentations and epidemiological studies. To evaluate the adverse effects of ambient air in summer and winter a multi-cellular lung model (16HBE14o-, macrophages, and dendritic cells) was exposed in a mobile cell exposure system. Cells were exposed on up to three consecutive days each 12 h to ambient air from Fribourg, Switzerland, during summer and winter seasons. Higher particle number, particulate matter mass, and nitrogen oxide levels were observed in winter ambient air compared to summer. Good cell viability was seen in cells exposed to summer air and short-term winter air, but cells exposed three days to winter air were compromised. Exposure of summer ambient air revealed no significant upregulation of oxidative stress or pro-inflammatory genes. On the opposite, the winter ambient air exposure led to an increased oxidative stress after two exposure days, and an increase in three assessed pro-inflammatory genes already after 12 h of exposure. We found that even with a short exposure time of 12 h adverse effects in vitro were observed only during exposure to winter but not summer ambient air. With this work we have demonstrated that our simple, fast, and cost-effective approach can be used to assess (adverse) effects of ambient air.

In-vitro diagnosis

DEFF Research Database (Denmark)

Poulsen, Lars K.

2001-01-01

This review on in-vitro diagnostic methods focuses on the use of methods to perform risk assessment on foods. Based on the International Life Science Institute (ILSI) risk decision tree, the methods are discussed and three scenarios are suggested: (i) testing for a well-known allergen; (ii) testing...

Design Optimization and In Vitro-In Vivo Evaluation of Orally Dissolving Strips of Clobazam

Directory of Open Access Journals (Sweden)

Rajni Bala

2014-01-01

Full Text Available Clobazam orally dissolving strips were prepared by solvent casting method. A full 32 factorial design was applied for optimization using different concentration of film forming polymer and disintegrating agent as independent variable and disintegration time, % cumulative drug release, and tensile strength as dependent variable. In addition the prepared films were also evaluated for surface pH, folding endurance, and content uniformity. The optimized film formulation showing the maximum in vitro drug release, satisfactory in vitro disintegration time, and tensile strength was selected for bioavailability study and compared with a reference marketed product (frisium5 tablets in rabbits. Formulation (F6 was selected by the Design-expert software which exhibited DT (24 sec, TS (2.85 N/cm2, and in vitro drug release (96.6%. Statistical evaluation revealed no significant difference between the bioavailability parameters of the test film (F6 and the reference product. The mean ratio values (test/reference of Cmax (95.87%, tmax (71.42%, AUC0−t (98.125%, and AUC0−∞ (99.213% indicated that the two formulae exhibited comparable plasma level-time profiles.

Design Optimization and In Vitro-In Vivo Evaluation of Orally Dissolving Strips of Clobazam

Science.gov (United States)

Bala, Rajni; Khanna, Sushil; Pawar, Pravin

2014-01-01

Clobazam orally dissolving strips were prepared by solvent casting method. A full 32 factorial design was applied for optimization using different concentration of film forming polymer and disintegrating agent as independent variable and disintegration time, % cumulative drug release, and tensile strength as dependent variable. In addition the prepared films were also evaluated for surface pH, folding endurance, and content uniformity. The optimized film formulation showing the maximum in vitro drug release, satisfactory in vitro disintegration time, and tensile strength was selected for bioavailability study and compared with a reference marketed product (frisium5 tablets) in rabbits. Formulation (F6) was selected by the Design-expert software which exhibited DT (24 sec), TS (2.85 N/cm2), and in vitro drug release (96.6%). Statistical evaluation revealed no significant difference between the bioavailability parameters of the test film (F6) and the reference product. The mean ratio values (test/reference) of C max (95.87%), t max (71.42%), AUC0−t (98.125%), and AUC0−∞ (99.213%) indicated that the two formulae exhibited comparable plasma level-time profiles. PMID:25328709

Extreme testing of undiluted e-cigarette aerosol in vitro using an Ames air-agar-interface technique.

Science.gov (United States)

Thorne, D; Hollings, M; Seymour, A; Adamson, J; Dalrymple, A; Ballantyne, M; Gaca, M

2018-04-01

There is a growing consensus that e-cigarettes hold the potential for reducing the harm associated with cigarette smoking. Recently published studies have reported in vitro testing of e-cigarettes, demonstrating reduced toxicological and biological effects. Few studies however have reported the use of e-cigarettes under extreme testing conditions. To assess the full mutagenic potential of a commercially available electronic-cigarette (Vype ePen), this study investigated the delivery of aerosol under extreme conditions, using a scaled-down 35 mm plate Ames bacterial reverse mutagenicity assay. S. typhimurium strains TA98, TA100, TA97, TA104 and E. coli WP2 uvrA pKM101 with or without metabolic activation (S9), were employed. Using a modified Vitrocell VC 10 exposure system 0, 180, 360, 540, 720 or 900 puffs of undiluted e-cigarette aerosol was generated and delivered to bacterial cultures aligned to reported human consumption data. The results demonstrate that no mutagenic activity was observed in any strain under any test condition even when exposed to 900 puffs of undiluted e-cigarette aerosols +/- S9. Positive control responses were observed in all strains +/- S9. Nicotine assessments demonstrated an increased and consistent aerosol delivery, with calculated maximum doses of ∼1 mg/mL delivery of nicotine. These data demonstrate the validity of this unique testing approach and adds further information to the growing weight of evidence that e-cigarettes offer substantially reduced exposure when compared to conventional cigarette smoke. For future in vitro assessments of next generation tobacco and nicotine products, the generation, delivery and testing of undiluted aerosols can now be considered. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

[In vitro activity of ertapenem against clinical bacterial isolates in 69 Spanish medical centers (E-test study)].

Science.gov (United States)

Gobernado, M; Sanz-Rodríguez, C; Villanueva, R; Torroba, L; Redondo, E; González-Esteban, J

2007-12-01

This study was conducted to assess the in vitro activity of ertapenem against clinical bacterial isolates from patients with community-acquired intra-abdominal and lower tract respiratory infections in Spain in 2003. As the study was conducted before the marketing of ertapenem, it was also useful to define a baseline susceptibility pattern for ertapenem in each of the participating hospitals for later surveillance studies. Each partipating site identified a variable number of aerobic and facultative bacteria isolated from patients with community-acquired intra-abdominal infection or pneumonia using standard procedures. E-test strips were used for determining the minimum inhibitory concentration (MIC) of ertapenem, while for other antimicrobials either quantitative dilution techniques or qualitative diffusion procedures were used according to each microbiology laboratory's routine practice. MIC breakpoints for categorization of susceptibility provided by the CLSI were used for interpreting MIC values. A total of 2,901 recent clinical isolates from patients with community-acquired intra-abdominal infection or pneumonia hospitalized in 69 Spanish medical centers were tested. These isolates included 2,039 Gram-negative bacteria (1,646 Enterobacteriaceae, 216 Haemophilus, 123 non-fermenting Gram-negative bacteria [NFGNB] and 54 others) and 862 Gram-positive bacteria (556 pneumococci, 159 staphylococci, 96 streptococci other than S. pneumoniae, 44 enterococci and 7 others). Ertapenem was very active in vitro against Enterobacteriaceae (99.8% susceptible), Haemophilus (96.3% susceptible), pneumococci (99.6% susceptible, of which 31% were penicillin non-susceptible strains), streptococci other than S. pneumoniae (99.0% susceptible) and methicillin-susceptible staphylococci (94.8% susceptible). For other Gram-positive and Gram-negative pathogens for which ertapenem susceptible breakpoints have not been defined, MIC(90) values were 0.38 and 0.064 mg/l, respectively. As

In Vitro Antibiotic Susceptibilities of Burkholderia mallei (Causative Agent of Glanders) Determined by Broth Microdilution and E-Test

Science.gov (United States)

Heine, Henry S.; England, Marilyn J.; Waag, David M.; Byrne, W. Russell

2001-01-01

In vitro susceptibilities to 28 antibiotics were determined for 11 strains of Burkholderia mallei by the broth microdilution method. The B. mallei strains demonstrated susceptibility to aminoglycosides, macrolides, quinolones, doxycycline, piperacillin, ceftazidime, and imipenem. For comparison and evaluation, 17 antibiotic susceptibilities were also determined by the E-test. E-test values were always lower than the broth dilution values. Establishing and comparing antibiotic susceptibilities of specific B. mallei strains will provide reference information for assessing new antibiotic agents. PMID:11408233

In vitro trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

Science.gov (United States)

Brun, R; Bühler, Y; Sandmeier, U; Kaminsky, R; Bacchi, C J; Rattendi, D; Lane, S; Croft, S L; Snowdon, D; Yardley, V; Caravatti, G; Frei, J; Stanek, J; Mett, H

1996-01-01

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for in vitro antitrypanosomal activities and cytotoxicities for human cells. One-third of the compounds tested showed trypanocidal activity at concentrations below 0.5 microM after an incubation period of 72 h. Structure-activity analysis revealed that bicyclic compounds with homocyclic rings and unmodified termini were the most active compounds. Results obtained in three laboratories employing different methods and trypanosome populations consistently ranked compound CGP 40215A highest. This compound had a 50% inhibitory concentration of 0.0045 microM for Trypanosoma brucei rhodesiense, was also active against other trypanosome species, including a multidrug-resistant Trypanosoma brucei brucei, and was significantly less toxic than other compounds tested for a human adenocarcinoma cell line, with a 50% inhibitory concentration of 1.14 mM. The effect of CGP 40215A was time and dose dependent, and low concentrations of the compound required exposure times of > 2 days to exert trypanocidal activity. Compounds were inactive against Leishmania donovani and Trypanosoma cruzi amastigotes in murine macrophages in vitro. PMID:8726017

Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella

International Nuclear Information System (INIS)

Lazzari, Giovanna; Tessaro, Irene; Crotti, Gabriella; Galli, Cesare; Hoffmann, Sebastian; Bremer, Susanne; Pellizzer, Cristian

2008-01-01

Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility

Radioactively labelled substances for in vivo and in vitro tests from the time of their production up to their use in nuclear medicine

International Nuclear Information System (INIS)

Riccato, M.T.

1976-01-01

Most important production steps and control tests applied to radionuclides and radioactive labelled compounds used in nuclear medicine are described. Radiopharmaceutical products are subdivided in products for therapy and for in vivo and in vitro diagnostics tests. (orig./HP) [de

Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

Science.gov (United States)

Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

2016-06-01

Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

A comparison of finite element analysis with in vitro bond strength tests of the bracket-cement-enamel system

NARCIS (Netherlands)

Algera, T.J.; Feilzer, A.J.; Prahl-Andersen, B.; Kleverlaan, C.J.

2011-01-01

The aim of this study was to determine the in vitro shear bond strength (SBS) and tensile bond strength (TBS) of 45 metal brackets bonded with Transbond XT to bovine enamel. The SBS was determined by loading the short and the long sides of the bracket base. Testing took place after storage of the

Are In Vitro Methods for the Detection of Endocrine Potentials in the Aquatic Environment Predictive for In Vivo Effects? Outcomes of the Projects SchussenAktiv and SchussenAktivplus in the Lake Constance Area, Germany

Science.gov (United States)

Henneberg, Anja; Bender, Katrin; Blaha, Ludek; Giebner, Sabrina; Kuch, Bertram; Köhler, Heinz-R.; Maier, Diana; Oehlmann, Jörg; Richter, Doreen; Scheurer, Marco; Schulte-Oehlmann, Ulrike; Sieratowicz, Agnes; Ziebart, Simone; Triebskorn, Rita

2014-01-01

Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption

Are in vitro methods for the detection of endocrine potentials in the aquatic environment predictive for in vivo effects? Outcomes of the Projects SchussenAktiv and SchussenAktivplus in the Lake Constance Area, Germany.

Directory of Open Access Journals (Sweden)

Anja Henneberg

Full Text Available Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1 chemical analyses, (2 in vitro bioassays, and (3 in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7 and reporter gene assays (human cell line HeLa-9903 and MDA-kb2. In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario to water downstream of a wastewater outfall (Schussen River or to water from a reference site (Argen River to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus and spirlin (Alburnoides bipunctatus, were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests and an occasional endocrine action (vitellogenin levels in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index. We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine

In vitro toxicity of infusion sets depends on their composition, storage time and storage conditions.

Science.gov (United States)

Kozlovskaya, Luba; Popilski, Hen; Gorenbein, Pavel; Stepensky, David

2015-07-15

Disposable medical devices release toxic leachables during their clinical use. Specifically, the individual parts of the infusion sets (the drip chamber, tube, flashball and injection site) are composed of numerous chemical compounds that can reach the patients' systemic circulation and induce local and systemic toxic effects. We aimed to reveal the relative in vitro toxicity of infusion sets from the leading vendors that are used in Israel, and to determine its dependence on their design and storage time/conditions. We found that leachates of the rubber parts were more toxic than those of the other parts of the infusion sets. The measured toxicity was affected by the experimental settings: the cells, medium composition, exposure duration, and the type of assay applied for toxicity assessment. We recommend to use the capillary endothelium cells for in vitro toxicity testing of the infusion sets, and refrain from the use of the MTT test which is insufficiently reliable, and can lead to artefacts and incorrect conclusions. Further investigation is needed to identify the toxic leachables from the individual parts of the infusion sets, and to reveal the risk of their toxicity during the clinical use of the infusion sets. Copyright © 2015 Elsevier B.V. All rights reserved.

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

Science.gov (United States)

Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

2017-10-17

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

In vitro disposition profiling of heterocyclic compounds.

Science.gov (United States)

Keemink, Janneke; Wuyts, Benjamin; Nicolaï, Johan; Jonghe, Steven De; Stella, Alessandro; Herdewijn, Piet; Augustijns, Patrick; Annaert, Pieter

2015-08-01

Compound libraries that are screened for biological activity commonly contain heterocycles. Besides potency, drug-like properties need to be evaluated to ensure in vivo efficacy of test compounds. In this context, we determined hepatic and intestinal disposition profiles for 17 heterocyclic compounds. All studied compounds showed rapid uptake in suspended rat hepatocytes, whereas metabolism was poor and the rate-limiting step in hepatic elimination. In vitro assays demonstrated a relatively low solubility and high intestinal permeability. Based on these in vitro data, heterocycles were categorized in the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS) to predict disposition characteristics before clinical data are available. Our findings emphasized the importance to use hepatocytes in addition to microsomes to study metabolism, since the latter lack non-microsomal enzymes and cellular context. Moreover, intracellular exposure should be considered to gain insight in the relevant fraction of the compound available at the enzymatic site. Finally, the study reveals discrepancies associated with the classification of heterocycles in BCS versus BDDCS. These probably originate from the binary character of both systems. Copyright © 2015 Elsevier B.V. All rights reserved.

[In Vitro and In Vivo Biocompatibility of a Novel, 3-Dimensional Cellulose Matrix Structure].

Science.gov (United States)

Dunda, S E; Ranker, M; Pallua, N; Machens, H-G; Ravichandran, A; Schantz, J-T

2015-12-01

Biological and physical characteristics of matrices are one essential factor in creating bioartificial tissue. In this study, a new 3-dimensional cellulose matrix (Xellulin(®)) was tested in terms of biocompatibility and applicability for tissue engineering in vitro and in vivo. The tested matrix Xellulin(®) is a natural hydrological gel-matrix containing bacterial cellulose and water. To evaluate the cell biocompatibilty, cell adherence and proliferation characteristics in vitro, the matrix was cultured with human fibroblasts. Further in vivo studies were carried out by transplanting preadipocytes of 4- to 6-week-old Wistar rats with 3 different conditions: a) Xellulin(®) including 500 000 preadipocytes subcutaneous, b) Xellulin(®) including 500 000 preadipocytes within an in vivo bioreactor chamber, c) Xellulin(®) without cells subcutaneous as control. After explantation on day 14 histomorphological and immunohistochemical evaluations were performed. In vitro study revealed an excellent biocompatibility with good cell adherence of the fibroblasts on the matrix and evidence of cell proliferation and creation of a 3-dimensional cell network. In vivo neocapillarisation could be shown in all groups with evidence of erythrocytes (H/E staining) and endothelial vascular cells (RECA-1-staining). A significantly higher vascular density was shown in vascularised bioreactor group (18.4 vessels/100 000 µm(2) (group b) vs. 8.1 (group a), pmatrix was noticed. The promising in vitro results concerning cell adherence and proliferation on the tested matrix could be confirmed in vivo with an evidence of 3-dimensional neocapillarisation. Cell survival was higher in the vascularised group, but without significance. Long-term tests (28-42 days) need to be carried out to evaluate long-term cell survival and the matrix stability. Furthermore, studies concerning the implementation of the matrix within anatomic structures as well as long-term biocompatibility are needed. �

Forecasting gastrointestinal precipitation and oral pharmacokinetics of dantrolene in dogs using an in vitro precipitation testing coupled with in silico modeling and simulation.

Science.gov (United States)

Kambayashi, Atsushi; Dressman, Jennifer B

2017-10-01

The aim of the current research was to determine the precipitation kinetics of dantrolene sodium using canine biorelevant in vitro testing and to model the precipitation kinetics by appropriately coupling the data with an in silico tool adapted for dogs. The precipitation profiles of dantrolene sodium solutions were obtained with the in vitro paddle apparatus at a revolution rate of 50rpm. The in silico prediction tool was designed using STELLA software and the predicted plasma concentration profiles of dantrolene using the in vitro precipitation data were compared with the observed in vivo pharmacokinetics in beagle dogs. The plasma profiles of dantrolene, which served as a model weakly acidic drug which precipitates in the upper gastrointestinal tract, was successfully predicted using the in vitro precipitation testing coupled with the in silico modeling and simulation approach. The approach was subsequently used to forecast the effect of pharmaceutical excipients (HPMC/PG) on the ability of the drug to supersaturate in the gut and the resulting pharmacokinetics. The agreement of the simulated pharmacokinetics with the observed values confirms the ability of canine biorelevant media to predict oral performance of enhanced dosage forms in dogs. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vitro Antidiabetic Effects and Antioxidant Potential of Cassia nemophila Pods

Directory of Open Access Journals (Sweden)

Gauhar Rehman

2018-01-01

Full Text Available The antidiabetic and antioxidant potential of ethanolic extract of Cassia nemophila pod (EECNP was evaluated by three in vitro assays, including yeast glucose uptake assay, glucose adsorption assay, and DPPH radical scavenging activity. The result revealed that the extracts have enhanced the uptake of glucose through the plasma membrane of yeast cells. A linear increase in glucose uptake by yeast cells was noticed with gradual increase in the concentration of the test samples. Moreover, the adsorption capacity of the EECNP was directly proportional to the molar concentration of glucose. Also, the DPPH radical scavenging capacity of the extract was increased to a maximum value of 43.3% at 80 μg/ml, which was then decreased to 41.9% at 100 μg/ml. From the results, it was concluded that EECNP possess good antidiabetic and antioxidant properties as shown by in vitro assays.

In vitro chemo-sensitivity assay guided chemotherapy is associated with prolonged overall survival in cancer patients.

Science.gov (United States)

Udelnow, Andrej; Schönfęlder, Manfred; Würl, Peter; Halloul, Zuhir; Meyer, Frank; Lippert, Hans; Mroczkowski, Paweł

2013-06-01

The overall survival (OS) of patients suffering From various tumour entities was correlated with the results of in vitro-chemosensitivity assay (CSA) of the in vivo applied drugs. Tumour specimen (n=611) were dissected in 514 patients and incubated for primary tumour cell culture. The histocytological regression assay was performed 5 days after adding chemotherapeutic substances to the cell cultures. n=329 patients undergoing chemotherapy were included in the in vitro/in vivo associations. OS was assessed and in vitro response groups compared using survival analysis. Furthermore Cox-regression analysis was performed on OS including CSA, age, TNM classification and treatment course. The growth rate of the primary was 73-96% depending on tumour entity. The in-vitro response rate varied with histology and drugs (e.g. 8-18% for methotrexate and 33-83% for epirubicine). OS was significantly prolonged for patients treated with in vitro effective drugs compared to empiric therapy (log-rank-test, p=0.0435). Cox-regression revealed that application of in vitro effective drugs, residual tumour and postoperative radiotherapy determined the death risk independently. When patients were treated with drugs effective in our CSA, OS was significantly prolonged compared to empiric therapy. CSA guided chemotherapy should be compared to empiric treatment by a prospective randomized trial.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests.

Science.gov (United States)

Argyri, Anthoula A; Zoumpopoulou, Georgia; Karatzas, Kimon-Andreas G; Tsakalidou, Effie; Nychas, George-John E; Panagou, Efstathios Z; Tassou, Chrysoula C

2013-04-01

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes

In vitro evaluation of biomimetic chitosan–calcium phosphate scaffolds with potential application in bone tissue engineering

International Nuclear Information System (INIS)

Tanase, C E; Popa, M I; Sartoris, A; Unger, R E; Kirkpatrick, C J; Verestiuc, L

2013-01-01

This work reports on the physicochemical properties and in vitro cytotoxicity assessment of chitosan–calcium phosphate (Cs–CP) scaffolds for bone tissue engineering, which were synthesized by a novel biomimetic co-precipitation method. X-ray diffraction (XRD) along with scanning electron microscopy (SEM) analysis confirmed the porous morphology of the scaffolds and the amorphous nature of the inorganic phase with different crystallite sizes and the formation of various forms of calcium phosphate. Compressive mechanical testing revealed that the Young's modulus of the biomaterials is in the range of human trabecular bone. In vitro tests were performed on the biomaterials for up to 14 days to study the behavior of the osteoblast-like human cell line (MG63), primary human osteoblasts (HOS) and human dermal microvascular endothelial cells (HDMEC). The cytotoxicity was evaluated by the MTS assay for cell metabolism and the detection of membrane integrity (lactate dehydrogenase-LDH release). An expression of the vascular endothelial growth factor (VEGF) in the cell supernatants was quantified by ELISA. Cell viability gave values close to untreated controls for MG63 and HOS, while in the case of HDMEC the viability after 2 weeks in the cell culture was between 80–90%. The cytotoxicity induced by the Cs–CP scaffolds on MG63, HOS and HDMEC in vitro was evaluated by the amount of LDH released, which is a sensitive and accurate marker for cellular toxicity. The increased levels of VEGF obtained in the osteoblast culture highlights its important role in the regulation of vascularization and bone remodeling. The biological responses of the Cs–CP scaffolds demonstrate a similar proliferation and differentiation characteristics of the cells comparable to the controls. These results reveal that biomimetic Cs–CP composite scaffolds are promising biomaterials for bone tissue engineering; their in vivo response remains to be tested. (paper)

In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing : Application of a generic PBK modelling approach

NARCIS (Netherlands)

Fragki, Styliani; Piersma, Aldert H; Rorije, Emiel; Zeilmaker, Marco J

2017-01-01

Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at

In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing: Application of a generic PBK modelling approach.

NARCIS (Netherlands)

Fragki, Styliani; Piersma, Aldert H; Rorije, Emiel; Zeilmaker, Marco J

2017-01-01

Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at

Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

OpenAIRE

Lenaerts, Anne J.; Gruppo, Veronica; Marietta, Karen S.; Johnson, Christine M.; Driscoll, Diane K.; Tompkins, Nicholas M.; Rose, Jerry D.; Reynolds, Robert C.; Orme, Ian M.

2005-01-01

This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of...

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test.

Science.gov (United States)

Alves, Eloísa Nunes; Presgrave, Rosaura de Farias; Presgrave, Octávio Augusto França; Sabagh, Fernanda Peres; de Freitas, João Carlos Borges Rolim; Corrado, Alexandre P

2008-07-01

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

Effects of Achyrocline satureioides Inflorescence Extracts against Pathogenic Intestinal Bacteria: Chemical Characterization, In Vitro Tests, and In Vivo Evaluation

Directory of Open Access Journals (Sweden)

Karla Suzana Moresco

2017-01-01

Full Text Available Three Achyrocline satureioides (AS inflorescences extracts were characterized: (i a freeze-dried extract prepared from the aqueous extractive solution and (ii a freeze-dried and (iii a spray-dried extract prepared from hydroethanol extractive solution (80% ethanol. The chemical profile, antioxidant potential, and antimicrobial activity against intestinal pathogenic bacteria of AS extracts were evaluated. In vitro antioxidant activity was determined by the total reactive antioxidant potential (TRAP assay. In vivo analysis and characterization of intestinal microbiota were performed in male Wistar rats (saline versus treated animals with AS dried extracts by high-throughput sequencing analysis: metabarcoding. Antimicrobial activity was tested in vitro by the disc diffusion tests. Moisture content of the extracts ranged from 10 to 15% and 5.7 to 17 mg kg−1 of fluorine. AS exhibited antioxidant activity, especially in its freeze-dried form which also exhibited a wide spectrum of antimicrobial activity against intestinal pathogenic bacteria greater than those observed by the antibiotic, amoxicillin, when tested against Bacillus cereus and Staphylococcus aureus. Antioxidant and antimicrobial activities of AS extracts seemed to be positively correlated with the present amount of flavonoids. These findings suggest a potential use of AS as a coadjuvant agent for treating bacterial-induced intestinal diseases with high rates of antibiotic resistance.

Comparative in vitro study for orthodontic adhesives relatively to sorption and solubility

Science.gov (United States)

Muntean, A.; Mesaros, A.; Festila, D.; Moldovan, M.; Boboia, S.; Mesaros, M.

2015-12-01

Water sorption and solubility correspond to undesirable physical characteristics because it may cause micro leakage and dissolution for composite materials used for orthodontic attachment bonding. The aim of this study was to evaluate the performance of four composite materials employed in orthodontic as adhesives, relatively to water and 50% alcoholic solution, by means of in vitro tests of sorption and solubility. We used an experimental composite sealer SO® (ICCRR Cluj Napoca) and 3 commercial products already on the market: Blugloo® (Ormco), Opal Bond MV® (Ultradent) and Bond It® (DB orthodontics). Data were recorded and specific statistic tests were performed, revealing significant differences for all materials relatively to tested solutions. The materials expressed an adequate performance in terms of sorption and solubility, offering various alternatives for orthodontists.

The 13th quality control survey for radioisotopes in vitro tests in Japan, 1991

Energy Technology Data Exchange (ETDEWEB)

1992-11-01

This paper presents the results of the 13th quality control nationwide survey. Of 568 hospitals performing RI in vitro tests as of December 1991, 280 (49.3%) participated in this survey. The following 37 items were examined: ACTH, 17[alpha]-hydroxyprogesterone, [alpha]-fetoprotein, aldosterone, [beta]2-microglobulin, CA 125, CA 15-3, CA 19-9, calcitonin, CEA, cortisol, C-peptide, digoxin, esteradiol, ferritin, free triiodothyronine (T[sub 3]), free thyroxine (T[sub 4]), FSH, gastrin, GH, glucagon, IgE, insulin, LH, PAP, progesterone, prolactin, PTH, somatomedin C, T[sub 3], T[sub 4], T[sub 3] uptake, TBG, testosterone, thyroglobulin, tissue polypeptide antigen, and TSH. Reliability and safety of measurement values were evaluated based on protein components of serum samples, spontaneous reliability test, time-dependent reliability test, repeated freezing-dissolution test, and serial change test after freezing. 'Within kit variation' between facilities was great especially for ACTH, CA 15-3, CA 19-9, free T[sub 4], calcitonin, gastrin, and testosterone, and was favorable for [beta][sub 2]-microglobulin, CA 125, digoxin, cortisol, IgE, T[sub 3], T[sub 4], T[sub 3] uptake, and TBG. It was noted that there was a great difference in measurement values of free T[sub 3] and free-T[sub 4] between RIA kits and non-isotopic kits. (N.K.).

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

Science.gov (United States)

Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime

2016-08-01

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions

DEFF Research Database (Denmark)

Østrup, Olga; Olbricht, Gayla; Østrup, Esben

2013-01-01

produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos showed striking overlap in functional annotation of transcripts during the EGA, suggesting conserved basic mechanisms...... a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell...... from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos...

Microleakage of composite crowns luted on CAD/CAM-milled human molars: a new method for standardized in vitro tests.

Science.gov (United States)

Schlenz, Maximiliane Amelie; Schmidt, Alexander; Rehmann, Peter; Niem, Thomas; Wöstmann, Bernd

2018-04-24

To investigate debonding of full crowns made of CAD/CAM composites, CAD/CAM technology was applied to manufacture standardized test abutments to increase the reproducibility of human teeth used in in vitro studies. A virtual test abutment and the corresponding virtual crown were designed and two STL data sets were generated. Sixty-four human third molars and CAD/CAM blocks were milled using a CNC machine. Crowns of four different composite blocks (Lava Ultimate (LU), Brilliant Crios (BC), Cerasmart (CS), Experimental (EX)) were adhesively bonded with their corresponding luting system (LU: Scotchbond Universal/RelyX Ultimate; BC: One Coat 7 Universal/DuoCem; CS: G-PremioBond/G-Cem LinkForce; EX: Experimental-Bond/Experimental-Luting-Cement). Half of the specimens were chemical-cured (CC) and the others were light-cured (LC). Afterwards, specimens were artificially aged in a chewing simulator (WL-tec, 1 million cycles, 50-500 N, 2 Hz, 37 °C). Finally, a dye penetration test was used to detect debonding. For inspection, the specimens were sliced, and penetration depth was measured with a digital microscope. Data were analyzed with the Mann-Whitney U test. No cases of total debonding were observed after cyclic loading. However, the LC specimens showed a significantly lower amount of leakage than the CC ones (p CAD/CAM technology in highly standardized test abutments for in vitro testing. For CAD/CAM composites, light curing should be performed. The success of a restoration depends on the long-term sealing ability of the luting materials, which avoids debonding along with microleakage. For CAD/CAM composites, separate light curing of the adhesive and luting composite is highly recommended.

Case Report: Ocular Myasthenia Gravis Associated with In Vitro Fertilization Procedures.

Science.gov (United States)

Yoo, Yung Ju; Han, Sang Beom; Yang, Hee Kyung; Hwang, Jeong-Min

2018-05-01

Ocular myasthenia gravis is a localized form of myasthenia gravis, which is a postsynaptic disorder of the neuromuscular junction that causes fluctuating weakness of extraocular muscles resulting from autoimmune mechanisms. In women with myasthenia, changes in sex hormone levels and administration of corticosteroids can trigger or worsen symptoms of myasthenia gravis. To describe a case of seronegative ocular myasthenia gravis whose first symptom appeared a day after in vitro fertilization procedure. A 37-year-old woman suddenly developed mild ptosis and fluctuating diplopia that worsened in the evening. Before the development of symptoms, she had undergone in vitro fertilization procedure and had taken oral steroids. Ocular motility examination revealed an intermittent exotropia in primary gaze at both distance and near. The neostigmine test confirmed her diagnosis as ocular myasthenia gravis. When taking a history for young women with sudden onset of binocular diplopia, steroids and sex hormones should be taken into account, which may trigger or exacerbate symptoms of ocular myasthenia gravis.

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

Directory of Open Access Journals (Sweden)

Carsten Geiss

2017-10-01

Full Text Available Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

In vitro human digestion test to monitor the dissolution of silver nanoparticles

International Nuclear Information System (INIS)

Bove, P; Sabella, S; Malvindi, M A

2017-01-01

Nanotechnology is a scientific revolution that the food industry has experienced over the last years. Widely employed as food additives and/or food contact materials in consumer products, silver nanoparticles are an example of this innovation. However, their increasing use makes also likely the human ingestion, thus requiring a proper risk analysis. In this framework, a comprehensive characterization of biotransformation of silver nanoparticles in biological fluids is fundamental for the regulatory needs. Herein, we aimed at studying the dissolution behaviour of silver nanoparticles using an in vitro test, which simulates the human oral ingestion of NPs during their passage through the gastrointestinal tract. The nanoparticle suspensions were characterized in the different digestion phases using several techniques to follow the changes of key physical properties ( e.g. , size, surface charge and plasmon peak) and to quantify the biotransformed products arisen by the process, as for example free silver ions. (paper)

In vitro testing of defense reactions in zygotic and somatic embryos of Abies numidica

Directory of Open Access Journals (Sweden)

Jiří Hřib

2011-01-01

Full Text Available Defense of desiccated cotyledonary somatic embryos and mature zygotic embryos of Abies numidica was tested in vitro by dual cultures with tester, fungus Phaeolus schweinitzii. Both types of embryos expressed defense reactions manifested by inhibited growth of fungal tester towards the embryos. Mycelial growth was described by logistic sigmoid growth model with a single asymptote. Mutual comparisons of mycelial growth in presence of zygotic and somatic embryos showed significant differences in parameters of mycelium growth curves towards the embryos. Larger defense reactions were observed in zygotic embryos relative to somatic embryos and unlimited control cultivations without embryo. The possible role of auxin in the defense response of plant embryos is discussed.

In vitro toxicity test of nano-sized magnesium oxide synthesized via solid-phase transformation

Science.gov (United States)

Zheng, Jun; Zhou, Wei

2018-04-01

Nano-sized magnesium oxide (MgO) has been a promising potential material for biomedical pharmaceuticals. In the present investigation, MgO nanoparticles synthesized through in-situ solid-phase transformation based on the previous work (nano-Mg(OH)2 prepared by precipitation technique) using magnesium nitrate and sodium hydroxide. The phase structure and morphology of the MgO nanoparticles are characterized by X-ray powder diffraction (XRD), selected area electronic diffraction (SAED) and transmission electron microscopy (TEM) respectively. In vitro hemolysis tests are adopted to evaluate the toxicity of the synthesized nano-MgO. The results evident that nano-MgO with lower concentration is slightly hemolytic, and with concentration increasing nano-MgO exhibit dose-responsive hemolysis.

Prenatal developmental toxicity testing of petroleum substances: Application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo.

Science.gov (United States)

Kamelia, Lenny; Louisse, Jochem; de Haan, Laura; Rietjens, Ivonne M C M; Boogaard, Peter J

2017-10-01

Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3-7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R 2 =0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

2-Aminoanthracene, 5-fluorouracil, colchicine, benzo[a]pyrene, cadmium chloride and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster ovary (CHO) cells at Covance Laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

Science.gov (United States)

Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, Debbie; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

2010-10-29

The reference genotoxic agents 2-aminoanthracene (a metabolism dependent weak clastogen), 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation), cadmium chloride (an inorganic carcinogen), and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster ovary (CHO) cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked positive increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

Molecular analysis of radiation-induced mutations in vitro

International Nuclear Information System (INIS)

Kronenberg, A.

1996-01-01

This review will focus on the nature of specific locus mutations detected in mammalian cells exposed in vitro to different types of ionizing radiations. Ionizing radiation has been shown to produce a wide variety of heritable alterations in DNA. These range from single base pair substitutions to stable loss or translocation of large portions of whole chromosomes. Data will be reviewed for certain test systems that reveal different mutation spectra. Techniques for the analysis of molecular alterations include applications of the polymerase chain reaction, some of which may be coupled with DNA sequence analysis, and a variety of hybridization-based techniques. The complexity of large scale rearrangements is approached with cytogenetic techniques including high resolution banding and various applications of the fluorescence in situ hybridization (FISH) technique. Radiation-induced mutant frequencies and mutation spectra are a function of the linkage constraints on the recovery of viable mutants for a given locus and test system. 44 refs

Duration of senescent cell survival in vitro as a characteristic of organism longevity, an additional to the proliferative potential of fibroblasts.

Science.gov (United States)

Yegorov, Yegor E; Zelenin, Alexander V

2003-04-24

More than 40 years have passed since the original publication by Hayflick and Moorhead led to the concept of the 'Hayflick limit' of the maximum number of divisions which somatic cells undergo in vitro. This concept is still regarded as a fundamental characteristic of species longevity. Here we want to emphasize another characteristic of somatic cells, namely, the duration of their survival in vitro in the non-dividing state after cessation of proliferation. This is suggested on the basis of results of recent experiments with so-called Japanese accelerated senescent mice. Results of these experiments reveal a good correlation between the longevity of the mice, the number of duplications of their fibroblasts in vitro, and the survival time of these cells in the non-dividing state. In routine culture conditions, cell survival time may be very long, as much as a few years. However, when the cells are grown under conditions of oxidative stress, cellular longevity is markedly shortened. This new test may serve as an additional marker of organismic longevity. The comparative value of both tests, the classical 'Hayflick limit' and the new test, is discussed.

Oxygen restriction as challenge test reveals early high-fat-diet-induced changes in glucose and lipid metabolism

NARCIS (Netherlands)

Duivenvoorde, L.P.M.; Schothorst, van E.M.; Derous, D.; Stelt, van der I.; Masania, J.; Rabbani, N.; Thornalley, P.J.; Keijer, J.

2015-01-01

Challenge tests stress homeostasis and may reveal deviations in health that remain masked under unchallenged conditions. Ideally, challenge tests are non-invasive and applicable in an early phase of an animal experiment. Oxygen restriction (OxR; based on ambient, mild normobaric hypoxia) is a

A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration : results of a multicentre analysis

NARCIS (Netherlands)

Hulsart-Billström, G.; Dawson, J.I.; Hofmann, S.; Müller, R.; Stoddart, M.J.; Alini, M.; Redl, H.; El Haj, A.; Brown, R.; Salih, V.; Hilborn, J.; Larsson, S.; Oreffo, R.O.C.

2016-01-01

New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility

In vitro bioactivity and cytotoxicity of chemically treated glass fibers

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Ângela Leão Andrade

2004-12-01

Full Text Available Samples of a commercial glass fiber FM® (Fiber Max were used to test the efficacy of a chemical sol-gel surface treatment to enhance their bioactivity. After treatment with tetraethoxysilane (TEOS, individual fiber samples were soaked into a simulated body fluid (SBF solution, from which they were removed at intervals of 5 and 10 days. Micrographs obtained by scanning electron microscopy (SEM analysis of samples chemically treated with TEOS revealed the formation of a hydroxyapatite (HA coating layer after 5 days into SBF solution. Fourier transform infrared spectroscopic (FTIR analyses confirmed that the coating layer has P-O vibration bands characteristic of HA. The in vitro cytotoxicity was evaluated using a direct contact test, minimum essential medium elution test (ISO 10993-5 and MTT assay. Fibers immersed in SBF and their extracts exhibited lower cytotoxicity than the controls not subjected to immersion, suggesting that SBF treatment improves the biocompatibility of the fiber.

In vitro testing to diagnose venom allergy and monitor immunotherapy: a placebo-controlled, crossover trial.

Science.gov (United States)

Brown, S G A; Haas, M A; Black, J A; Parameswaran, A; Woods, G M; Heddle, R J

2004-05-01

In people with a history of sting allergy, only prior reaction severity and older age are known to predict subsequent reaction risk. Furthermore, no diagnostic test other than a deliberate sting challenge has been found to identify people in whom venom immunotherapy (VIT) has been unsuccessful. We aimed to assess the utility of a number of in vitro tests to diagnose venom allergy and to monitor immunotherapy. During a double-blind randomized placebo-controlled crossover trial of Myrmecia pilosula ant VIT the following venom-specific tests were performed at enrolment, and at completion of treatment prior to a diagnostic sting challenge; leucocyte stimulation index (SI), IL-4 production, IgE RAST, histamine release test (HRT), leukotriene release test (LRT) and basophil activation test (BAT). Intradermal venom skin testing (VST) was also performed at trial entry. Only VST and HRT identified those at risk of sting anaphylaxis in the placebo group. Although IgE RAST, leucocyte SI and IL-4 production, LRT and BAT all correlated well with intradermal VSTs, they did not predict sting challenge outcome. After successful VIT, venom-induced leucocyte IL-4 production tended to fall, whereas IgE RAST increased and a natural decline in HRT reactivity was reversed. A confounding seasonal affect on laboratory results was suspected. The HRT warrants further assessment for diagnosis of venom allergy. Uninformative performance of the commercially available LRT and BAT tests may be due to pre-incubation with IL-3. None of the tests evaluated appear to be reliable markers of successful VIT.

In vitro activity of Origanum vulgare essential oil against Candida species

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Marlete Brum Cleff

2010-03-01

Full Text Available The aim of this study was to evaluate the in vitro activity of the essential oil extracted from Origanum vulgare against sixteen Candida species isolates. Standard strains tested comprised C. albicans (ATCC strains 44858, 4053, 18804 and 3691, C. parapsilosis (ATCC 22019, C. krusei (ATCC 34135, C. lusitaniae (ATCC 34449 and C. dubliniensis (ATCC MY646. Six Candida albicans isolates from the vaginal mucous membrane of female dogs, one isolate from the cutaneous tegument of a dog and one isolate of a capuchin monkey were tested in parallel. A broth microdilution technique (CLSI was used, and the inoculum concentration was adjusted to 5 x 10(6 CFU mL-1. The essential oil was obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography. Susceptibility was expressed as Minimal Inhibitory Concentration (MIC and Minimal Fungicidal Concentration (MFC. All isolates tested in vitro were sensitive to O. vulgare essential oil. The chromatographic analysis revealed that the main compounds present in the essential oil were 4-terpineol (47.95%, carvacrol (9.42%, thymol (8.42% and □-terpineol (7.57%. C. albicans isolates obtained from animal mucous membranes exhibited MIC and MFC values of 2.72 µL mL-1 and 5 µL mL-1, respectively. MIC and MFC values for C. albicans standard strains were 2.97 µL mL-1 and 3.54 µL mL-1, respectively. The MIC and MFC for non-albicans species were 2.10 µL mL-1 and 2.97 µL mL-1, respectively. The antifungal activity of O. vulgare essential oil against Candida spp. observed in vitro suggests its administration may represent an alternative treatment for candidiasis.

Bacterial challenge of NISSHO ultrafilter ETF 609: results of in vitro testing.

Science.gov (United States)

Krautzig, S; Lonnemann, G; Shaldon, S; Koch, K M

1996-07-01

In hemodialysis, a certain degree of bacterial contamination on the dialysate side is a regular finding. Concern has been growing that this contamination may lead to a chronic inflammatory response in the patient. Ultrafiltration of dialysate can be used to reduce bacterial content and levels of cytokine-inducing substances upstream of the patient's dialyzer. The aim of this study was to test in vitro the rejection capacity of a polysulfone hollow-fiber ultrafilter (ETF 609, NISSHO Co., Osaka, Japan) challenged with bacterial filtrates derived from Pseudomonas aeruginosa PA103. Results showed a reduction of interleukin-1 beta-inducing activity (measured on peripheral blood mononuclear cells) from 5,035 +/- 394 pg/ml prefilter to nondetectable levels postfilter and endotoxin levels (limulus amebocyte lysate assay) of 4,167 +/- 1,079 versus 12 +/- 2 pg/ml, respectively. In conclusion, ultrafiltration of dialysate with the polysulfone ultrafilter ETF 609 leads to a potent reduction of cytokine-inducing activity.

Testing in Vitro of an Apifitoterapeutic Formula Against Nosema spp.

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Vasilica Savu

2015-05-01

Full Text Available Nosema, a parasitic disease that affects adult honey bees, has a directly correlation with the losses of bee colonies, until to depopulation. The target of our study was to determine the antinosema action of an apifitoterapeutic formula that was obtained in an earlier phase of our researches. In the present study, we have had two experiences (F and N formed by clinically healthy bees. The experimental bees have received, in vitro, naturally infested honey (7 spores by Nosema spp / field. The first experience (F, I-IX groups was treated with apifitoterapeutic formula (10 ml/ honey kg, for 10 days (from T1 to T2 moment, while the second experience (N, with X-XVIII groups was infested with naturally infested honey, for 20 days (from T1 to T2 moment. The first experience (F showed 22% positive diagnosed bees, while the second experience (N showed 89% positive diagnosed bees. In the first experience, the infestation degree was very weak (group I and weak (group III, while the other groups were negative. The antiparasitic formula has showed, in laboratory conditions, a positive impact on experimental honey bees, with an efficiency over 78%. In the further, testing prophylactically and therapeutically will be conducted on bee families.

Absorbable magnesium-based stent: physiological factors to consider for in vitro degradation assessments

OpenAIRE

Wang, Juan; Smith, Christopher E.; Sankar, Jagannathan; Yun, Yeoheung; Huang, Nan

2015-01-01

Absorbable metals have been widely tested in various in vitro settings using cells to evaluate their possible suitability as an implant material. However, there exists a gap between in vivo and in vitro test results for absorbable materials. A lot of traditional in vitro assessments for permanent materials are no longer applicable to absorbable metallic implants. A key step is to identify and test the relevant microenvironment and parameters in test systems, which should be adapted according ...

Evaluation of Fatigue Behavior in Dental Implants from In Vitro Clinical Tests: A Systematic Review

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Rosa Rojo

2018-05-01

Full Text Available In the area of dentistry, there is a wide variety of designs of dental implant and materials, especially titanium, which aims to avoid failures and increase their clinical durability. The purpose of this review was to evaluate fatigue behavior in different connections and implant materials, as well as their loading conditions and response to failure. In vitro tests under normal and dynamic loading conditions evaluating fatigue at implant and abutment connection were included. A search was conducted in PubMed, Scopus, and Science Direct. Data extraction was performed independently by two reviewers. The quality of selected studies was assessed using the Cochrane Handbook proposed by the tool for clinical trials. Nineteen studies were included. Fourteen studies had an unclear risk and five had high risk of bias. Due to the heterogeneity of the data and the evaluation of the quality of the studies, meta-analysis could not be performed. Evidence from this study suggests that both internal and morse taper connections presented a better behavior to failure. However, it is necessary to unify criteria in the methodological design of in vitro studies, following methodological guidelines and establishing conditions that allow the homogenization of designs in ISO (International Organization for Standardization standards.

In vitro evaluation of matrix metalloproteinases as predictive testing for nickel, a model sensitizing agent

International Nuclear Information System (INIS)

Lamberti, Monica; Perfetto, Brunella; Costabile, Teresa; Canozo, Nunzia; Baroni, Adone; Liotti, Francesco; Sannolo, Nicola; Giuliano, Mariateresa

2004-01-01

The identification of potential damage due to chemical exposure in the workplace is a major health and regulatory concern. Traditional tests that measure both sensitization and elicitation responses require the use of animals. An alternative to this widespread use of experimental animals could have a crucial impact on risk assessment, especially for the preliminary screening of new molecules. We developed an in vitro model for the screening of potential toxic compounds. Human keratinocytes (HaCat) were used as target cells while matrix metalloproteinases (MMP) were selected as responders because they are key enzymes involved in extracellular matrix (ECM) degradation in physiological and pathological conditions. Chemical exposure was performed using nickel sulphate as a positive tester. Nickel contact induced upregulation of MMP-2 and IL-8 mRNA production. Molecular activation occurred even at very low nickel concentrations even though no phenotypic changes were observed. MMP-9 accumulation was found in the medium of treated cells with respect to controls. These observations led to the hypothesis that even minimal exposure can accumulate transcriptional activity resulting in long-term clinical signs after contact. Our simple in vitro model can be applied as a useful preliminary complement to the animal studies to screen the effects of new potential toxic compounds

Darwin's concepts in a test tube: parallels between organismal and in vitro evolution.

Science.gov (United States)

Díaz Arenas, Carolina; Lehman, Niles

2009-02-01

The evolutionary process as imagined by Darwin 150 years ago is evident not only in nature but also in the manner in which naked nucleic acids and proteins experience the "survival of the fittest" in the test tube during in vitro evolution. This review highlights some of the most apparent evolutionary patterns, such as directional selection, purifying selection, disruptive selection, and iterative evolution (recurrence), and draws parallels between what happens in the wild with whole organisms and what happens in the lab with molecules. Advances in molecular selection techniques, particularly with catalytic RNAs and DNAs, have accelerated in the last 20 years to the point where soon any sort of complex differential hereditary event that one can ascribe to natural populations will be observable in molecular populations, and exploitation of these events can even lead to practical applications in some cases.

In Vitro Sensitivity Test in Antibiotics from the Fermentation Process in a Sugar-Alcohol Plant in the State of Paraná, Brazil

OpenAIRE

Lopes, Murilo Brandão; Faculdade de Apucarana – FAP; Silva, Thaís Medeiros Boldrin; Faculdade Metropolitana de Maringá – UNIFAMMA

2011-01-01

Since the development of different types of microorganisms is common during the fermentation process in sugar-alcohol plants, due to the processing states of prime matter, microbiological control is mandatory. In vitro sensitivity test is highly important for the fermentation process at sugar-alcohol plants since the type of antibiotic with the best antibacterial activity is evaluated. The test classifies antibiotics through their effects, namely, efficient, less efficient, slightly efficient...

In vitro fatigue tests and in silico finite element analysis of dental implants with different fixture/abutment joint types using computer-aided design models.

Science.gov (United States)

Yamaguchi, Satoshi; Yamanishi, Yasufumi; Machado, Lucas S; Matsumoto, Shuji; Tovar, Nick; Coelho, Paulo G; Thompson, Van P; Imazato, Satoshi

2018-01-01

The aim of this study was to evaluate fatigue resistance of dental fixtures with two different fixture-abutment connections by in vitro fatigue testing and in silico three-dimensional finite element analysis (3D FEA) using original computer-aided design (CAD) models. Dental implant fixtures with external connection (EX) or internal connection (IN) abutments were fabricated from original CAD models using grade IV titanium and step-stress accelerated life testing was performed. Fatigue cycles and loads were assessed by Weibull analysis, and fatigue cracking was observed by micro-computed tomography and a stereomicroscope with high dynamic range software. Using the same CAD models, displacement vectors of implant components were also analyzed by 3D FEA. Angles of the fractured line occurring at fixture platforms in vitro and of displacement vectors corresponding to the fractured line in silico were compared by two-way ANOVA. Fatigue testing showed significantly greater reliability for IN than EX (pimplant fixture platforms. FEA demonstrated that crack lines of both implant systems in vitro were observed in the same direction as displacement vectors of the implant fixtures in silico. In silico displacement vectors in the implant fixture are insightful for geometric development of dental implants to reduce complex interactions leading to fatigue failure. Copyright © 2017 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Combined in vivo and in vitro approach for the characterization of penicillin-specific polyclonal lymphocyte reactivity: tolerance tests with safe penicillins instead of challenge with culprit drugs.

Science.gov (United States)

Sachs, B; Al Masaoudi, T; Merk, H F; Erdmann, S

2004-10-01

Amino-penicillins are a major cause of delayed-type reactions to penicillins. The aim of this study was to establish a diagnostic approach for the characterization of the individual penicillin-specific polyclonal lymphocyte reactivity in order to detect side chain-specific sensitization to amino-penicillins. Patients can then be advised to undergo a tolerance test with safe penicillins instead of provocation with culprit penicillins for confirmation of penicillin allergy. We investigated penicillin-specific polyclonal lymphocyte reactivity in nine patients with delayed-type reactions to amino-penicillins by a combined in vivo (patch, prick and intracutaneous tests with delayed readings) and in vitro (lymphocyte transformation test, LTT) approach. A combination of LTT and skin tests improved the sensitivity for the characterization of penicillin-specific polyclonal lymphocyte reactivity and allowed the detection of three different patterns of lymphocyte reactivity. Four patients showed a side chain-specific sensitization to amino-penicillins in vivo and in vitro and were advised to undergo tolerance tests with safe penicillins. Two patients agreed and were exposed to parenteral benzyl-penicillin and oral phenoxymethyl-penicillin which they tolerated without complications. These data suggest that a combined in vivo and in vitro approach is helpful for the detection of side chain-specific sensitization to amino-penicillins. Patients with such sensitization are very likely to tolerate safe penicillins, thereby expanding their therapeutic options when antibiotic treatment is required.

Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro.

Science.gov (United States)

Visan, Anke; Hayess, Katrin; Sittner, Dana; Pohl, Elena E; Riebeling, Christian; Slawik, Birgitta; Gulich, Konrad; Oelgeschläger, Michael; Luch, Andreas; Seiler, Andrea E M

2012-10-01

Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and γ-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test; Phenotypage de la reparation de l'ADN de lignees Xeroderma pigmentosum, par un test in vitro multiparametrique

Energy Technology Data Exchange (ETDEWEB)

Raffin, A.L.

2009-06-15

DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

Deciphering CAPTCHAs: what a Turing test reveals about human cognition.

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Thomas Hannagan

Full Text Available Turning Turing's logic on its head, we used widespread letter-based Turing Tests found on the internet (CAPTCHAs to shed light on human cognition. We examined the basis of the human ability to solve CAPTCHAs, where machines fail. We asked whether this is due to our use of slow-acting inferential processes that would not be available to machines, or whether fast-acting automatic orthographic processing in humans has superior robustness to shape variations. A masked priming lexical decision experiment revealed efficient processing of CAPTCHA words in conditions that rule out the use of slow inferential processing. This shows that the human superiority in solving CAPTCHAs builds on a high degree of invariance to location and continuous transforms, which is achieved during the very early stages of visual word recognition in skilled readers.

An in vitro drug sensitivity test using a higher 3H-TdR incorporation and a modified human tumor stem cell assay

International Nuclear Information System (INIS)

Wang Enzhong

1991-01-01

An in vitro drug sensitivity test was developed to evaluate the lethal effects of drugs on human pulmonary carcinoma cells (HPCC). This method was a variant and combination of Human Tumor Stem (HTSCA) and a short-term test using 3 H-TdR incorporation. It consisted of a cell containing liquid top layer and a soft agar bottom layer in 24-well microplates. The medium was RPMI 1640 supplemented with 20% malignant pleural effusion, which could enhance 3 H-TdR incorporation into malignant cells. When 50%, 40%, 30% and 30% of cell survival rate defined as sensitivity-threshold for VCR, MMC, DDP and ADM respectively, in the vitro effectiveness were close to those of clinical single-drug treatment in HPCC by Wright et al. This method was also compared with HTSCA in ten human lung cancer cell lines and four pulmonary carcinoma tissues. The agreement rates were 83% and 100% respectively. Thus we presume this system is more useful for oncological clinics than the others

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

Science.gov (United States)

Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

2015-06-01

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

The 21th quality control survey for radioisotope in vitro tests in Japan, 1999

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Shishiba, Yoshimasa; Shimizu, Taeko [Toranomon Hospital, Tokyo (Japan); Ichihara, Kiyoshi; Kousaka, Tadako; Kobayashi, Hisae; Tachibana, Katsuhiko; Tsushima, Toshio; Hoshino, Minoru; Mori, Mikio

2000-10-01

The quality control survey in the title has been conducted every year from 1978 to improve the quality of the in vitro tests and this report gives the survey results done in 1999. The survey was performed in 133 facilities out of 283 in Japan, which involved 18 national and public university hospitals, 16 private university hospitals, 19 national and public hospitals, 18 private hospitals, 41 hygiene test institutes and 21 reagent manufacturers. Tests examined were on 6 substances related to functions of pituitary, 5 of thyroid function, 1 of parathyroid, 4 of digestive-pancreas, 5 of gonad-placenta, 4 of adrenal and 1 of renal-blood pressure regulation, on IgE, on digoxin and on 11 tumor-related substances. Tests were done on 2 - 3 samples supplied from the Committee and for reproducibility for within-day, between-day, effects of freeze-thaw and periodic variation after thawing. Methods involved those using non-radioisotopes like enzyme immunoassay as well as those using radioactive isotopes like radioimmunoassay. Results given by the mean, standard deviation and coefficient of variation for each kit, were analyzed for within-kit (between-facility) variation and between-kit (between-reagent) variation by one way analysis of variance. Findings were similar to those previously reported without the particular new problem; however, the committee considered that the survey should be done with human serum as a matrix especially for samples with a high matrix effect in future. (K.H.)

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

International Nuclear Information System (INIS)

Park, Margriet V.D.Z.; Annema, Wijtske; Salvati, Anna; Lesniak, Anna; Elsaesser, Andreas; Barnes, Clifford; McKerr, George; Howard, C. Vyvyan; Lynch, Iseult; Dawson, Kenneth A.; Piersma, Aldert H.; Jong, Wim H. de

2009-01-01

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 μg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa.

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Robert A Harrison

2017-10-01

Full Text Available Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa.We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a 'gold standard' comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom.None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro

In vitro drug sensitivity testing of tumor cells from patients with non-Hodgkin's lymphoma using the fluorometric microculture cytotoxicity assay.

Science.gov (United States)

Nygren, P; Hagberg, H; Glimelius, B; Sundström, C; Kristensen, J; Christiansen, I; Larsson, R

1994-01-01

Tumor cell drug sensitivity is an important determinant of chemotherapy response. Its measurement in vitro would aid in therapy individualization and new drug development. The fluorometric microculture cytotoxicity assay (FMCA), based on production by viable cells of fluorescent fluorescein after 3 days of culture, was used for cytotoxic drug sensitivity testing of 73 samples of tumor cells from patients with non-Hodgkin's lymphoma (NHL). The technical success rate was 92%, and FMCA data showed good correlation to the Disc assay. NHL samples were considerably more drug sensitive than were samples from in vivo resistant tumors. There was no obvious difference in drug sensitivity for high- vs. low-grade or untreated vs. previously treated low-grade NHL. For 26 patients, clinical outcome was correlated to in vitro response giving a sensitivity and specificity of 93 and 48%, respectively. Cross-resistance between standard drugs was frequent in vitro. Resistance modulators potentiated the effect of vincristine and doxorubicin in 10-29% of the samples, most frequently from previously treated patients. The FMCA seems to report clinically relevant drug sensitivity data for NHL, and thus it could serve as a tool for optimization of chemotherapy in the future.

Filling the concept with data: integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing.

Science.gov (United States)

Natsch, Andreas; Emter, Roger; Ellis, Graham

2009-01-01

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

Science.gov (United States)

Sakaguchi, Hitoshi; Ashikaga, Takao; Miyazawa, Masaaki; Kosaka, Nanae; Ito, Yuichi; Yoneyama, Katsurako; Sono, Sakiko; Itagaki, Hiroshi; Toyoda, Hidekazu; Suzuki, Hiroyuki

2009-04-01

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

AN EFFECTIVE WAY TO CARRY OUT MASS IN VITRO PROPAGATION OF POTENTILLA ALBA L.

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L. I. Tikhomirova

2016-04-01

Full Text Available Despite the plant’s extensive area of distribution, Potentilla alba L. natural resources are scarce and cannot meet the modern needs of the pharmaceutical industry. Because of the mass preparation of medical raw materials by using P. alba, it entered into the list of rare and endangered species plants of the Red Data Book of the Republic of Belarus. This plant is not represented in the wild flora of Western Siberia, but there is a great need for developing a method for the mass propagation of P. alba using in vitro culture in order to obtain a high-quality planting material. At the explant stage, the technique of the P. alba introduction into in vitro culture is developed. This paper reveals the morphogenetic features of the development of P. alba explants of different types and the regenerative capacity of the tissue culture. At the micropropagation stage, the optimum culture media and the growth conditions for the regenerated plants are selected. At the stage of test-tube plants rooting and transferring them into ex vitro conditions, the most effective means of adaptation to non-sterile conditions in hydroponics are proposed.

Rocuronium is more hepatotoxic than succinylcholine in vitro.

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Sauer, Martin; Piel, Ines; Haubner, Cristof; Richter, Georg; Mann, Miriam; Nöldge-Schomburg, Gabriele; Mencke, Thomas

2017-09-01

The development of liver failure is a major problem in critically ill patients. The hepatotoxicity of many drugs, as one important reason for liver failure, is poorly screened for in human models. Rocuronium and succinylcholine are neuromuscular blocking agents used for tracheal intubation and for rapid-sequence induction. We used an in-vitro test with a permanent cell line and compared rocuronium and succinylcholine for hepatotoxicity. In-vitro study. A basic science laboratory, University Hospital Rostock, Germany. The basic test compound is the permanent human liver cell line HepG2/C3A. In a standardised microtitre plate assay the toxicity of different concentrations of rocuronium, succinylcholine and plasma control was tested. After two incubation periods of 3 days, the viability of cells (XTT test, lactate dehydrogenase release and trypan blue staining), micro-albumin synthesis and the cytochrome 1A2 activity (metabolism of ethoxyresorufin) were measured. Differences between rocuronium and succinylcholine were assessed using the Kruskal-Wallis one-way test and two-tailed Mann-Whitney U test. Rocuronium, but not succinylcholine, led to a significant dose-dependent decrease of viability, albumin synthesis and cytochrome 1A2 activity of test cells. An in-vitro test with a cell line showed hepatotoxicity of rocuronium that was dose-dependent. Further studies are needed to investigate the underlying mechanisms of the effects of rocuronium on hepatic cellular integrity. Not suitable.

The utility of in vitro solubility testing in assessment of uranium exposure

International Nuclear Information System (INIS)

Eidson, A.F.; Damon, E.G.; Hahn, F.F.; Griffith, W.C. Jr.

1989-01-01

Assessment of accidental exposures in the uranium industry requires estimation of retention and excretion rates using bioassay measurements. This task is difficult if the solubility of the inhaled uranium compound is unknown. In our research, Beagle dogs were exposed to aerosols of commercial uranium milling products containing either pure ammonium diuranate (ADU) or U 3 0 8 . Dogs were exposed to ADU aerosols to achieve a median retained body burden of 0.058 mg U per kg body weight, or to U 3 0 8 aerosols to achieve a median retained body burden of 0.28 mg U per kg. A biokinetic model was applied to simulate retention and excretion of the two forms of uranium in vivo. Comparison of published in vitro dissolution data and modelling results with information from accidental human exposures showed that in vitro dissolution studies are necessary to characterise the differential solubilities of uranium compounds, and indicate the potential for kidney toxicity. Because variability in pulmonary clearance and urinary excretion rates is high among individual people, in vitro dissolution results are only marginally useful for estimating urinary excretion rates. (author)

Genotoxic and Antigenotoxic Assessment of Chios Mastic Oil by the In Vitro Micronucleus Test on Human Lymphocytes and the In Vivo Wing Somatic Test on Drosophila.

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Dimitris Vlastos

Full Text Available Chios mastic oil (CMO, the essential oil derived from Pistacia lentiscus (L. var. chia (Duham, has generated considerable interest because of its antimicrobial, anticancer, antioxidant and other beneficial properties. In the present study, the potential genotoxic activity of CMO as well as its antigenotoxic properties against the mutagenic agent mitomycin-C (MMC were evaluated by employing the in vitro Cytokinesis Block MicroNucleus (CBMN assay and the in vivo Somatic Mutation And Recombination Test (SMART. In the in vitro experiments, lymphocytes were treated with 0.01, 0.05 and 0.10% (v/v of CMO with or without 0.05 μg/ml MMC, while in the in vivo assay Drosophila larvae were fed with 0.05, 0.10, 0.50 and 1.00% (v/v of CMO with or without 2.50 μg/ml MMC. CMO did not significantly increase the frequency of micronuclei (MN or total wing spots, indicating lack of mutagenic or recombinogenic activity. However, the in vitro analysis suggested cytotoxic activity of CMO. The simultaneous administration of MMC with CMO did not alter considerably the frequencies of MMC-induced MN and wing spots showing that CMO doesn't exert antigenotoxic or antirecombinogenic action. Therefore, CMO could be considered as a safe product in terms of genotoxic potential. Even though it could not afford any protection against DNA damage, at least under our experimental conditions, its cytotoxic potential could be of interest.

In vivo testing of chemicals that undergo substantial biotransformation: An opportunity to advance in vitro-in vivo metabolism extrapolation procedures for fish

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This is one of several keynote presentations given at the beginning of a one-day workshop on in vivo testing with fish. The purpose of this talk is to describe recent progress on using in vitro data to predict metabolism impacts on chemical accumulation by fish. The focus of th...

Absorbable magnesium-based stent: physiological factors to consider for in vitro degradation assessments.

Science.gov (United States)

Wang, Juan; Smith, Christopher E; Sankar, Jagannathan; Yun, Yeoheung; Huang, Nan

2015-03-01

Absorbable metals have been widely tested in various in vitro settings using cells to evaluate their possible suitability as an implant material. However, there exists a gap between in vivo and in vitro test results for absorbable materials. A lot of traditional in vitro assessments for permanent materials are no longer applicable to absorbable metallic implants. A key step is to identify and test the relevant microenvironment and parameters in test systems, which should be adapted according to the specific application. New test methods are necessary to reduce the difference between in vivo and in vitro test results and provide more accurate information to better understand absorbable metallic implants. In this investigative review, we strive to summarize the latest test methods for characterizing absorbable magnesium-based stent for bioabsorption/biodegradation behavior in the mimicking vascular environments. Also, this article comprehensively discusses the direction of test standardization for absorbable stents to paint a more accurate picture of the in vivo condition around implants to determine the most important parameters and their dynamic interactions.

Biofilms of vaginal Lactobacillus in vitro test.

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Wei, Xiao-Yu; Zhang, Rui; Xiao, Bing-Bing; Liao, Qin-Ping

2017-01-01

This paper focuses on biofilms of Lactobacillus spp. - a type of normal flora isolated from healthy human vaginas of women of childbearing age; thereupon, it broadens the research scope of investigation of vaginal normal flora. The static slide culture method was adopted to foster biofilms, marked by specific fluorescence staining. Laser scanning confocal and scanning electron microscopy were used to observe the microstructure of the biofilms. Photographs taken from the microstructure were analysed to calculate the density of the biofilms. The body of Lactobacillus spp., though red, turned yellow when interacting with the green extracellular polysaccharides. The structure of the biofilm and aquaporin within the biofilm were imaged. Lactobacillus density increases over time. This study provides convincing evidence that Lactobacillus can form biofilms and grow over time in vitro. This finding establishes an important and necessary condition for selecting proper strains for the pharmaceutics of vaginal ecology.

Studies of rhodamine-123: effect on rat prostate cancer and human prostate cancer cells in vitro.

Science.gov (United States)

Arcadi, J A; Narayan, K S; Techy, G; Ng, C P; Saroufeem, R M; Jones, L W

1995-06-01

The effect of the lipophilic, cationic dye, Rhodamine-123 (Rh-123), on prostate cancer in rats, and on three tumor cell lines in vitro is reported here. The general toxicity of Rh-123 in mice has been found to be minimal. Lobund-Wistar (L-W) rats with the autochthonous prostate cancer of Pollard were treated for six doses with Rh-123 at a dose of 15 mg/kg subcutaneously every other day. Microscopic examination of the tumors revealed cellular and acinar destruction. The effectiveness of Rh-123 as a cytotoxic agent was tested by clonogenic and viability assays in vitro with three human prostate cancer cell lines. Severe (60-95%) growth inhibition was observed following Rh-123 exposure for 2-5 days at doses as low as 1.6 micrograms/ml in all three prostate cancer cell lines.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4: proposing a new lipid formulation performance classification system.

Science.gov (United States)

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Calderone, Marilyn; Igonin, Annabel; Jule, Eduardo; Vertommen, Jan; Blundell, Ross; Benameur, Hassan; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2014-08-01

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF performance, including the degree of supersaturation generated on dispersion/digestion. Stresses evaluated included decreasing LBF concentration (↓LBF), increasing bile salt, and decreasing pH. Their capacity to stress LBFs was dependent on LBF composition and drug type: ↓LBF was a stressor to medium-chain glyceride-rich LBFs, but not more hydrophilic surfactant-rich LBFs, whereas decreasing pH stressed tolfenamic acid LBFs, but not fenofibrate LBFs. Lastly, a new Performance Classification System, that is, LBF composition independent, is proposed to promote standardized LBF comparisons, encourage robust LBF development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance grading. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Retention of mandibular advancement devices in the treatment of obstructive sleep apnea: an in vitro pilot study.

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Vanderveken, Olivier M; Van de Heyning, Paul; Braem, Marc J

2014-05-01

In order for a mandibular advancement device (MAD) to be efficacious, it must remain seated on the teeth during sleep. Quantitative data on the retentive characteristics of MADs are currently unavailable. The present pilot study is the first to describe an in vitro setup testing the retentive characteristics of different monobloc MADs. A hydraulic cyclic test machine was used with MADs seated on dental casts to measure retention forces upon removal of the MADs. A custom-made monobloc (CM-mono), a thermoplastic monobloc (TP-mono), and a thermoplastic duobloc (TP-duo) configured as a monobloc were tested. Two protrusions were investigated, representing 25 and 65% of the maximal protrusion. The effects of the type of MAD, duration of the test, and amount of protrusion on removal forces were measured. The measured removal forces of all three MADs tested differed significantly, with the TP-duo showing the highest values (P tested. However, only the TP-duo showed a significant reduction in time in removal forces for both protrusion positions (P tested (P = 0.0074). This in vitro pilot study reveals significant differences in retention forces for the MADs tested. The findings are consistent with clinical effects of nightly loss of MADs as reported in the literature and are within the range of reported physiological mouth-opening forces. Future research is needed to determine the key design features of MADs that explain these differences.

[Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

Science.gov (United States)

Sklifas, A N; Zhalimov, V K; Temnov, A A; Kukushkin, N I

2012-01-01

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays Efeito da trealose na seleção de leveduras para uso como probióticos utilizando testes in vitro e in vivo

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Flaviano S. Martins

2008-03-01

Full Text Available Probiotics are viable defined microorganisms (bacteria or yeasts that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.Probióticos são definidos como microrganismos (bactérias e leveduras que exercem um efeito benéfico na saúde do hospedeiro quando ingeridos em quantidades adequadas. A seleção desses agentes bioterapêuticos normalmente é feita por testes in vitro simulando o ambiente gastrointestinal que determina a capacidade de sobrevivência no trato digestivo. Neste trabalho, a possibilidade de extrapolação dos dados obtidos nos testes in vitro para as condições in vivo foi estudada utilizando cinco linhagens de Saccharomyces cerevisiae isoladas da floresta Atlântica brasileira. O conteúdo de trealose e a sobrevivência após a exposição a diversos estresses fisiológicos geralmente encontrados no trato gastrointestinal de humanos foram determinados para as cinco linhagens e os resultados comparados com a Saccharomyces boulardii, um probiótico conhecido. Esses resultados

A ring test of a wireless in vitro gas production system

DEFF Research Database (Denmark)

Cornou, Cecile; Storm, Ida Marie Lindhardt Drejer; Hindrichsen, Ida Katarina Auf der M.

2013-01-01

) or sheep (SP). Curves, corrected for blanks, were fitted using an exponential regression model with a lag time. The following variables were considered: (i) GP24 and GP48: raw values at 24 and 48 h (mL/g DM), corrected for blanks; (ii) A: asymptotic GP (mL/g DM); (iii) T1/2: time when half A is produced (h......); (iv) GPMR: maximum predicted GP rate (mL/h); (v) L: lag time (h). A mixed model including laboratories as random effect was used. A significant interaction between substrate and laboratories was found for all variables except A. The most repeatable and reproducible results were observed for A and GP48......The in vitro gas production (GP) technique has been widely used for feed evaluation. However, variability in results limits useful comparisons. Results from a ring test undertaken in four laboratories (Italy - IT, Spain - SP, Wales - WA and Denmark - DK) using the same wireless equipment (ANKOM...

The use of indium-111 platelet scintigraphy in man: comparisons with in vitro tests and in vivo platelet function--a five year experience

International Nuclear Information System (INIS)

Ezikowitz, M.D.; Ferri, P.; Pope, C.; Smith, E.O.; Snyder, E.L.

1985-01-01

In this paper, the authors present data collected from 540 patients between July 1978 and July 1983. They discuss briefly the method they employed for labeling platelets, emphasizing in vivo and in vitro markers of platelet function used for quality control of platelet preparation. They discuss the application of their technique for identification of mural left ventrical trombi, and review the disparity between in vivo and in vitro tests of platelet function. Then they deal with diagnosis of subacute bacterial endocarditis, deep veonus thrombosis, coronary trombi, left arterial masses, and the use of tomographic imaging. Finally they report changes in platelet function in stored platelets from normal volunteers

Comparative In Vitro Biological Toxicity of Four Kinds of Air Pollution Particles.

Science.gov (United States)

Shin, Han-Jae; Cho, Hyun Gi; Park, Chang Kyun; Park, Ki Hong; Lim, Heung Bin

2017-10-01

Accumulating epidemiological evidence indicates that exposure to fine air pollution particles (APPs) is associated with a variety of adverse health effects. However, the exact physiochemical properties and biological toxicities of fine APPs are still not well characterized. We collected four types of fine particle (FP) (diesel exhaust particles [DEPs], natural organic combustion [NOC] ash, synthetic organic combustion [SOC] ash, and yellow sand dust [YSD]) and investigated their physicochemical properties and in vitro biological toxicity. DEPs were almost entirely composed of ultrafine particles (UFPs), while the NOC, SOC, and YSD particles were a mixture of UFPs and FPs. The main elements in the DEPs, NOC ash, SOC ash, and YSD were black carbon, silicon, black carbon, and silicon, respectively. DEPs exhibited dose-dependent mutagenicity even at a low dose in Salmonella typhimurium TA 98 and 100 strains in an Ames test for genotoxicity. However, NOC, SOC, and YSD particles did not show any mutagenicity at high doses. The neutral red uptake assay to test cell viability revealed that DEPs showed dose-dependent potent cytotoxicity even at a low concentration. The toxicity of DEPs was relatively higher than that of NOC, SOC, and YSD particles. Therefore, these results indicate that among the four FPs, DEPs showed the highest in vitro biological toxicity. Additional comprehensive research studies such as chemical analysis and in vivo acute and chronic inhalation toxicity tests are necessary to determine and clarify the effects of this air contaminant on human health.

Validation of a numerical FSI simulation of an aortic BMHV by in vitro PIV experiments.

Science.gov (United States)

Annerel, S; Claessens, T; Degroote, J; Segers, P; Vierendeels, J

2014-08-01

In this paper, a validation of a recently developed fluid-structure interaction (FSI) coupling algorithm to simulate numerically the dynamics of an aortic bileaflet mechanical heart valve (BMHV) is performed. This validation is done by comparing the numerical simulation results with in vitro experiments. For the in vitro experiments, the leaflet kinematics and flow fields are obtained via the particle image velocimetry (PIV) technique. Subsequently, the same case is numerically simulated by the coupling algorithm and the resulting leaflet kinematics and flow fields are obtained. Finally, the results are compared, revealing great similarity in leaflet motion and flow fields between the numerical simulation and the experimental test. Therefore, it is concluded that the developed algorithm is able to capture very accurately all the major leaflet kinematics and dynamics and can be used to study and optimize the design of BMHVs. Copyright © 2014 IPEM. Published by Elsevier Ltd. All rights reserved.

Ficus septica plant extracts for treating Dengue virus in vitro

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Nan-Chieh Huang

2017-06-01

Full Text Available Dengue virus types 1-4 (DENV-1-4 are positive-strand RNA viruses with an envelope that belongs to the Flaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. In order to discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an enveloped virus such as DENV.

Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

International Nuclear Information System (INIS)

Raffin, A.L.

2009-06-01

DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

Integration of QSAR and in vitro toxicology.

Science.gov (United States)

Barratt, M D

1998-01-01

The principles of quantitative structure-activity relationships (QSAR) are based on the premise that the properties of a chemical are implicit in its molecular structure. Therefore, if a mechanistic hypothesis can be proposed linking a group of related chemicals with a particular toxic end point, the hypothesis can be used to define relevant parameters to establish a QSAR. Ways in which QSAR and in vitro toxicology can complement each other in development of alternatives to live animal experiments are described and illustrated by examples from acute toxicological end points. Integration of QSAR and in vitro methods is examined in the context of assessing mechanistic competence and improving the design of in vitro assays and the development of prediction models. The nature of biological variability is explored together with its implications for the selection of sets of chemicals for test development, optimization, and validation. Methods are described to support the use of data from in vivo tests that do not meet today's stringent requirements of acceptability. Integration of QSAR and in vitro methods into strategic approaches for the replacement, reduction, and refinement of the use of animals is described with examples. PMID:9599692

Induction of the acrosome reaction test to in vitro estimate embryo production in Nelore cattle

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M.Z. Costa

2010-08-01

Full Text Available The effectiveness of induction of the acrosome reaction (AR test as a parameter to in vitro estimate embryo production (IVP in Nelore breed and the AR pattern by the Trypan Blue/Giemsa (TB stain were evaluated. Frozen semen samples from ten Nelore bulls were submitted to AR induction and were also evaluated for cleavage and blastocyst rates. The treatments utilized for AR induction were: control (TALP medium, TH (TALP medium + 10μg heparin, TL (TALP medium + 100μg lysophosphatidylcholine and THL (TALP medium + 10μg heparin + 100μg lysophosphatidylcholine. Sperm acrosomal status and viability were evaluated by TB staining at 0 and after 4h incubation at 38°C. The results obtained for AR presented a significant difference (P<0.05 in the percentage of acrosome reacted live sperm after 4h of incubation in the treatments that received heparin. The cleavage and blastocyst rates were 60% and 38% respectively and a significant difference was observed among bulls (P<0.05. It was founded a satisfactory model to estimate the cleavage and blastocyst rates by AR induction test. Therefore, it can be concluded that the induction of the AR test is a valuable tool to predict the IVP in Nelore breed.

Characterization of new eye drops with choline salicylate and assessment of their irritancy by in vitro short time exposure tests.

Science.gov (United States)

Wroblewska, Katarzyna; Kucinska, Małgorzata; Murias, Marek; Lulek, Janina

2015-09-01

The aim of our study was to examine the irritation potential of new eye drops containing 2% choline salicylate (CS) as an active pharmaceutical ingredient (API) and various polymers increasing eye drop viscosity (hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone). The standard method for assessing the potential of irritating substances has been the Draize rabbit eye test. However the European Centre for Validation of Alternative Methods and the Coordinating Committee for Validation of Alternative Methods recommend, short time exposure (STE) in vitro tests as an alternative method for assessing eye irritation. The eye irritation potential was determined using cytotoxicity test methods for rabbit corneal cell line (SIRC) after 5 min exposure. The viability of cells was determined using two cytotoxicity assays: MTT and Neutral Red Uptake. According to the irritation rankings for the short time exposure test, all tested eye drops are classified as non-irritating (cell viability >70%).

In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and gelatin: A comparative study

International Nuclear Information System (INIS)

Gomes, S.R.; Rodrigues, G.; Martins, G.G.; Roberto, M.A.; Mafra, M.; Henriques, C.M.R.

2015-01-01

Many polymers have been investigated with respect to their use in skin tissue engineering. However, directly comparable data on the role played by different polymers in assisting skin wound healing requires their in vitro and in vivo evaluation under the same conditions. Therefore, we performed a study in order to compare the performance of electrospun nanofiber mats from three different polymers concerning cell–scaffold interaction and wound healing promotion. A polyester (polycaprolactone, PCL), a protein (gelatin from cold water fish skin, GEL) and a polysaccharide (chitosan, CS) were the polymers chosen. Gelatin nanofibers were crosslinked with glutaraldehyde vapor. The scaffolds were characterized physico-chemically, in vitro by seeding with human fetal fibroblasts, HFFF2, and used in vivo as skin substitutes in a rat wound model with total skin removal. In vitro tests revealed that cells adhered and proliferated in all scaffolds. However, cells deep into the scaffold were only observed in the PCL and CS scaffolds. In in vivo tests CS scaffolds had the highest impact on the healing process by decreasing the extent of wound contraction and enhancing the production of a neodermis and re-epithelialization of the wound. - Highlights: • We produced and compared the properties of electrospun PCL, CS and fish GEL. • In vitro, cells adhered and proliferated better on GEL scaffolds. • Deep cell migration was observed in the PCL and CS matrices. • In vivo, both CS and GEL matrices integrated well within the wounds. • Only CS effectively blocked, although only partially, the contraction phenomenon

In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and gelatin: A comparative study

Energy Technology Data Exchange (ETDEWEB)

Gomes, S.R., E-mail: srrg@campus.fct.unl.pt [Centro de Física e Investigação Tecnológica/Departamento de Física Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Rodrigues, G. [Centro de Biologia Ambiental/Departamento de Biologia Animal Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa (Portugal); Martins, G.G. [Centro de Biologia Ambiental/Departamento de Biologia Animal Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa (Portugal); Instituto Gulbenkian de Ciência, R. da Quinta Grande, 6, 2780-156 Oeiras (Portugal); Roberto, M.A. [Departamento de Cirurgia Plástica e Reconstrutiva e Unidade de Queimados, Hospital de São José, Rua José António Serrano, 1150-199 Lisboa (Portugal); Mafra, M. [Serviço de Anatomia Patológica, Hospital de São José, Rua José António Serrano, 1150-199 Lisboa (Portugal); Henriques, C.M.R. [Centro de Física e Investigação Tecnológica/Departamento de Física Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); and others

2015-01-01

Many polymers have been investigated with respect to their use in skin tissue engineering. However, directly comparable data on the role played by different polymers in assisting skin wound healing requires their in vitro and in vivo evaluation under the same conditions. Therefore, we performed a study in order to compare the performance of electrospun nanofiber mats from three different polymers concerning cell–scaffold interaction and wound healing promotion. A polyester (polycaprolactone, PCL), a protein (gelatin from cold water fish skin, GEL) and a polysaccharide (chitosan, CS) were the polymers chosen. Gelatin nanofibers were crosslinked with glutaraldehyde vapor. The scaffolds were characterized physico-chemically, in vitro by seeding with human fetal fibroblasts, HFFF2, and used in vivo as skin substitutes in a rat wound model with total skin removal. In vitro tests revealed that cells adhered and proliferated in all scaffolds. However, cells deep into the scaffold were only observed in the PCL and CS scaffolds. In in vivo tests CS scaffolds had the highest impact on the healing process by decreasing the extent of wound contraction and enhancing the production of a neodermis and re-epithelialization of the wound. - Highlights: • We produced and compared the properties of electrospun PCL, CS and fish GEL. • In vitro, cells adhered and proliferated better on GEL scaffolds. • Deep cell migration was observed in the PCL and CS matrices. • In vivo, both CS and GEL matrices integrated well within the wounds. • Only CS effectively blocked, although only partially, the contraction phenomenon.

Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription1

Science.gov (United States)

Ido, Ayaka; Iwata, Shinya; Iwata, Yuka; Igarashi, Hisako; Hamada, Takahiro; Sonobe, Seiji; Sugiura, Masahiro; Yukawa, Yasushi

2016-01-01

In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin). PMID:26662274

In vitro activity of tigecycline and comparators against carbapenem-resistant Enterobacteriaceae in Africa-Middle East countries: TEST 2007-2012.

Science.gov (United States)

Renteria, M I; Biedenbach, D J; Bouchillon, S K; Hoban, D J; Raghubir, N; Sajben, P

2014-09-01

Multidrug-resistant (MDR) Enterobacteriaceae are an emerging concern for healthcare providers. Infections caused by MDR pathogens are associated with increased costs, length of hospital stay, and morbidity and mortality rates. Carbapenem-resistant Enterobacteriaceae (CRE) continue to increase, and infections with these organisms are observed worldwide not only as hospital-acquired infections but also as community-acquired infections. Increasing antimicrobial resistance dictates the need for continued surveillance studies of common and MDR pathogens. The Tigecycline Evaluation Surveillance Trial (TEST) examined the susceptibility of pathogens isolated in Africa and the Middle East from 2007 to 2012. A total of 4155 Enterobacteriaceae isolates were evaluated to determine the in vitro activity and changes in resistance patterns for tigecycline and comparators. Carbapenem resistance was found in 191 (4.6%) of the isolates tested. Klebsiella pneumoniae was the most common CRE (64.9%), followed by Enterobacter cloacae (14.1%) and Escherichia coli (9.9%). Tigecycline MIC 90 values (minimum inhibitory concentration required to inhibit 90% of the isolates) were 2μg/mL against all of these enteric species, with susceptibility rates of 96.8%, 92.6% and 100%, respectively. Tigecycline had in vitro activity against CRE, with a 95.3% susceptibility rate. Copyright © 2014 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

In Vitro Testing of Scaffolds for Mesenchymal Stem Cell-Based Meniscus Tissue Engineering—Introducing a New Biocompatibility Scoring System

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Felix P. Achatz

2016-04-01

Full Text Available A combination of mesenchymal stem cells (MSCs and scaffolds seems to be a promising approach for meniscus repair. To facilitate the search for an appropriate scaffold material a reliable and objective in vitro testing system is essential. This paper introduces a new scoring for this purpose and analyzes a hyaluronic acid (HA gelatin composite scaffold and a polyurethane scaffold in combination with MSCs for tissue engineering of meniscus. The pore quality and interconnectivity of pores of a HA gelatin composite scaffold and a polyurethane scaffold were analyzed by surface photography and Berliner-Blau-BSA-solution vacuum filling. Further the two scaffold materials were vacuum-filled with human MSCs and analyzed by histology and immunohistochemistry after 21 days in chondrogenic media to determine cell distribution and cell survival as well as proteoglycan production, collagen type I and II content. The polyurethane scaffold showed better results than the hyaluronic acid gelatin composite scaffold, with signs of central necrosis in the HA gelatin composite scaffolds. The polyurethane scaffold showed good porosity, excellent pore interconnectivity, good cell distribution and cell survival, as well as an extensive content of proteoglycans and collagen type II. The polyurethane scaffold seems to be a promising biomaterial for a mesenchymal stem cell-based tissue engineering approach for meniscal repair. The new score could be applied as a new standard for in vitro scaffold testing.

Molecular ecotoxicology of nanosilver guided using in vitro prognosis

DEFF Research Database (Denmark)

Hayashi, Yuya; Heckmann, Lars-Henrik; Engelmann, Péter

2012-01-01

To study the molecular and cellular basis of silver nanoparticle (AgNP) toxicity, we here used a recently established in vitro model of earthworm coelomocytes in comparison to the conventional in vivo molecular ecotoxicology approach. Compared to the latter where the test organisms are exposed...... to NPs of interest held in an environmental matrix, in vitro models benefit from the ease of controlling exposure conditions in a defined set of biochemical milieus that NPs may encounter. The AgNPs tested in the present study originated from the same source, but to enhance the colloidal stability...... in the in vitro test media the NPs were pre-treated with serum proteins. In addition to physical characterisation of AgNPs, the active silver ion fraction was measured (in serum-supplemented cell culture medium and in soil pore-water). Using flow cytometry and atomic absorption spectrophotometry, we show...

In vitro comparison of Günther Tulip and Celect filters: testing filtering efficiency and pressure drop.

Science.gov (United States)

Nicolas, M; Malvé, M; Peña, E; Martínez, M A; Leask, R

2015-02-05

In this study, the trapping ability of the Günther Tulip and Celect inferior vena cava filters was evaluated. Thrombus capture rates of the filters were tested in vitro in horizontal position with thrombus diameters of 3 and 6mm and tube diameter of 19mm. The filters were tested in centered and tilted positions. Sets of 30 clots were injected into the model and the same process was repeated 20 times for each different condition simulated. Pressure drop experienced along the system was also measured and the percentage of clots captured was recorded. The Günther Tulip filter showed superiority in all cases, trapping almost 100% of 6mm clots both in an eccentric and tilted position and trapping 81.7% of the 3mm clots in a centered position and 69.3% in a maximum tilted position. The efficiency of all filters tested decreased as the size of the embolus decreased and as the filter was tilted. The injection of 6 clots raised the pressure drop to 4.1mmHg, which is a reasonable value that does not cause the obstruction of blood flow through the system. Copyright © 2014 Elsevier Ltd. All rights reserved.

Testing pollen of single and stacked insect-resistant Bt-maize on in vitro reared honey bee larvae.

Science.gov (United States)

Hendriksma, Harmen P; Härtel, Stephan; Steffan-Dewenter, Ingolf

2011-01-01

The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.

A novel physiological testing device to study knee biomechanics in vitro

NARCIS (Netherlands)

van de Bunt, Fabian; Emanuel, Kaj S.; Wijffels, Thomas; Kooren, Peter N.; Kingma, Idsart; Smit, Theodoor H.

2017-01-01

Background To properly study knee kinetics, kinematics and the effects of injury and surgical treatment in vitro, the knee should be constrained as little as possible, while imposing physiological loads. A novel dynamic biomechanical knee system (BKS) is presented here. The aim of this study was to

A novel physiological testing device to study knee biomechanics in vitro

NARCIS (Netherlands)

van de Bunt, Fabian; Emanuel, Kaj S.; Wijffels, Thomas; Kooren, Peter N.; Kingma, Idsart; Smit, Theodoor H.

2017-01-01

Background: To properly study knee kinetics, kinematics and the effects of injury and surgical treatment in vitro, the knee should be constrained as little as possible, while imposing physiological loads. A novel dynamic biomechanical knee system (BKS) is presented here. The aim of this study was to

Effect of ionizing radiation on platelet function in vitro

International Nuclear Information System (INIS)

Kalovidouris, A.E.; Papayannis, A.G.

1981-01-01

The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300x10 9 /l) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 μmol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet function tests. (Auth.)

MRI with intraoral orthodontic appliance-a comparative in vitro and in vivo study of image artefacts at 1.5 T.

Science.gov (United States)

Zachriat, C; Asbach, P; Blankenstein, K I; Peroz, I; Blankenstein, F H

2015-01-01

We investigated artefacts caused from orthodontic appliances at 1.5-T MRI of the head and neck region and whether the image quality can be improved utilizing the artefact-minimizing sequence WARP. In vitro tests were performed by phantom measurements of different orthodontic devices applying different types of MR sequences [echoplanar imaging (EPI), turbo spin echo (TSE) and TSE-WARP, gradient echo (GRE)]. Two independent readers determined after calibration the level of artefacts. Subsequently, the interobserver agreement was calculated. The measurement of artefacts was based on the American Society for Testing Materials Standard F 2119-07. For in vivo imaging, one test person was scanned with an inserted multibracket appliance. The level of artefacts for 27 target regions was evaluated. In vitro: ceramic brackets and ferromagnetic steel brackets produced artefact radii up to 1.12 and 7.40 cm, respectively. WARP reduced these artefacts by an average of 32.7%. The Bland-Altman-Plot indicated that maximum measurement differences of 3 mm have to be expected with two calibrated observers. In vivo: the EPI sequence for brain imaging was not analysable. The TSE sequence of the brain did not demonstrate artefacts except for the nasal cavity. Conversely, the TSE sequence of the cervical spine revealed severe artefacts in the midface region. The GRE sequence appeared to be more susceptible to artefacts than did the TSE sequence. In vitro measurements allow an estimation of the in vivo artefact size. Orthodontic appliances may often remain intraorally when performing MRI. WARP showed a more significant effect in vitro than in vivo.

Development in Assay Methods for in Vitro Antimalarial Drug Efficacy Testing: A Systematic Review

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Shweta Sinha

2017-10-01

Full Text Available The emergence and spread of drug resistance are the major challenges in malaria eradication mission. Besides various strategies laid down by World Health Organization, such as vector management, source reduction, early case detection, prompt treatment, and development of new diagnostics and vaccines, nevertheless the need for new and efficacious drugs against malaria has become a critical priority on the global malaria research agenda. At several screening stages, millions of compounds are screened (1,000–2,000,000 compounds per screening campaign, before pre-clinical trials to select optimum lead. Carrying out in vitro screening of antimalarials is very difficult as different assay methods are subject to numerous sources of variability across different laboratories around the globe. Despite this, in vitro screening is an essential part of antimalarial drug development as it enables to resource various confounding factors such as host immune response and drug–drug interaction. Therefore, in this article, we try to illustrate the basic necessity behind in vitro study and how new methods are developed and subsequently adopted for high-throughput antimalarial drug screening and its application in achieving the next level of in vitro screening based on the current approaches (such as stem cells.

In vitro screening for population variability in toxicity of pesticide-containing mixtures

Science.gov (United States)

Abdo, Nour; Wetmore, Barbara A.; Chappell, Grace A.; Shea, Damian; Wright, Fred A.; Rusyna, Ivan

2016-01-01

Population-based human in vitro models offer exceptional opportunities for evaluating the potential hazard and mode of action of chemicals, as well as variability in responses to toxic insults among individuals. This study was designed to test the hypothesis that comparative population genomics with efficient in vitro experimental design can be used for evaluation of the potential for hazard, mode of action, and the extent of population variability in responses to chemical mixtures. We selected 146 lymphoblast cell lines from 4 ancestrally and geographically diverse human populations based on the availability of genome sequence and basal RNA-seq data. Cells were exposed to two pesticide mixtures – an environmental surface water sample comprised primarily of organochlorine pesticides and a laboratory-prepared mixture of 36 currently used pesticides – in concentration response and evaluated for cytotoxicity. On average, the two mixtures exhibited a similar range of in vitro cytotoxicity and showed considerable inter-individual variability across screened cell lines. However, when in vitroto-in vivo extrapolation (IVIVE) coupled with reverse dosimetry was employed to convert the in vitro cytotoxic concentrations to oral equivalent doses and compared to the upper bound of predicted human exposure, we found that a nominally more cytotoxic chlorinated pesticide mixture is expected to have greater margin of safety (more than 5 orders of magnitude) as compared to the current use pesticide mixture (less than 2 orders of magnitude) due primarily to differences in exposure predictions. Multivariate genome-wide association mapping revealed an association between the toxicity of current use pesticide mixture and a polymorphism in rs1947825 in C17orf54. We conclude that a combination of in vitro human population-based cytotoxicity screening followed by dosimetric adjustment and comparative population genomics analyses enables quantitative evaluation of human health hazard

Phosphite fertilisers as inhibitors of Hymenoscyphus fraxineus (anamorph Chalara fraxinea growth in tests in vitro

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Tkaczyk Miłosz

2017-03-01

Full Text Available This study is designed to test the potential for reducing the growth of the mycelium of the fungus Hymenoscyphus fraxineus (anamorph Chalara fraxinea by using phosphite preparations at various concentrations in vitro. The study shows that adding pure phosphite to potato dextrose agar media inhibits the development of the fungus, but if the preparation is applied in the form of ammonium phosphite (Actifos, the growth of fungus will be accelerated. Probably the addition of nitrogen contained in the product Actifos has positive effect on the mycelial growth, but pure phosphite restricts its development. These studies are preliminary and only show the potential use of phosphite to reduce the development of H. fraxineus; however, to completely confirm its operation, further research is needed in this area.

In Vitro Activities of New Antimicrobials against Nocardia brasiliensis

Science.gov (United States)

Vera-Cabrera, Lucio; Gonzalez, Eva; Choi, Sung H.; Welsh, Oliverio

2004-01-01

The in vitro sensitivities of 30 strains of Nocardia brasiliensis to DA-7867, gatifloxacin, moxifloxacin, and BMS-284756 (garenoxacin) were determined using the broth microdilution method. All N. brasiliensis strains were sensitive to these antimicrobials. The most active drug in vitro was DA-7867, with a MIC at which 90% of the isolates tested were inhibited of 0.03 μg/ml and a MIC at which 50% of the isolates tested were inhibited of 0.06 μg/ml. PMID:14742215

Comparative expression analysis reveals lineage relationships between human and murine gliomas and a dominance of glial signatures during tumor propagation in vitro.

Science.gov (United States)

Henriquez, Nico V; Forshew, Tim; Tatevossian, Ruth; Ellis, Matthew; Richard-Loendt, Angela; Rogers, Hazel; Jacques, Thomas S; Reitboeck, Pablo Garcia; Pearce, Kerra; Sheer, Denise; Grundy, Richard G; Brandner, Sebastian

2013-09-15

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor. ©2013 AACR.

Toward development of an in vitro model of methamphetamine-induced dopamine nerve terminal toxicity.

Science.gov (United States)

Kim, S; Westphalen, R; Callahan, B; Hatzidimitriou, G; Yuan, J; Ricaurte, G A

2000-05-01

To develop an in vitro model of methamphetamine (METH)-induced dopamine (DA) neurotoxicity, striatal synaptosomes were incubated at 37 degrees C with METH for different periods of time (10-80 min), washed once, then tested for DA transporter function at 37 degrees C. METH produced time- and dose-dependent reductions in the V(max) of DA uptake, without producing any change in K(m). Incubation of synaptosomes with the DA neurotoxins 1-methyl-4-phenyl-pyridinium ion, 6-hydroxydopamine, and amphetamine under similar conditions produced comparable effects. In contrast, incubation with fenfluramine, a serotonin neurotoxin, did not. METH-induced decreases in DA uptake were selective, insofar as striatal glutamate uptake was unaffected. Various DA transporter blockers (cocaine, methylphenidate, and bupropion) afforded complete protection against METH-induced decreases in DA uptake, without producing any effect themselves. METH's effects were also temperature dependent, with greater decreases in DA uptake occurring at higher temperatures. Tests for residual drug revealed small amounts (0.1-0.2 microM) of remaining METH, but kinetic studies indicated that decreases in DA uptake were not likely to be due to METH acting as a competitive inhibitor of DA uptake. Decreases in the V(max) of DA uptake were not accompanied by decreases in B(max) of [(3)H]WIN 35,428 binding, possibly because there is no mechanism for removing damaged DA nerve endings from the in vitro preparation Collectively, these results give good support to the development of a valid in vitro model that may prove helpful for elucidating the mechanisms underlying METH-induced DA neurotoxicity.

Sacarose e período de cultivo in vitro na aclimatização ex vitro de ginseng brasileiro (Pfaffia glomerata Spreng. Pedersen Sucrose and duration of in vitro growth on ex vitro acclimatization of Brazilian ginseng (Pfaffia glomerata Spreng. Pedersen

Directory of Open Access Journals (Sweden)

Etiane Caldeira Skrebsky

2004-10-01

photosynthetic capacity of plants cultivated in vitro, an extra source of carbohydrates is required to supply their metabolic demands. The growth rate of roots and shoots of P. glomerata are different during the in vitro cultivation. The objectives of this study were to evaluate the effect of sucrose levels and the duration of in vitro growth on ex vitro acclimatization of P. glomerata seedlings. Treatments consisted of a bifactorial combination (5x2 of five sucrose levels (15, 30, 45, 60 and 75g L-1 and two periods of in vitro cultivation (25 and 32 days after inoculation. In the in vitro cultivation, the experimental unit consisted of a test tube containing 10mL of MS medium and a nodal segment, from in vitro cultivated plants, of 1,0cm in length without leaves. The acclimatization scheme consisted of ex vitro cultivation by four successive phases: (i opening of the test tube and subsequent cultivation in growth room for three days; (ii seedlings subcultivation to Plantmax substrate and cultivation in growth room for 21 days; (iii seedlings transfer to partially shady natural environment for 24 days; and (iv seedling subcultivation to soil under field condition. The greatest plant growth by increasing the sucrose availability (levels among 45 and 60g L-1 during the in vitro growth contributed to acclimatization. Regardless of duration of the in vitro growth, the seedlings obtained and efficient acclimatization. The acclimatization procedure was 100% efficient on seedling production of P. glomerata.

In Vitro and In Vivo Activities of Essential Oil from the Seed of Anethum graveolens L. against Candida spp.

Directory of Open Access Journals (Sweden)

Hong Zeng

2011-01-01

Full Text Available The essential oil produced from the seed of Anethum graveolens L. (Umbelliferae was tested in vitro and in vivo anti-Candida activity. The microbroth dilution method was used in the minimal inhibitory concentration (MIC, according to M27-A3 of the guidelines of the Clinical and Laboratory Standard Institute (CLSI. And then, efficacy evaluation of essential oil in the prophylaxis and treatment of experimental vaginal candidiasis was performed in immunosuppressed mice. The anti-Candida activity was analyzed by microbiological and histological techniques and was compared with that of fluconazole (FCZ. The results showed essential oil was active in vitro against all tested strains, with MICs ranging from 0.312 μL/mL (for C. tropicalis, C. parapsilosis, and C. krusei to 0.625 μL/mL (for 6 isolated C. albicans strains. Essential oil (2% v/v was highly efficacious in accelerating C. albicans 09-1555 clearance from experimentally infected mice vagina by prophylaxis and therapeutic treatments. In both therapeutic efficacy and prophylaxis studies, the histological findings confirmed the microbiological results. The experimental results revealed that the tested essential oil is effective against vulvovaginal candidiasis in immunosuppressed mice.

Target-specific support vector machine scoring in structure-based virtual screening: computational validation, in vitro testing in kinases, and effects on lung cancer cell proliferation.

Science.gov (United States)

Li, Liwei; Khanna, May; Jo, Inha; Wang, Fang; Ashpole, Nicole M; Hudmon, Andy; Meroueh, Samy O

2011-04-25

We assess the performance of our previously reported structure-based support vector machine target-specific scoring function across 41 targets, 40 among them from the Directory of Useful Decoys (DUD). The area under the curve of receiver operating characteristic plots (ROC-AUC) revealed that scoring with SVM-SP resulted in consistently better enrichment over all target families, outperforming Glide and other scoring functions, most notably among kinases. In addition, SVM-SP performance showed little variation among protein classes, exhibited excellent performance in a test case using a homology model, and in some cases showed high enrichment even with few structures used to train a model. We put SVM-SP to the test by virtual screening 1125 compounds against two kinases, EGFR and CaMKII. Among the top 25 EGFR compounds, three compounds (1-3) inhibited kinase activity in vitro with IC₅₀ of 58, 2, and 10 μM. In cell cultures, compounds 1-3 inhibited nonsmall cell lung carcinoma (H1299) cancer cell proliferation with similar IC₅₀ values for compound 3. For CaMKII, one compound inhibited kinase activity in a dose-dependent manner among 20 tested with an IC₅₀ of 48 μM. These results are encouraging given that our in-house library consists of compounds that emerged from virtual screening of other targets with pockets that are different from typical ATP binding sites found in kinases. In light of the importance of kinases in chemical biology, these findings could have implications in future efforts to identify chemical probes of kinases within the human kinome.

Rapid urease test (RUT) for evaluation of urease activity in oral bacteria in vitro and in supragingival dental plaque ex vivo.

Science.gov (United States)

Dahlén, Gunnar; Hassan, Haidar; Blomqvist, Susanne; Carlén, Anette

2018-05-18

Urease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo. The RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 μl of RUT test solution in the well of a micro-plate to which a 1 μl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 μl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test. The strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher scores compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46). The rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease

Mechanisms of cell death induced by infusion sets leachables in in vitro experimental settings.

Science.gov (United States)

Kozlovskaya, Luba; Stepensky, David

2015-01-30

Leachable materials that are released from infusion sets during their use can induce local and systemic toxic effects. We studied the mechanisms and kinetics of cell death induced by infusion sets leachates in vitro using L-929 and bEnd. 3 cells. Changes in cell morphology and metabolic activity were determined using light microscopy and the MTT test, respectively. Detailed analysis of the mechanisms of cell death was performed using membrane integrity and caspases 3 and 7 activity tests, annexin V-FITC/7-AAD analysis by FACS, and DAPI nuclear staining followed by confocal microscopy. Infusion sets released toxic leachables and induced toxic effects. Latex flashball was the most toxic part of the studied infusion sets, and it potently induced cell oncosis via increased permeability of the cell membrane. Latex-induced decrease in cells metabolic activity and cell death were not accompanied by activation of caspases 3 and 7, changes in nuclear morphology, or substantial annexin V-FITC cell staining. Leachables from the tube part of the infusion sets were less toxic, and induced some biochemical changes without altering the cells morphology. Further studies are needed to reveal the in vivo toxicity of infusion sets and its correlation with the results of in vitro toxicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Testing for developmental neurotoxicity using a battery of in vitro assays for key cellular events in neurodevelopment.

Science.gov (United States)

Harrill, Joshua A; Freudenrich, Theresa; Wallace, Kathleen; Ball, Kenneth; Shafer, Timothy J; Mundy, William R

2018-04-05

Medium- to high-throughput in vitro assays that recapitulate the critical processes of nervous system development have been proposed as a means to facilitate rapid testing and identification of chemicals which may affect brain development. In vivo neurodevelopment is a complex progression of distinct cellular processes. Therefore, batteries of in vitro assays that model and quantify effects on a variety of neurodevelopmental processes have the potential to identify chemicals which may affect brain development at different developmental stages. In the present study, the results of concentration-response screening of 67 reference chemicals in a battery of high content imaging and microplate reader-based assays that evaluate neural progenitor cell proliferation, neural proginitor cell apoptosis, neurite initiation/outgrowth, neurite maturation and synaptogenesis are summarized and compared. The assay battery had a high degree of combined sensitivity (87%) for categorizing chemicals known to affect neurodevelopment as active and a moderate degree of combined specificity (71%) for categorizing chemicals not associated with affects on neurodevelopment as inactive. The combined sensitivity of the assay battery was higher compared to any individual assay while the combined specificity of the assay battery was lower compared to any individual assay. When selectivity of effects for a neurodevelopmental endpoint as compared to general cytotoxicity was taken into account, the combined sensitivity of the assay battery decreased (68%) while the combined specificity increased (93%). The identity and potency of chemicals identified as active varied across the assay battery, underscoring the need for use of a combination of diverse in vitro models to comprehensively screen chemicals and identify those which potentially affect neurodevelopment. Overall, these data indicate that a battery of assays which address many different processes in nervous system development may be used to

Testing Pollen of Single and Stacked Insect-Resistant Bt-Maize on In vitro Reared Honey Bee Larvae

Science.gov (United States)

Hendriksma, Harmen P.; Härtel, Stephan; Steffan-Dewenter, Ingolf

2011-01-01

The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops. PMID:22194811

Testing pollen of single and stacked insect-resistant Bt-maize on in vitro reared honey bee larvae.

Directory of Open Access Journals (Sweden)

Harmen P Hendriksma

Full Text Available The ecologically and economic important honey bee (Apis mellifera is a key non-target arthropod species in environmental risk assessment (ERA of genetically modified (GM crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.

In vitro studies reveal antiurolithic effect of Terminalia arjuna using quantitative morphological information from computerized microscopy

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A. Mittal

2015-10-01

Full Text Available ABSTRACT Purpose: For most cases, urolithiasis is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate (CaOx deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE of Terminalia arjuna (T. arjuna. Materials and Methods: The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software ‘Image-Pro Plus 7.0’ of Olympus upright microscope (BX53. Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro. Results: Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl radicals with an IC50 at 13.1µg/mL. Conclusions: The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.

Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle.

Science.gov (United States)

Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M

2015-11-30

This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect. Copyright © 2015 Elsevier B.V. All rights reserved.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Science.gov (United States)

Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

2016-01-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

FREQUENCY OF CHROMOSOMAL ABERRATIONS AND MICRONUCLEI IN HORSE LYMPHOCYTES FOLLOWING IN VITRO EXPOSURE TO LOW DOSE IONISING RADIATION

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Dunja Rukavina

2012-07-01

Full Text Available Ionising radiation is known to cause chromosomal instability, which is observed as increased frequency of chromosomal aberration and micronuclei. These are listed as reliable criteria in biological dosimetry. Numerous experiments conducted on both animal and plant models demonstrated that increase in radiation dosage is followed by increased mutation frequency, and that mutations occur even at the lowest exposure. We used horse blood in vitro irradiated by low doses of ionizing radiation. Cultivation of peripheral blood lymphocytes and micronucleus test were used as biomarkers of genetic damage. The observed aberrations were recorded and classified in accordance with the International System of Cytogenetic Nomenclature. Micronuclei were identified on the basis of criteria proposed by Fenech et al. (8. Analysis of chromosomal aberration showed increased frequency of aberrations in blood cultures exposed to 0,1 Gy and 0,2 Gy compared to the controls. Microscopic analysis of chromosomal damage in in vitro micronucleus test revealed that the applied radiation dose induced micronuclei while no binucleated cells with micronuclei were found in lymphocytes that were not irradiated. In this paper we analysed the influence of low dose ionising radiation on frequency of chromosomal aberration and micronuclei in horse lymphocytes following in vitro exposure to X-rays (0,1 Gy and 0,2 Gy. Key words: chromosomal aberrations, micronuclei, ionising radiation, horse lymphocytes

Application of rosula-formation tests for determining man lymphocyte radiosensitivity

International Nuclear Information System (INIS)

Shchilik, Ts.; Krushevskij, E.; Endrzhejchak, V.

1982-01-01

Radiosensitivity of subpopulation of lymphocytes-T-lymphocytes and B-lymphocytes was studied to diagnose acute radiation disease as well as if radiosensitivity of any of them is more effective indication of irradiation as compared with absolute lymphocyte quantity. The investigations were carried on in vitro using blood of healthy men-donors at the age of 21-25. It is shown that absolute quantity of cells forming AE rosette in perapheral blood is a much better indication of irradiation as compared with absolute quantity of lymphocytes. Considerable significance of tests of rosette formation especially AE test is underlined. High test sensitivity and relative simplicity of accomplishment permit authors to recommend it for diagnostic purposes when revealing acute radiation disease including the stages of medicinal evacuation

Transfer of hydrophobic contaminants in urban runoff particles to benthic organisms estimated by an in vitro bioaccessibility test

DEFF Research Database (Denmark)

Nakajima, F.; Saito, K.; Isozaki, Y.

2006-01-01

An in vitro bioaccessibility test was applied for assessing the transfer of polycyclic aromatic hydrocarbons (PAHs) present in road dust, into benthic organisms living in a receiving water body. The road dust is supposed to be urban runoff particles under wet weather conditions. Sodium dodecyl...... sulfate (SDS) solution was used as a hypothetical gut fluid. Pyrene, fluoranthene and phenanthrene were the main PAH species in the SIDS extractable fraction of road dust, as well as the whole extract. Benzo(ghi)perylene showed relatively low concentrations in the SIDS extract in spite of a high...... concentration in the original dust. The PAH composition in benthic organisms (polychaetes) did not correspond with that of the surrounding sediment and the PAHs detected were also detected in high concentrations in the SDS extract of road dust. When testing the toxicity of the extracted contaminants...

In vivo and in vitro dermal penetration of lipophilic and hydrophilic pesticides in mice

International Nuclear Information System (INIS)

Grissom, R.E. Jr.; Brownie, C.; Guthrie, F.E.

1987-01-01

Dermal absorption is a major portal of entry for a wide variety of potentially toxic substances. In vivo and in vitro investigations assessing penetration of topically applied xenobiotics using both human and other animals have been conducted. Current ethical considerations have drastically curtailed the testing of xenobiotics in human volunteers; consequently, dermal penetration in humans is usually estimated from in vivo tests in animals and in vitro tests using either human or animal skin. In order for in vitro penetration results to be meaningful, there needs to be close relationship with in vivo data. The objective of the present study was to investigate the relationship between in vivo and in vitro penetration of both hydrophilic and lipophilic 14 C-labelled compounds in mice

Evaluation of antinociceptive, in-vivo & in-vitro anti-inflammatory activity of ethanolic extract of Curcuma zedoaria rhizome.

Science.gov (United States)

Ullah, H M Arif; Zaman, Sayera; Juhara, Fatematuj; Akter, Lucky; Tareq, Syed Mohammed; Masum, Emranul Haque; Bhattacharjee, Rajib

2014-09-22

The present study was aimed to investigate the antinociceptive and anti-inflammatory activity of the Curcuma zedoaria (family Zingiberaceae) ethanolic rhizome extract in laboratory using both in vitro and in vivo methods so as to justify its traditional use in the above mentioned pathological conditions. Phytochemical screening was done to find the presence of various secondary metabolites of the plant. In vivo antinociceptive activity was performed employing the hot plate method, acidic acid induced writhing test and formalin induced writhing test on Swiss albino mice at doses of 250 and 500 mg/kg body weight. Anti-inflammatory activity test was done on Long Evans rats at two different doses (250 and 500 mg/kg body weight) by using carrageenan induced paw edema test. Finally in vitro anti-inflammatory test by protein-denaturation method was followed. Data were analyzed by one-way analysis of variance (ANOVA) and Dunnett's t-test was used as the test of significance. P value <0.05 was considered as the minimum level of significance. Phytochemical screening revealed presence of tannins, saponins, flavonoids, gums & carbohydrates, steroids, alkaloids, reducing sugars and terpenoids in the extract. In the hot plate method, the extract increased the reaction time of heat sensation significantly to 61.99% and 78.22% at the doses of 250 and 500 mg/kg BW respectively. In acetic acid induced writhing test, the percent inhibition of writhing response by the extract was 48.28% and 54.02% at 250 and 500 mg/kg doses respectively (p < 0.001). The extract also significantly inhibited the licking response in both the early phase (64.49%, p < 0.01) and the late phase (62.37%, p < 0.01) in formalin induced writhing test. The extract significantly (p < 0.05, p < 0.01 and p < 0.001) inhibited carrageenan induced inflammatory response in rats in a dose related manner. In in-vitro anti-inflammatory test, the extract significantly inhibited protein denaturation of 77.15, 64.43, 53

In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants

International Nuclear Information System (INIS)

Carney, D.N.; Mitchell, J.B.; Kinsella, T.J.

1983-01-01

The in vitro response to radiation and chemotherapeutic drugs of cell lines established from 7 patients with small cell (SC) lung cancer were tested using a soft agarose clonogenic assay. Five cell lines retained the typical morphological and biochemical amine precursor uptake decarboxylation characteristics of SC, while two cell lines had undergone ''transformation'' to large cell (LC) morphological variants with loss of amine precursor uptake decarboxylation cell characteristics of SC. The radiation survival curves for the SC lines were characterized by D0 values ranging from 51 to 140 rads and extrapolation values (n) ranging from 1.0 to 3.3. While the D0 values of the radiation survival curves of the LC variants were similar (91 and 80 rads), the extrapolation values were 5.6 and 11.1 In vitro chemosensitivity testing of the cell lines revealed an excellent correlation between prior treatment status of the patient and in vitro sensitivity or resistance. No correlation was observed between in vitro chemosensitivity and radiation response. These data suggest that transformation of SC to LC with loss of amine precursor uptake and decarboxylation characteristics is associated with a marked increase in radiation resistance (n) in vitro. The observation of a 2- to 5-fold increase in survival of the LC compared to the SC lines following 200 rads suggests that the use of larger daily radiation fractions and/or radiation-sensitizing drugs might lead to a significantly greater clinical response in patients with LC morphology. This clinical approach may have a major impact on patient response and survival

[In vitro metabolism of fenbendazole prodrug].

Science.gov (United States)

Wen, Ai-Dan; Duan, Li-Ping; Liu, Cong-Shan; Tao, Yi; Xue, Jian; Wu, Ning-Bo; Jiang, Bin; Zhang, Hao-Bing

2013-02-01

Synthesized fenbendazole prodrug N-methoxycarbonyl-N'-(2-nitro-4-phenylthiophenyl) thiourea (MPT) was analyzed in vitro in artificial gastric juice, intestinal juice and mouse liver homogenate model by using HPLC method, and metabolic curve was then generated. MPT was tested against Echinococcus granulosus protoscolices in vitro. The result showed that MPT could be metabolized in the three biological media, and to the active compound fenbendazole in liver homogenate, with a metabolic rate of 7.92%. Besides, the prodrug showed a weak activity against E. granulosus protoscolices with a mortality of 45.9%.

Testing in Vitro of an Apifitoterapeutic Formula Against Nosema spp.

OpenAIRE

Vasilica Savu; Ion Rădoi; Maria Magdici; Silvia Patruica; Nicoleta Ion; Agripina Sapcaliu

2015-01-01

Nosema, a parasitic disease that affects adult honey bees, has a directly correlation with the losses of bee colonies, until to depopulation. The target of our study was to determine the antinosema action of an apifitoterapeutic formula that was obtained in an earlier phase of our researches. In the present study, we have had two experiences (F and N) formed by clinically healthy bees. The experimental bees have received, in vitro, naturally infested honey (7 spores by Nosema spp / field). Th...

MRI with intraoral orthodontic appliance—a comparative in vitro and in vivo study of image artefacts at 1.5 T

Science.gov (United States)

Zachriat, C; Asbach, P; Blankenstein, K I; Peroz, I

2015-01-01

Objectives: We investigated artefacts caused from orthodontic appliances at 1.5-T MRI of the head and neck region and whether the image quality can be improved utilizing the artefact-minimizing sequence WARP. Methods: In vitro tests were performed by phantom measurements of different orthodontic devices applying different types of MR sequences [echoplanar imaging (EPI), turbo spin echo (TSE) and TSE-WARP, gradient echo (GRE)]. Two independent readers determined after calibration the level of artefacts. Subsequently, the interobserver agreement was calculated. The measurement of artefacts was based on the American Society for Testing Materials Standard F 2119-07. For in vivo imaging, one test person was scanned with an inserted multibracket appliance. The level of artefacts for 27 target regions was evaluated. Results: In vitro: ceramic brackets and ferromagnetic steel brackets produced artefact radii up to 1.12 and 7.40 cm, respectively. WARP reduced these artefacts by an average of 32.7%. The Bland–Altman-Plot indicated that maximum measurement differences of 3 mm have to be expected with two calibrated observers. In vivo: the EPI sequence for brain imaging was not analysable. The TSE sequence of the brain did not demonstrate artefacts except for the nasal cavity. Conversely, the TSE sequence of the cervical spine revealed severe artefacts in the midface region. The GRE sequence appeared to be more susceptible to artefacts than did the TSE sequence. Conclusions: In vitro measurements allow an estimation of the in vivo artefact size. Orthodontic appliances may often remain intraorally when performing MRI. WARP showed a more significant effect in vitro than in vivo. PMID:25734243

Highly Stabilized Curcumin Nanoparticles Tested in an In Vitro Blood–Brain Barrier Model and in Alzheimer’s Disease Tg2576 Mice

OpenAIRE

Cheng, Kwok Kin; Yeung, Chin Fung; Ho, Shuk Wai; Chow, Shing Fung; Chow, Albert H. L.; Baum, Larry

2012-01-01

The therapeutic effects of curcumin in treating Alzheimer’s disease (AD) depend on the ability to penetrate the blood–brain barrier. The latest nanoparticle technology can help to improve the bioavailability of curcumin, which is affected by the final particle size and stability. We developed a stable curcumin nanoparticle formulation to test in vitro and in AD model Tg2576 mice. Flash nanoprecipitation of curcumin, polyethylene glycol-polylactic acid co-block polymer, and polyvinylpyrrolidon...

Earthworms and in vitro physiologically-based extraction tests : complementary tools for a holistic approach towards understanding risk at arsenic-contaminated sites

OpenAIRE

Button, Mark; Watts, Michael J.; Cave, Mark R.; Harrington, Chris F.; Jenkin, Gawen R.T.

2009-01-01

The relationship of the total arsenic content of a soil and its bioaccumulation by earthworms (Lumbricus rubellus and Dendrodrilus rubidus) to the arsenic fraction bioaccessible to humans, measured using an in vitro physiologically-based extraction test (PBET), was investigated. Soil and earthworm samples were collected at 24 sites at the former arsenic mine at the Devon Great Consols (DGC) in southwest England (UK), along with an uncontaminated site in Nottingham, UK, for comparison. Analysi...

Neuroblast of the grasshopper embryo as a new mutagen test system. Pt. 1. In vitro radiosensitivity

Energy Technology Data Exchange (ETDEWEB)

Liang, J C; Gaulden, M E [Texas Univ., Dallas (USA). Dept. of Radiology

1982-04-01

The neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer) are being studied to determine their suitability for detecting environmental clastogens (chromosome-breaking agents). They are very sensitive to the induction of chromosome breakage by radiation in vitro. Their sensitvity, 0.011 break/cell/R, is 4-5 times higher than pollen mother cells of Tradescantia (micronuclei), 10 times higher than either human lymphocytes or Chinese hamster cells (metaphase chromosome aberrations), and 15 times higher than mouse erythroblasts (micronuclei). Furthermore, they have no spontaneous chromosome breakage, which facilitates the detection of agents that break chromosomes. The present study shows that Chortophaga embryos maintain normal mitotic activity in vitro for 5 cell cycles at 38/sup 0/C (20 h), and that neuroblasts of embryos grown in vitro have the same radiosensitivity as those of embryos in vivo. Thus in vitro exposure of grasshopper embryos is a promising method for obtaining data on the response of neuroblasts to chemical clastogens.

In vitro-in vivo extrapolation: estimation of human serum concentrations of chemicals equivalent to cytotoxic concentrations in vitro

International Nuclear Information System (INIS)

Guelden, Michael; Seibert, Hasso

2003-01-01

In the present study an extrapolation model for estimating serum concentrations of chemicals equivalent to in vitro effective concentrations is developed and applied to median cytotoxic concentrations (EC 50 ) determined in vitro. Nominal concentrations of a chemical in serum and in vitro are regarded as equivalent, if they result in the same aqueous concentration of the unbound form. The algorithm used is based on equilibrium distribution and requires albumin binding data, the octanol-water partition coefficient (K ow ), and the albumin concentrations and lipid volume fractions in vitro and in serum. The chemicals studied cover wide ranges of cytotoxic potency (EC 50 : 2.5-530000 μM) and lipophilicity (log K ow : -5 to 7). Their albumin binding characteristics have been determined by means of an in vitro cytotoxicity test as described previously. The equivalent serum concentrations of 19 of the 33 compounds investigated, having high protein binding and/or lipophilicity, were substantially higher than the EC 50 -values, by factors of 2.5-58. Prominent deviations between the equivalent nominal concentrations in serum and in vitro were largely restricted to chemicals with higher cytotoxic potency (EC 50 ≤1000 μM). The results suggest that estimates of equivalent serum concentrations based on in vitro data are robust for chemicals with low lipophilicity (log K ow ≤2) and low potency (EC 50 >1000 μM). With more potent chemicals or those with higher lipophilicity partitioning into lipids and/or binding to serum proteins have to be taken into account when estimating in vivo serum concentrations equivalent to in vitro effective concentrations

Spermicidal activity of the hexane extract of Piper longum: an in vitro study.

Science.gov (United States)

Sarwar, Abu Hasnath Md Golam; Nirala, Ranjeet Kumar; Arif, Mohammed; Khillare, Beena; Thakur, Sonu Chand

2015-01-01

This study was carried out to assess the spermicidal action of hexane extract from the fruits of Piper longum Linn. The sperm immobilisation studies showed that 20 mg/mL of hexane extract was able to immobilise sperms completely within 20 s. The sperm revival test revealed that the effects were spermicidal as sperm immobilisation effect was irreversible. There was also a significant reduction in sperm viability in the treated group in comparison to the control. The hypo-osmotic swelling of these sperms was significantly reduced, indicating that the hexane extract may probably cause injury to the sperm plasma membrane. Hence, this study showed that the hexane extract of P. longum possesses potential contraceptive spermicidal activity in vitro.

Evaluation of in vitro antioxidant and anti-inflmmatory activities of Ximenia americana extracts

Directory of Open Access Journals (Sweden)

Arun Kashivishwanath Shettar

2015-11-01

Full Text Available Objective: To evaluate in vitro antioxidant and anti-inflammatory activity of Ximenia americana extracts. Methods: Herbal extraction was done by Soxhlet extraction method with increasing polarity of solvents viz., chloroform, ethyl acetate, methanol, ethanol and water. Phytochemical analysis was done using different biochemical tests. Antioxidant potential of plant extracts were analyzed by ferric ion reducing antioxidant power, phosphomolybdenum and 2,2-diphenyl-1- picrylhydrazyl, and anti-inflammatory activity by using protein denaturation in vitro bioassay. Total phenolic content of each extract was also determined to assess their corresponding effect on antioxidant capacity of plant. Results: Phytochemical analysis showed that each solvent extract contained broad spectrum of secondary metabolites, phenolic compounds, flavonoids, tannins and glycosides, whereas compared to other solvent extracts, chloroform extract showed negative result for phenolic compounds whereas aqueous extract exhibited the highest phenolic content and the significant antioxidant capacity based on the test performed. Out of all extracts, methanol extract showed high anti-inflammatory activity. Conclusions: The present study revealed that different solvent extracts of Ximenia americana leaves contain broad spectrum of bioactive compounds. Results confirm that aqueous extract exhibited high antioxidant activity and methanol extract exhibited high antiinflammatory activity. Further study requires purification, characterization and structural elucidation of phenolic compounds in both extracts that may help in the development of new phytopharmaceuticals.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation.

Science.gov (United States)

Vogler, Amy J; Nottingham, Roxanne; Busch, Joseph D; Sahl, Jason W; Shuey, Megan M; Foster, Jeffrey T; Schupp, James M; Smith, Susan R; Rocke, Tonie E; Keim, Paul; Wagner, David M

2016-11-01

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used. Copyright © 2016 Elsevier B.V. All rights reserved.

The VITRO Score (Von Willebrand Factor Antigen/Thrombocyte Ratio as a New Marker for Clinically Significant Portal Hypertension in Comparison to Other Non-Invasive Parameters of Fibrosis Including ELF Test.

Directory of Open Access Journals (Sweden)

Stephanie Hametner

Full Text Available Clinically significant portal hypertension (CSPH, defined as hepatic venous pressure gradient (HVPG ≥10 mmHg, causes major complications. HVPG is not always available, so a non-invasive tool to diagnose CSPH would be useful. VWF-Ag can be used to diagnose. Using the VITRO score (the VWF-Ag/platelet ratio instead of VWF-Ag itself improves the diagnostic accuracy of detecting cirrhosis/ fibrosis in HCV patients.This study tested the diagnostic accuracy of VITRO score detecting CSPH compared to HVPG measurement.All patients underwent HVPG testing and were categorised as CSPH or no CSPH. The following patient data were determined: CPS, D'Amico stage, VITRO score, APRI and transient elastography (TE.The analysis included 236 patients; 170 (72% were male, and the median age was 57.9 (35.2-76.3; 95% CI. Disease aetiology included ALD (39.4%, HCV (23.4%, NASH (12.3%, other (8.1% and unknown (11.9%. The CPS showed 140 patients (59.3% with CPS A; 56 (23.7% with CPS B; and 18 (7.6% with CPS C. 136 patients (57.6% had compensated and 100 (42.4% had decompensated cirrhosis; 83.9% had HVPG ≥10 mmHg. The VWF-Ag and the VITRO score increased significantly with worsening HVPG categories (P<0.0001. ROC analysis was performed for the detection of CSPH and showed AUC values of 0.92 for TE, 0.86 for VITRO score, 0.79 for VWF-Ag, 0.68 for ELF and 0.62 for APRI.The VITRO score is an easy way to diagnose CSPH independently of CPS in routine clinical work and may improve the management of patients with cirrhosis.

In vitro efficacy of doripenem against pseudomonas aeruginosa and acinetobacter baumannii by e-test

International Nuclear Information System (INIS)

Gilani, M.; Munir, T.; Latif, M.; Rehman, S.

2015-01-01

To assess the in vitro efficacy of doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii using Epsilometer strips. Study Design: Cross-sectional study. Place and Duration of Study: Department of Microbiology, Army Medical College, Rawalpindi and National University of Sciences and Technology, Islamabad, from May 2014 to September 2014. Methodology: A total of 60 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa collected from various clinical samples received from Military Hospital were included in the study. The specimens were inoculated onto blood, MacConkey and chocolate agars. The isolates were identified using Gram staining, motility, catalase test, oxidase test and API 20NE (Biomeriux, France). Organisms identified as Acinetobacter baumannii and Pseudomonas aeruginosa were included in the study. Bacterial suspensions equivalent to 0.5 McFarland turbidity standard of the isolates were prepared and applied on Mueller Hinton agar. Epsilometer strip was placed in the center of the plate and incubated for 18-24 hours. Minimum Inhibitory Concentration (MIC) was taken to be the point where the epsilon intersected the E-strip. MIC of all the isolates was noted. Results: For Pseudomonas aeruginosa isolates, MIC50 was 12 micro g/mL and MIC90 was 32 micro g/mL. For Acinetobacter baumannii MIC 50 and MIC90 was 32 micro g/mL. Conclusion: Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting. (author)

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

Science.gov (United States)

Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

2007-06-01

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver

Way forward in case of a false positive in vitro genotoxicity result for a cosmetic substance?

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Doktorova, Tatyana Y; Ates, Gamze; Vinken, Mathieu; Vanhaecke, Tamara; Rogiers, Vera

2014-02-01

The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a high sensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant positive results (i.e. low specificity) thereby imposing the need for additional follow-up by in vitro and/or in vivo genotoxicity tests. This could have a major impact on the cosmetic industry in Europe, seen the imposed animal testing and marketing bans on cosmetics and their ingredients. Afflicted, but safe substances could therefore be lost. Using the example of triclosan, a cosmetic preservative, we describe here the potential applicability of a human toxicogenomics-based in vitro assay as a potential mechanistically based follow-up test for positive in vitro genotoxicity results. Triclosan shows a positive in vitro chromosomal aberration test, but is negative during in vivo follow-up tests. Toxicogenomics analysis unequivocally shows that triclosan is identified as a compound acting through non-DNA reactive mechanisms. This proof-of-principle study illustrates the potential of genome-wide transcriptomics data in combination with in vitro experimentation as a possible weight-of-evidence follow-up approach for de-risking a positive outcome in a standard mutagenicity/genotoxicity battery. As such a substantial number of cosmetic compounds wrongly identified as genotoxicants could be saved for the future. Copyright © 2013 Elsevier Ltd. All rights reserved.

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

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Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

XPS analysis of aluminosilicate microspheres bioactivity tested in vitro

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Todea, M.; Vanea, E. [Faculty of Physics and Institute of Interdisciplinary Research on Bio-Nano-Sciences, Babes Bolyai University, Cluj-Napoca 400084 (Romania); Bran, S. [University of Medicine and Pharmacy “Iuliu Haţieganu”, Department of Cranio-Maxillofacial Surgery, 400029 Cluj-Napoca (Romania); Berce, P. [Technical University of Cluj-Napoca, Faculty of Machine Building and National Centre of Rapid Prototyping, 400641 Cluj-Napoca (Romania); Simon, S., E-mail: simons@phys.ubbcluj.ro [Faculty of Physics and Institute of Interdisciplinary Research on Bio-Nano-Sciences, Babes Bolyai University, Cluj-Napoca 400084 (Romania)

2013-04-01

The study aims to characterize surface properties of aluminosilicate microspheres incorporating yttrium, with potential biomedical applications. Micrometric particles of spherical shape were obtained by spray drying method. The behavior of aluminosilicate microspheres without yttrium and with yttrium was investigated under in vitro conditions, by seven days incubation in simulated body fluid (SBF). The surface elemental composition and the atomic environments on outermost layer of the microspheres, prior to and after incubation in SBF were evaluated by X-ray photoelectron spectroscopy (XPS) in order to investigate their bioactivity. The results were analyzed to underline the effect of yttrium addition on surface properties of the aluminosilicate microspheres and implicitly on the behavior of the samples in simulated body environments.

Autophagy in the test tube: In vitro reconstitution of aspects of autophagosome biogenesis.

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Rao, Yijian; Matscheko, Nena; Wollert, Thomas

2016-06-01

Autophagy is a versatile recycling pathway that delivers cytoplasmic contents to lysosomal compartments for degradation. It involves the formation of a cup-shaped membrane that expands to capture cargo. After the cargo has been entirely enclosed, the membrane is sealed to generate a double-membrane-enclosed compartment, termed the autophagosome. Depending on the physiological state of the cell, the cargo is selected either specifically or non-specifically. The process involves a highly conserved set of autophagy-related proteins. Reconstitution of their action on model membranes in vitro has contributed tremendously to our understanding of autophagosome biogenesis. This review will focus on various in vitro techniques that have been employed to decipher the function of the autophagic core machinery. © 2016 Federation of European Biochemical Societies.

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.

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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.

Do senescence markers correlate in vitro and in situ within individual human donors?

DEFF Research Database (Denmark)

Waaijer, Mariëtte E C; Gunn, David A; van Heemst, Diana

2018-01-01

Little is known on how well senescence markers in vitro and in situ correlate within individual donors. We studied correlations between the same and different in vitro markers. Furthermore, we tested correlations between in vitro markers with in situ p16INK4a positivity.From 100 donors (20-91 yea...

Functional and hypoglycemic properties of nopal cladodes (O. ficus-indica) at different maturity stages using in vitro and in vivo tests.

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Nuñez-López, María A; Paredes-López, Octavio; Reynoso-Camacho, Rosalía

2013-11-20

Nopal (Opuntia ficus-indica) cladodes are recommended for their therapeutic properties; their maturity stage may affect their biological properties. Cladodes of three maturity stages, from the same crop and location, were dehydrated and evaluated for some of their physicochemical and nutritional characteristics and antidiabetic properties. The flours of small and medium cladodes (SCF and MCF, respectively) had higher contents of dietary fiber, water absorption, swelling, and viscosity compared to those of the large cladode flour (LCF). Streptozotocin-induced diabetic rats, treated with MCF and SCF (doses of 50 mg/kg body weight), showed reduction of postprandial blood glucose on 46.0 and 23.6%, respectively (p < 0.05), in relation to the control; and LCF had no significant effect. In vitro, glucose diffusion tests showed similar ranking by the two former samples, whereas the latter was close to the control. Cladode maturity stages showed different fiber content and produced suspensions with differences in viscosity, which may affect in vitro and in vivo glucose responses.

To test or not to test: A cross-sectional survey of the psychosocial determinants of self-testing for cholesterol, glucose, and HIV

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Dinant Geert-Jan

2011-02-01

Full Text Available Abstract Background Although self-tests are increasingly available and widely used, it is not clear whether their use is beneficial to the users, and little is known concerning the determinants of self-test use. The aim of this study was to identify the determinants of self-test use for cholesterol, glucose, and HIV, and to examine whether these are similar across these tests. Self-testing was defined as using in-vitro tests on body materials, initiated by consumers with the aim of diagnosing a particular disorder, condition, or risk factor for disease. Methods A cross-sectional Internet survey was conducted among 513 self-testers and 600 non-testers, assessing possible determinants of self-test use. The structured questionnaire was based on the Health Belief Model, Theory of Planned Behavior, and Protection Motivation Theory. Data were analyzed by means of logistic regression. Results The results revealed that perceived benefits and self-efficacy were significantly associated with self-testing for all three conditions. Other psychosocial determinants, e.g. gender, cues to action, perceived barriers, subjective norm, and moral obligation, seemed to be more test-specific. Conclusions Psychosocial determinants of self-testing are not identical for all tests and therefore information about self-testing needs to be tailored to a specific test. The general public should not only be informed about advantages of self-test use but also about the disadvantages. Designers of information about self-testing should address all aspects related to self-testing to stimulate informed decision making which, in turn, will result in more effective self-test use.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

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Sørli, Jorid B; Huang, Yishi; Da Silva, Emilie; Hansen, Jitka S; Zuo, Yi Y; Frederiksen, Marie; Nørgaard, Asger W; Ebbehøj, Niels E; Larsen, Søren T; Hougaard, Karin S

2018-01-01

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

Summary. The 19th quality control survey for radioisotope in vitro tests in Japan, 1997

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Shishiba, Yoshimasa; Shimizu, Taeko [Toranomon Hospital, Tokyo (Japan); Ichihara, Kiyoshi; Kousaka, Tadako; Kobayashi, Hisae; Tsushima, Toshio; Hoshino, Minoru; Mori, Mikio

1998-11-01

This summary concerns results of the survey in the title performed by the Subcommittee for Radioisotope in vitro Test in the Medical and Pharmaceutical Committee, Japan Radioisotope Association. The survey was conducted in 161 facilities in Japan including public and private hospitals, health institutes and reagent manufacturers. Samples were sent to the facilities and subjected to examinations of intra- and inter-day reproducibility, freeze-thawing effects and periodical changes of the measured values. Methods employed were RIA, IRMA, EIA, ELISA, LPIA, PAMIA, FIA, FPIA, NIA, LAT, CLIA and ECLIA. Results were analyzed by variation coefficients in the kit and between the kits. Samples were for: growth hormone, somatomedin C, follicle stimulating hormone, luteinizing hormone, prolactin, thyroid stimulating hormone, triiodothyronine, thyroxine, thyroxine binding globulin, calcitonin, insulin, C-peptide, glucagon, gastrin, testosterone, estradiol, progesterone, {beta}human chorionic gonadotropin, 17{alpha}-hydroxyprogesterone, aldosterone, cortisol, dehydroepiandrosterone, renin, immunogloblin E, digoxin, {alpha}-fetoprotein, carcinoembryonic antigen, tissue polypeptide antigen, CA125, CA19-9, CA15-3, prostatic acid phosphatase, prostate specific antigen, {beta}{sub 2}-microgloblin and ferritin. Results were found rather unsatistactory for some products in the clinical practice, necessary for improvement of detection for some tests and needed for some kits for solving the problems of difference between kits and of matrix effect. (K.H)

Myelomonocytic THP-1 cells for in vitro testing of immunomodulatory properties of nanoparticles.

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Schroecksnadel, Sebastian; Jenny, Marcel; Fuchs, Dietmar

2011-02-01

The use of nanoparticles for new therapeutic and diagnostics options represents a new risk for individuals exposed to such compounds. The myelomonocytic cell line THP-1 could be a useful alternative to human peripheral blood mononuclear cells (PBMC) to test for effects of drugs and compounds. Stimulation degree of cells can be monitored by measurement of neopterin and/or the kynurenine to tryptophan ratio. The method is robust and reproducible in the range of 0.1-1.0 microg/ml of LPS. However, compared to the PBMC assay it will not reveal any effect on the T-cell interaction.

In vitro repopulation of haemopoietic stem cells after irradiation

International Nuclear Information System (INIS)

Mori, K.J.; Kumagai, Keiko; Seto, Akira; Ito, Yohei

1981-01-01

A culture system was designed in which proliferation of the haemopoietic stem cells was supported by adherent 'stromal' cell colonies. Application of the culture system to studies on kinetic behaviour of the haemopoietic stem cells after irradiation revealed; i) bone marrow stromal cells were radiosensitive with D 0 = 95R, when measured as the capability to proliferate and form adherent cell colonies in vitro, ii) radiosensitivity of the pluripotent stem cells (CFUs) in vitro was within the range of the in vivo sensitivity, iii) irradiated bone marrow cells under in vitro condition could repopulate at the same rate as those under in vivo condition, thereby suggesting that the function related to the support of haemopoiesis was radioresistant, iv) concentrations of both CFUs and granulocyte-macrophage precursor cells (CFUc) were higher in the irradiated cultures than those in unirradiated control culture at 3 weeks after irradiation. (author)

Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

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Jertta-Riina Sarkanen

2011-01-01

Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

In Vitro Dosimetry of Silver Nanoparticles

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An important issue for interpreting in vitro nanomaterial testing is quantifying the dose absorbed by target cells. Considerations include the concentration added to the culture and the proportion of the applied dose that is absorbed by the target cells. Rapid and efficient techn...

PHARMACOLOGICAL IN VITRO MODELS IN PRE-CLINICAL DRUG TESTING - EXAMPLE OF hSERT TRANSFECTED HUMAN EMBRYONIC KIDNEY CELLS

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Mihajlo Jakovljević

2012-06-01

Full Text Available Preclinical drug testing should be considered an important stage during examinations of its efficiency and safety in any likely indication observed. Purpose of the process is acquisition of substantial amount of particular drug-related data before approaching clinical trials in humans. Historical preclinical testing relied on available testing in microbe cultures and animal models. During recent decades laboratory techniques of human cell lines cultivation have been developed and improved. These provide unique possibility of drug acting mechanism testing in a simplified environment lacking basic homeostatic mechanisms. Some examples of these are measuring drug impact to biochemical transport, signaling or anabolic processes. Humane cell lines of embrional kidney 293 are an example of easy-to-grow and disseminate and quite endurable cell line. This methodological article notices some of the details of HEK293 cells cultivation and breading. We took transfection as an example of in vitro model creation for drug testing. Transfection refers to gene introduction into HEK293 cellular genome in order to achieve membrane expression of coded protein. In our case it would be human serotonin transporter. Article contains description of one particular methodological approach in measuring human serotonin transporter expression. The role and importance of serotonin pump in affective disorders genesis was already widely recognized. Aim of the paper was to emphasize feasibility of cell cultivation and its advantages in comparison with alternative traditional methods.

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

Science.gov (United States)

Burra, D D; Mühlenbock, P; Andreasson, E

2015-09-01

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

A simple in vitro test tube method for estimating the bioavailability of phosphorus in feed ingredients for swine.

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Bollinger, David W; Tsunoda, Atsushi; Ledoux, David R; Ellersieck, Mark R; Veum, Trygve L

2004-04-07

A simplified in vitro test tube (TT) method was developed to estimate the percentage of available P in feed ingredients for swine. The entire digestion procedure with the TT method consists of three consecutive enzymatic digestions carried out in a 50-mL conical test tube: (1) Pre-digestion with endo-xylanase and beta-glucanase for 1 h, (2) peptic digestion for 2 h, and (3) pancreatic digestion for 2 or 4 h. The TT method is simpler and much easier to perform compared to the dialysis tubing (DT) method, because dialysis tubing is not used. Reducing sample size from 1.0 to 0.25 g for the TT method improved results. In conclusion, the accuracy and validity of the TT method is equal to that of our more complicated DT method (r = 0.97, P < 0.001), designed to mimic the digestive system of swine, for estimating the availability of P in plant-origin feed ingredients.

Prediction of Chemical Carcinogenicity in Rodents from in vitro Genetic Toxicity Assays

Science.gov (United States)

Tennant, Raymond W.; Margolin, Barry H.; Shelby, Michael D.; Zeiger, Errol; Haseman, Joseph K.; Spalding, Judson; Caspary, William; Resnick, Michael; Stasiewicz, Stanley; Anderson, Beth; Minor, Robert

1987-05-01

Four widely used in vitro assays for genetic toxicity were evaluated for their ability to predict the carcinogenicity of selected chemicals in rodents. These assays were mutagenesis in Salmonella and mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Seventy-three chemicals recently tested in 2-year carcinogenicity studies conducted by the National Cancer Institute and the National Toxicology Program were used in this evaluation. Test results from the four in vitro assays did not show significant differences in individual concordance with the rodent carcinogenicity results; the concordance of each assay was approximately 60 percent. Within the limits of this study there was no evidence of complementarity among the four assays, and no battery of tests constructed from these assays improved substantially on the overall performance of the Salmonella assay. The in vitro assays which represented a range of three cell types and four end points did show substantial agreement among themselves, indicating that chemicals positive in one in vitro assay tended to be positive in the other in vitro assays. To help put this project into its proper context, we emphasize certain features of the study: 1) Standard protocols were used to mimic the major use of STTs worldwide--screening for mutagens and carcinogens; no attempt was made to optimize protocols for specific chemicals. 2) The 73 NTP chemicals and their 60% incidence of carcinogenicity are probably not representative of the universe of chemicals but rather reflect the recent chemical selection process for the NTP carcinogenicity assay. 3) The small, diverse group of chemicals precludes a meaningful evaluation of the predictive utility of chemical structure information. 4) The NTP is currently testing these same 73 chemicals in two in vivo STTs for chromosomal effects. 5) Complete data for an additional group of 30 to 40 NTP chemicals will be gathered on

[First Argentine consensus guidelines for in vitro antimicrobial susceptibility testing of clinically relevant anaerobic bacteria in humans/ Anaerobic Subcommittee of the Asociación Argentina de Microbiología].

Science.gov (United States)

Legaria, María C; Bianchini, Hebe M; Castello, Liliana; Carloni, Graciela; Di Martino, Ana; Fernández Canigia, Liliana; Litterio, Mirta; Rollet, Raquel; Rossetti, Adelaida; Predari, Silvia C

2011-01-01

Through time, anaerobic bacteria have shown good susceptibility to clinically useful antianaerobic agents. Nevertheless, the antimicrobial resistance profile of most of the anaerobic species related to severe infections in humans has been modified in the last years and different kinds of resistance to the most active agents have emerged, making their effectiveness less predictable. With the aim of finding an answer and for the purpose of facilitating the detection of anaerobic antimicrobial resistance, the Anaerobic Subcommittee of the Asociación Argentina de Microbiología developed the First Argentine consensus guidelines for in vitro antimicrobial susceptibility testing of clinically relevant anaerobic bacteria in humans. This document resulted from the compatibilization of the Clinical and Laboratory Standards Institute recommendations, the international literature and the work and experience of the Subcommittee. The Consensus document provides a brief taxonomy review, and exposes why and when anaerobic antimicrobial susceptibility tests should be conducted, and which antimicrobial agents can be used according to the species involved. The recommendations on how to perform, read and interpret in vitro anaerobic antimicrobial susceptibility tests with each method are exposed. Finally, the antibiotic susceptibility profile, the classification of antibiotics according to their in vitro activities, the natural and acquired mechanisms of resistance, the emerging resistance and the regional antibiotic resistance profile of clinically relevant anaerobic species are shown.

Bioequivalence and in vitro antimicrobial activity between generic and brand-name levofloxacin.

Science.gov (United States)

Sun, Hsin-Yun; Liao, Hsiao-Wei; Sheng, Meng-Huei; Tai, Hui-Min; Kuo, Ching-Hua; Sheng, Wang-Huei

2016-07-01

Generic agents play a crucial role in reducing the cost of medical care in many countries. However, the therapeutic equivalence remains a great concern. Our study aims to assess the in vitro antimicrobial activity and bioequivalence between generic and brand-name levofloxacin. Enantiomeric purity test, dissolution test, and in vitro antimicrobial susceptibility against seven clinically important pathogens by the agar dilution method were employed to assess the similarity between four generic products and brand-name levofloxacin (Daiichi Sankyo). All the generic and brand-name levofloxacin passed enantiomeric purity test. The results of dissolution tests were not similar among the generic products and the brand-name levofloxacin. Compared with the generic products, the brand-name levofloxacin had the smallest mean variations (-25% to 13%) with reference standard (United States Pharmacopeia levofloxacin Reference Standards). Variations were observed particularly in dissolution profiles and in vitro activity between generic products and brand-name levofloxacin. Copyright © 2016 Elsevier Inc. All rights reserved.

Dimetilsulfóxido - DMSO no teste de sensibilidade microbiana in vitro em cepas de Rhodococcus equi isoladas de afecções pulmonares em potros

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Ribeiro Márcio Garcia

2001-01-01

Full Text Available Comparou-se a sensibilidade microbiana in vitro de isolados de Rhodococcus equi pelo teste padrão de difusão com discos, com o modificado, pela adição de 5% de dimetilsulfóxido-DMSO. Observou-se aumento da sensibilidade do R. equi no teste com DMSO, frente a aminoglicosídeos (canamicina, amicacina, estreptomicina e ao cloranfenicol, enquanto para a eritromicina e derivados ß-lactâmicos (penicilina G, cefalosporinas, amoxicilina, oxacilina, constatou-se redução da sensibilidade do agente.

Antioxidant properties of salmon (Salmo salar L.) protein fraction hydrolysates revealed following their ex vivo digestion and in vitro hydrolysis.

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Borawska, Justyna; Darewicz, Małgorzata; Pliszka, Monika; Vegarud, Gerd E

2016-06-01

Salmon (Salmo salar L.) myofibryllar protein (MP) and sarcoplasmic protein (SP) were digested with human gastric and duodenal juices and hydrolysed in vitro with commercial pepsin and Corolase PP. The digestion after duodenal juice/Corolase PP caused almost complete breakdown of peptide bonds in MP and SP. The DPPH(•) scavenging activity of proteins decreased during both ex vivo digestion and in vitro hydrolysis. The highest value of DPPH(•) scavenging activity was shown for the gastric digest of SP (8.88 ± 0.87%). The ABTS(+•) scavenging activity of MP and SP increased during digestion/hydrolysis. The duodenal digest of SP was characterised by the highest value of ABTS(+•) scavenging activity (72.7 ± 1.2%). In turn, the highest value of ferric-reducing power was determined for the gastric digest of SP (84.8 ± 0.2%). Salmon antioxidant peptides Phe-Ile-Lys-Lys, His-Leu, Ile-Tyr, Pro-His-Leu, Pro-Trp, Val-Pro-Trp were identified in both ex vivo digested and in vitro hydrolysed MP and SP. An antioxidant peptide, Val-Tyr, was additionally detected in the in vitro hydrolysate of SP. The results indicate the salmon myofibrillar and sarcoplasmic protein fractions as potential sources of antioxidant peptides that could be released in the gastrointestinal tract but their amino acid sequence and quantification vary. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Synthesis, β-haematin inhibition, and in vitro antimalarial testing of isocryptolepine analogues: SAR study of indolo[3,2-c]quinolines with various substituents at C2, C6, and N11.

Science.gov (United States)

Wang, Ning; Wicht, Kathryn J; Imai, Kento; Wang, Ming-Qi; Anh Ngoc, Tran; Kiguchi, Ryo; Kaiser, Marcel; Egan, Timothy J; Inokuchi, Tsutomu

2014-05-01

A series of indolo[3,2-c]quinolines were synthesized by modifying the side chains of the ω-aminoalkylamines at the C6 position and introducing substituents at the C2 position, such as F, Cl, Br, Me, MeO and NO2, and a methyl group at the N11 position for an SAR study. The in vitro antiplasmodial activities of the derivative agents against two different strains (CQS: NF54 and CQR: K1) and the cytotoxic activity against normal L6 cells were evaluated. The test results showed that compounds 6k and 6l containing the branched methyl groups of 3-aminopropylamino at C6 with a Cl atom at C2 exhibited a very low cytotoxicity with IC50 values above 4000 nM, high antimalarial activities with IC50 values of about 11 nM for CQS (NF54), IC50 values of about 17 nM for CQR (K1), and RI resistance indices of 1.6. Furthermore, the compounds were tested for β-haematic inhibition, and QSAR revealed an interesting linear correlation between the biological activity of CQS (NF54) and three contributing factors, namely solubility, hydrophilic surface area, and β-haematin inhibition for this series. In vivo testing of 6l showed a reduction in parasitaemia on day 4 with an activity of 38%. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long-term in-vitro precision of direct digital X-ray radiogrammetry

International Nuclear Information System (INIS)

Dhainaut, Alvilde; Hoff, Mari; Kaelvesten, Johan; Lydersen, Stian; Forslind, Kristina; Haugeberg, Glenn

2011-01-01

Digital X-ray radiogrammetry (DXR) calculates peripheral bone mineral density (BMD) from hand radiographs. The short-term precision for direct DXR has been reported to be highly satisfactory. However, long-term precision for this method has not been examined. Thus, the aim of this study was to examine the long-term in-vitro precision for the new direct digital version of DXR. The in-vitro precision for direct DXR was tested with cadaver phantoms on four different X-ray systems at baseline, 3 months, 6 months, and in one machine also at 12 months. At each time point, 31 measurements were performed. The in-vitro longitudinal precision for the four radiographic systems ranged from 0.22 to 0.43% expressed as coefficient of variation (CV%). The smallest detectable difference (SDD) ranged from 0.0034 to 0.0054 g/cm 2 . The in vitro long-term precision for direct DXR was comparable to the previous reported short-term in-vitro precision for all tested X-ray systems. These data show that DXR is a stable method for detecting small changes in bone density during 6-12 months of follow-up. (orig.)

Quality Attributes and In Vitro Bioequivalence of Different Brands of Amoxicillin Trihydrate Tablets.

Science.gov (United States)

Al-Tabakha, Moawia M; Fahelelbom, Khairi M S; Obaid, Dana Emad Eddin; Sayed, Sadik

2017-05-20

Bacterial resistance and antibiotic drug effectiveness can be related to administering generic products with a subtherapeutic dose or poor in vivo drug release. The aim of this study was to investigate whether locally marketed amoxicillin tablets have the required chemical and physical attributes, including in vitro bioequivalence performance. Five generic products (T1, T2, T3, T4, and T5) containing combination of amoxicillin trihydrate and potassium clavulanate as 1 g strength present in immediate release tablets were compared to the reference listed drug product Augmentin® (R) for weight variation, friability, resistance to crushing, and chemical content of amoxicillin. Difference (ƒ1) and similarity (ƒ2) factors were calculated to assess in vitro bioequivalence requirements. The tablets from different products have shown compliance with the pharmacopeial requirements of the performed tests. The measured resistance to crushing of tablets did not influence the dissolution time. Three generic products released more than 85% of amoxicillin by the first 15 min as did the reference product and were considered as bioequivalent products. T1 and T4 had ƒ1 values of 16.5% and 25.4% respectively and their ƒ2 values were 44.5 and 34.6 respectively, indicating failure to meet in vitro bioequivalence requirements. Tablet formulations can play an important role in achieving bioequivalence. Independent investigations such as this study serve as an important tool to reveal possible inferior or noncompliant products that may find their way to the market.

Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

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Yukio Kurihara

2014-12-01

Full Text Available Several transcription factors (TFs coordinate to regulate expression of specific genes at the transcriptional level. In Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant’s transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5 transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq instead of the in vivo chromatin immunoprecipitation (ChIP-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function.

Results of in vitro chemosensitivity assays

International Nuclear Information System (INIS)

Tanigawa, Nobuhiko; Morimoto, Hideki; Akita, Toshiaki; Inoue, Hiroshi; Tanaka, Takeo.

1986-01-01

The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60 % of specimens in HTCA and 58 % in SA produced significant growth in vitro. HTCA was 52 % (13/25) reliable for predicting in vivo sensitivity, and 95 % (36/38) reliable for in vivo resistance, whereas SA was 40 % (8/20) reliable for in vivo sensitivity and 88 % (21/24) for in vivo resistance. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48 % and 60 % in HTCA, and 46 % and 68 % in SA, respectively). (p < 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between : 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors. (J.P.N.)

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II).

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Groeber, F; Schober, L; Schmid, F F; Traube, A; Kolbus-Hernandez, S; Daton, K; Hoffmann, S; Petersohn, D; Schäfer-Korting, M; Walles, H; Mewes, K R

2016-10-01

To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus, the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

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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.

Ebselen inhibits the activity of acetylcholinesterase globular isoform G4 in vitro and attenuates scopolamine-induced amnesia in mice.

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Martini, Franciele; Pesarico, Ana P; Brüning, César A; Zeni, Gilson; Nogueira, Cristina W

2018-02-05

There is a well-known relationship between the cholinergic system and learning, memory, and other common cognitive processes. The process for researching and developing new drugs has lead researchers to repurpose older ones. This study investigated the effects of ebselen on the activity of acethylcholinesterase (AChE) isoforms in vitro and in an amnesia model induced by scopolamine in Swiss mice. In vitro, ebselen at concentrations equal or higher than 10 μM inhibited the activity of cortical and hippocampal G4/AChE, but not G1/AChE isoform. Treatment of mice with ebselen (50 mg/kg, i.p.) was effective against impairment of spatial recognition memory in both Y-maze and novel object recognition tests induced by scopolamine (1 mg/kg, i.p.). Ebselen (50 mg/kg) inhibited hippocampal AChE activity in mice. The present study demonstrates that ebselen inhibited the G4/AChE isoform in vitro and elicited an anti-amnesic effect in a mouse model induced by scopolamine. These findings reveal ebselen as a potential compound in terms of opening up valid therapeutic avenues for the treatment of memory impairment diseases. © 2018 Wiley Periodicals, Inc.

In vitro antimicrobial activity of methanolic leaf extract of Psidium guajava L.

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Dhiman, Anju; Nanda, Arun; Ahmad, Sayeed; Narasimhan, B.

2011-01-01

Aim: This study was designed to examine the chemical composition and in vitro antimicrobial potential of methanolic extract of Psidium guajava Linn (Myrtaceae). Materials and Methods: The inhibitory effect of methanolic extract of P. guajava was tested against three bacterial and two fungal strains by using the paper disc diffusion method. Results: The methanolic extract exhibited antibacterial activity against E. coli with minimum inhibitory concentration, 0.78 μg/ml, minimum bactericidal concentration of 50 μg/ml, and appreciable antifungal activity with minimum inhibitory concentration of 12.5 μg/ml. Preliminary phytochemical analysis of methanolic extract revealed the presence of antimicrobial compounds such as flavonoids, steroids, and tannins, which may contribute for the antimicrobial action of P. guajava. Conclusion: The extract was found to be bacteriostatic and fungistatic in action. PMID:21687350

In vitro antimicrobial activity of methanolic leaf extract of Psidium guajava L.

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Anju Dhiman

2011-01-01

Full Text Available Aim: This study was designed to examine the chemical composition and in vitro antimicrobial potential of methanolic extract of Psidium guajava Linn (Myrtaceae. Materials and Methods: The inhibitory effect of methanolic extract of P. guajava was tested against three bacterial and two fungal strains by using the paper disc diffusion method. Results: The methanolic extract exhibited antibacterial activity against E. coli with minimum inhibitory concentration, 0.78 μg/ml, minimum bactericidal concentration of 50 μg/ml, and appreciable antifungal activity with minimum inhibitory concentration of 12.5 μg/ml. Preliminary phytochemical analysis of methanolic extract revealed the presence of antimicrobial compounds such as flavonoids, steroids, and tannins, which may contribute for the antimicrobial action of P. guajava. Conclusion: The extract was found to be bacteriostatic and fungistatic in action.

In vitro biological performance of minerals substituted hydroxyapatite coating by pulsed electrodeposition method

Energy Technology Data Exchange (ETDEWEB)

Gopi, Dhanaraj, E-mail: dhanaraj_gopi@yahoo.com [Department of Chemistry, Periyar University, Salem 636 011, Tamilnadu (India); Centre for Nanoscience and Nanotechnology, Periyar University, Salem 636 011, Tamilnadu (India); Karthika, Arumugam; Nithiya, Subramani [Department of Chemistry, Periyar University, Salem 636 011, Tamilnadu (India); Kavitha, Louis [Centre for Nanoscience and Nanotechnology, Periyar University, Salem 636 011, Tamilnadu (India); Department of Physics, Periyar University, Salem 636 011, Tamilnadu (India)

2014-03-01

The present study deals with the optimization of minerals (Sr, Mg and Zn) substituted hydroxyapatite coatings (M-HAP) at different pulse on and off time (1 s, 2 s, 3 s and 4 s) by pulsed electrodeposition method. The formation of M-HAP coating was investigated using Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction studies (XRD). The morphological features and the content of Sr, Mg and Zn ions in M-HAP coated Ti–6Al–4V were investigated by high resolution scanning electron microscopy (HRSEM) and energy dispersive X-ray analysis (EDAX). The electrochemical studies were performed for M-HAP coated Ti–6Al–4V in simulated body fluid which exhibited better corrosion resistance at the prolonged pulse off time. The in vitro cell adhesion test revealed that the M-HAP coating is found appropriate for the formation of new cell growth which proves the enhanced biocompatible nature of the coating. Thus the M-HAP coating will serve as a potential candidate in orthopedic applications. - Highlights: • We successfully achieved minerals substituted HAP coatings on Ti alloy by PED method. • The M-HAP coated Ti alloy exhibited better bioresistivity in SBF. • The as-coated sample showed antimicrobial activity and better cell viability. • The in vitro test displayed the formation of new cell growth. • The M-HAP coating can serve as a better candidate in orthopedic applications.

Evaluation of five CAD/CAM materials by microstructural characterization and mechanical tests: a comparative in vitro study.

Science.gov (United States)

Sonmez, Nesrin; Gultekin, Pinar; Turp, Volkan; Akgungor, Gokhan; Sen, Deniz; Mijiritsky, Eitan

2018-01-08

Polymer infiltrated ceramics and nano-ceramic resins are the new restorative materials which have been developed in order to enhance the adverse properties of glass-matrix ceramics and resin composites. The aim of the present in vitro study was to evaluate the characteristics of various CAD/CAM materials through mechanical, microstructural, and SEM analysis. Five test groups (n = 22) were formed by using the indicated CAD/CAM blocks: VITA Enamic (VITA Zahnfabrik), Lava Ultimate (3 M ESPE), IPS e.max CAD (Ivoclar Vivadent), IPS Empress CAD (Ivoclar Vivadent), and VITA Mark II (VITA Zahnfabrik). Two specimens from each test group were used for XRD and EDS analysis. Remaining samples were divided into two subgroups (n = 10). One subgroup specimens were thermocycled (5 °C to 55 °C, 30s, 10,000 cycles) whereas the other were not. All of the specimens were evaluated in terms of flexural strength, Vickers hardness, and fracture toughness. Results were statistically analyzed using two-way ANOVA, one-way ANOVA, Tukey's HSD, and Student's t tests (α = .05). Fractured specimens were evaluated using SEM. The highest Vickers microhardness value was found for VITA Mark II (p CAD was found to have the highest flexural strength (p CAD was also higher than other tested block materials (p CAD groups. It should be realised that simulated aging process seem to affect ceramic-polymer composite materials more significantly than glass ceramics.

Growth factor release by vesicular phospholipid gels: in-vitro results and application for rotator cuff repair in a rat model.

Science.gov (United States)

Buchmann, Stefan; Sandmann, Gunther H; Walz, Lars; Reichel, Thomas; Beitzel, Knut; Wexel, Gabriele; Tian, Weiwei; Battmann, Achim; Vogt, Stephan; Winter, Gerhard; Imhoff, Andreas B

2015-04-10

Biological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model. G-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation (n = 15; control, placebo, 1 and 10 μg G-CSF/d) was performed. 6 weeks postoperatively the healing site was analyzed histologically (n = 9; H&E by modified MOVIN score/Collagen I/III) and biomechanically (n = 6). In vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 μg/d but not for G-CSF 10 μg/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 μg/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness. By use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties

In vitro inhibitory activity of essential oil vapors against Ascosphaera apis.

Science.gov (United States)

Kloucek, Pavel; Smid, Jakub; Flesar, Jaroslav; Havlik, Jaroslav; Titera, Dalibor; Rada, Vojtech; Drabek, Ondrej; Kokoska, Ladislav

2012-02-01

This work evaluates the in vitro inhibitory activity of 70 essential oils (EOs) in the vapor phase for the control of Chalkbrood disease caused by Ascosphaera apis Maassen ex Claussen (Olive et Spiltoir). Two wild strains isolated from infected honey bee colonies together with one standard collection strain were tested by the microatmosphere method. From 70 EOs, 39 exhibited an antifungal effect against A. apis standard and wild strains. The greatest antifungal action was observed for EO vapors from Armoracia rusticana, followed by Thymus vulgaris, Cymbopogon flexosus, Origanum vulgare and Allium sativum. An investigation of chemical composition by GC-MS revealed, that the most active EOs contained allyl isothiocyanate, citral, carvacrol and diallyl sulfides as the main constituents. The chemical composition plays a key role, as activities of different EOs from the same botanical species were different according to their composition.

In Vitro Antibiotic Susceptibility Studies Of Bacteria Associated With ...

African Journals Online (AJOL)

In vitro tests of the susceptibility of isolates of bacterial keratitis pathogens to antibiotics were carried out in this study. Staphylococcus aureus was the most frequently isolated organisms followed by Pseudomonas aeruginosa and Streptococcus pneumoniae. Antibiotic sensitivity testing showed a high susceptibility to ...

In Vitro Contracture Test Results and Anaesthetic Management of a Patient with Emery-Dreifuss Muscular Dystrophy for Cardiac Transplantation

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Frank Schuster

2012-01-01

Full Text Available Emery-Dreifuss muscular dystrophy (EDMD is a hereditary neuromuscular disorder characterized by slowly progressive muscle weakness, early contractures, and dilated cardiomyopathy. We reported an uneventful general anaesthesia using total intravenous anaesthesia (TIVA for cardiac transplantation in a 19-year-old woman suffering from EDMD. In vitro contracture test results of two pectoralis major muscle bundles of the patient suggest that exposition to triggering agents does not induce a pathological sarcoplasmic calcium release in the lamin A/C phenotype. However, due to the lack of evidence in the literature, we would recommend TIVA for patients with EDMD if general anaesthesia is required.

Group study of an "undercover" test for visuospatial neglect: invisible cancellation can reveal more neglect than standard cancellation.

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Wojciulik, E; Rorden, C; Clarke, K; Husain, M; Driver, J

2004-09-01

Visual neglect is a relatively common deficit after brain damage, particularly strokes. Cancellation tests provide standard clinical measures of neglect severity and deficits in daily life. A recent single-case study introduced a new variation on standard cancellation. Instead of making a visible mark on each target found, the patient made invisible marks (recorded with carbon paper underneath, for later scoring). Such invisible cancellation was found to reveal more neglect than cancellation with visible marks. Here we test the generality of this. Twenty three successive cases with suspected neglect each performed cancellation with visible or invisible marks. Neglect of contralesional targets was more pronounced with invisible marks. Indeed, about half of the patients only showed neglect in this version. For cases showing more neglect with invisible marks, stronger neglect of contralesional targets correlated with more revisits to ipsilesional targets for making additional invisible marks upon them. These results indicate that cancellation with invisible marks can reveal more neglect than standard cancellation with visible marks, while still providing a practical bedside test. Our observations may be consistent with recent proposals that demands on spatial working memory (required to keep track of previously found items only when marked invisibly) can exacerbate spatial neglect.

IN VITRO ANTIOXIDANT, TOTAL PHENOLIC AND FLAVONOID CONTENTS OF SIX ALLIUM SPECIES GROWING IN EGYPT

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Mahfouz Abdel-Gawad

2014-02-01

Full Text Available This study was designated to determine the total phenolic and flavonoid contents as well as evaluation the in vitro antioxidant activity of the defatted methanolic extracts of six Allium species growing in Egypt. Three of them are subspecies of Allium cepa L. (ssp. red onion, ssp. white onion and ssp. green onion, the other three species are Allium sativum L. (garlic, Allium porrum L. (leek and Allium kurrat L. (kurrat baladi. The results exhibited that A. cepa (ssp. red onion and A. porrum have the highest phenolic contents. On the other hand, in vitro antioxidant activity using three methods, 1, 1-diphenyl-2-picrylhydrazyl (DPPH radical, phosphomolybdate and reducing power assays revealed that A. cepa (ssp. red onion and A. porrum have high antioxidant activities. Moreover, there was positive correlation between the antioxidant activity and total phenolic contents of the tested Allium species. Therefore, the two plant species A. cepa (ssp. red onion and A. porrum were submitted to fractionation process using chloroform, ethyl acetate and n-butanol. The results showed that the ethyl acetate fractions of the two plants have high phenolic and flavonoid contents as well as have high antioxidant activities. Also, the preliminary phytochemical screening of the tested Allium species showed that A. cepa (ssp. red onion and A. porrum have high quantities of flavonoids, steroids, terpenoids and saponins.

In vitro bioactivity test of real dental implants according to ISO 23317

Czech Academy of Sciences Publication Activity Database

Kolafová, M.; Št’ovíček, J.; Strnad, J.; Zemek, Josef; Dybal, Jiří

2017-01-01

Roč. 32, č. 6 (2017), s. 1221-1230 ISSN 0882-2786 Institutional support: RVO:68378271 ; RVO:61389013 Keywords : dental implants * n vitro bioactivity * ISO 23317 * SBF * surface treatment Subject RIV: BM - Solid Matter Physics ; Magnetism; CD - Macromolecular Chemistry (UMCH-V) OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.); Polymer science (UMCH-V) Impact factor: 2.263, year: 2016

A combined metabolomic and phylogenetic study reveals putatively prebiotic effects of high molecular weight arabino-oligosaccharides when assessed by in vitro fermentation in bacterial communities derived from humans

DEFF Research Database (Denmark)

Sulek, Karolina; Vigsnæs, Louise Kristine; Schmidt, Line Rieck

2014-01-01

Prebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation...... of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied. By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, ... plant structures. Additionally, the combination of qPCR and LC–MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health....

In-vitro tensile testing machine for vibration study of fresh rabbit Achilles tendon

Science.gov (United States)

Revel, Gian M.; Scalise, Alessandro; Scalise, Lorenzo; Pianosi, Antonella

2001-10-01

A lot of people, overall athletic one suffer from tendinitis or complete rupture of the Achilles tendon. This structure becomes inflamed and damaged mainly from a variety of mechanical forces and sometimes due to metabolic problems, such as diabetes or arthritis. Over the past three decades extensive studies have been performed on the structural and mechanical properties of Achilles tendon trying to explain the constitutive equations to describe and foresee tendon behavior. Among the various mechanical parameters, the vibrational behavior is also of interest. Several investigations are performed in order to study how the Achilles tendon vibrations influence the response of the muscle proprioception and human posture. The present article describes how in vitro tensile experiments can be performed, taking into account the need to simulate physiological condition of Achilles tendon and thus approaching some opened problems in the design of the experimental set-up. A new system for evaluating tendon vibrations by non contact techniques is proposed. Preliminary simple elongation tests are made extracting the main mechanical parameters: stress and strain at different fixed stretches, in order to characterize the tissue. Finally, a vibration study is made at each pretensioned tendon level evaluating the oscillating curves caused by a small hammer.

Comparing in vitro activity of tigecycline by using the disk diffusion test, the manual microdilution method, and the VITEK 2 automated system.

Science.gov (United States)

Leal Castro, A L; Buitrago Gutierrez, G; Ovalle, V; Cortes, J A; Alvarez, C A

2010-01-01

Tigecycline is a broad spectrum antibiotic having activity against multiresistant isolates. In vitro susceptibility testing is difficult to perform with the use of traditional microbiological techniques. The aim of this study was to evaluate the disk diffusion test with three different Mueller-Hinton agar brands, and the Vitek 2 automated system in comparison with the standard broth microdilution method against 200 gram-negative isolates (Escherichia coil, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and Acinetobacter baumannii). Among Enterobacteriaceae, the Becton Dickinson agar had the lowest rate of minor (32.5%) and major errors (3.8%). No very major errors were found. For A. baumanni, the rate of minor and major errors was lower. A high rate of agreement (94%) was found between the broth microdilution method and the Vitek 2 system. Our results show that there are important differences between agars used for the disk diffusion test, and that Vitek 2 is a valid tool for susceptibility testing in clinical laboratories.

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models.

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Soyoung Kim

Full Text Available Many chemicals have been reported to induce phototoxicity. The absorbance of light energy within the sunlight range is a common characteristic of phototoxicity. The 3T3 NRU phototoxicity test (PT in 3T3 mouse skin fibroblasts has been used to identify the phototoxic potential induced by excited chemicals after exposure to ultra violet (UV. However, as phototoxicity may occur in ocular cells, it is necessary to develop a more suitable test for cornea-derived cells. In this study, we attempted to establish a new in vitro PT method in rabbit corneal cell lines (SIRC. We evaluated five ophthalmic agents, ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline, for their cytotoxic potential and in vitro phototoxicity. The results obtained using 3D human corneal models revealed that the UV-induced eye tissue toxicity by the test substances showed good correlation with those obtained using the in vitro phototoxicity test. However, the results from the 3D PT for ciprofloxacin, norfloxacin, and tetracycline in the 3D human cornea model were only partially comparable. Therefore, we suggest the SIRC cell line as a new phototoxicity test model; however, a sequential testing strategy, such as 3D PT, was also proposed to obtain relevant information for topical eye agents.

In vitro schistosomicidal and antiviral activities of Arctium lappa L. (Asteraceae) against Schistosoma mansoni and Herpes simplex virus-1.

Science.gov (United States)

Dias, Mirna Meana; Zuza, Ohana; Riani, Lorena R; de Faria Pinto, Priscila; Pinto, Pedro Luiz Silva; Silva, Marcos P; de Moraes, Josué; Ataíde, Ana Caroline Z; de Oliveira Silva, Fernanda; Cecílio, Alzira Batista; Da Silva Filho, Ademar A

2017-10-01

Schistosomiasis and herpes diseases represent serious issues to the healthcare systems, infecting a large number of people worldwide, mainly in developing countries. Arctium lappa L. (Asteraceae), known as "bardana" and "burdock", is a medicinal plant popularly used for several purposes, including as antiseptic. In this study, we evaluated the in vitro schistosomicidal and antiherpes activities of the crude extract of A. lappa, which have not yet been described. Fruits of A. lappa L. were extracted by maceration with ethanol: H 2 O (96:4 v/v) in order to obtain the hydroalcoholic extract of A. lappa (AL). In vitro schistosomicidal assays were assessed against adult worms of Schistosoma mansoni, while the in vitro antiviral activity of AL was evaluated on replication of Herpes simplex virus type-1 (HSV-1). Cell viability was measured by MTT assay, using Vero cells and chemical composition of AL was determined by qualitative UPLC-ESI-QTOF-MS analysis. UPLC-ESI-QTOF-MS analysis of AL revealed the presence of dibenzylbutyrolactone lignans, such as arctiin and arctigenin. Results showed that AL was not cytotoxic to Vero cells even when tested at 400μg/mL. qPCR results indicated a significant viral load decreased for all tested concentrations of AL (400, 50, and 3.125μg/mL), which showed similar antiviral effect to acyclovir (50μg/mL) when tested at 400μg/mL. Also, AL (400, 200, and 100μg/mL) caused 100% mortality and significantly reduction on motor activity of all adult worms of S. mansoni. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner after treatment with AL. This report provides the first evidence for the in vitro schistosomicidal and antiherpes activities of AL, opening the route to further schistosomicidal and antiviral studies with AL and their compounds, especially lignans. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Genome-wide allelotyping of a new in vitro model system reveals early events in breast cancer progression.

Science.gov (United States)

Li, Zheng; Meng, Zhen Hang; Sayeed, Aejaz; Shalaby, Refaat; Ljung, Britt-Marie; Dairkee, Shanaz H

2002-10-15

Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.

Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models

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Dominik Schreiber

2017-08-01

Full Text Available Inflammatory bowel diseases (IBD, which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1β, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml, TNF-α (10 ng/ml, and IL-1β (5 ng/ml. Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.

Cost effectiveness of ovarian reserve testing in in vitro fertilization: a Markov decision-analytic model.

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Moolenaar, Lobke M; Broekmans, Frank J M; van Disseldorp, Jeroen; Fauser, Bart C J M; Eijkemans, Marinus J C; Hompes, Peter G A; van der Veen, Fulco; Mol, Ben Willem J

2011-10-01

To compare the cost effectiveness of ovarian reserve testing in in vitro fertilization (IVF). A Markov decision model based on data from the literature and original patient data. Decision analytic framework. Computer-simulated cohort of subfertile women aged 20 to 45 years who are eligible for IVF. [1] No treatment, [2] up to three cycles of IVF limited to women under 41 years and no ovarian reserve testing, [3] up to three cycles of IVF with dose individualization of gonadotropins according to ovarian reserve, and [4] up to three cycles of IVF with ovarian reserve testing and exclusion of expected poor responders after the first cycle, with no treatment scenario as the reference scenario. Cumulative live birth over 1 year, total costs, and incremental cost-effectiveness ratios. The cumulative live birth was 9.0% in the no treatment scenario, 54.8% for scenario 2, 70.6% for scenario 3 and 51.9% for scenario 4. Absolute costs per woman for these scenarios were €0, €6,917, €6,678, and €5,892 for scenarios 1, 2, 3, and 4, respectively. Incremental cost-effectiveness ratios (ICER) for scenarios 2, 3, and 4 were €15,166, €10,837, and €13,743 per additional live birth. Sensitivity analysis showed the model to be robust over a wide range of values. Individualization of the follicle-stimulating hormone dose according to ovarian reserve is likely to be cost effective in women who are eligible for IVF, but this effectiveness needs to be confirmed in randomized clinical trials. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Development and validation of a dissolution test for diltiazem hydrochloride in immediate release capsules

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Taciane Ferreira Mendonça

2011-01-01

Full Text Available This work describes the development and validation of a dissolution test for 60 mg of diltiazem hydrochloride in immediate release capsules. The best dissolution in vitro profile was achieved using potassium phosphate buffer at pH 6.8 as the dissolution medium and paddle as the apparatus at 50 rpm. The drug concentrations in the dissolution media were determined by UV spectrophotometry and HPLC and a statistical analysis revealed that there were significant differences between HPLC and spectrophotometry. This study illustrates the importance of an official method for the dissolution test, since there is no official monograph for diltiazem hydrochloride in capsules.

Toxicity assessment of chemical contaminants;transition from in vitromethods to novel in vitro methods

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A.A. Farshad

2007-04-01

Full Text Available Exposure to occupational and environmental contaminants is a major contributor to human health problems. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there areapproximately 80, 000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from ethical, economical and scientific perspectives. Therefore, increasing the number of available industrial chemicals andnew products has created a demand for alternatives to animal methods for better safety evaluation. Recent toxicity studies have demonstrated that in vitro methods are capable of rapidly providing toxicity information. In this review, current toxicity test methods for risk evaluation of industrial chemical contaminants are presented. To evaluate the potential applications of  more recent test methods developed for toxicity testing of chemical contaminants are discussed. Although  to be considered more broadly for risk assessment of human chemical exposures. In vitro methods,in vitro toxicology methods cannot exactly mimic the biodynamics of the whole body, in vitro  relationships (QSARs and physiologically based toxicokinetic (PBTK models have a potentialtest systems in combination with the knowledge of quantitative structure activity.

In Vitro Evaluation of the Sensitivity of a Hyaluronic Acid PEG Cross-Linked to Bovine Testes Hyaluronidase

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Nicola Zerbinati

2018-01-01

Full Text Available Neauvia Intense is biocompatible, injectable hyaluronic acid (HA filler PEG cross-linked for facial soft-tissue augmentation that provides volume to tissues. The aim of the present study is to evaluate the sensitivity of Neauvia Intense in hyaluronidase from bovine testes in a time-course analysis. The test is based on the colourimetric determination of the N-acetyl – D - glucosamine (NAG released by the hyaluronidase in standardised conditions. The in vitro conditions involve the treatment of Neauvia Intense with a known concentration of the enzyme (6080U/ml. The NAG content was determined at different times to assess the kinetics of the degradation (1h, 3h, 6h, 24h, 48h, 72h, 120h, and 168h; the Ehrlich’s reagent was used for the colourimetric quantification, by the method described by Reissing and colleagues. The intensity of the violet colour developed after the chemical reaction was proportional to the NAG present in each sample. A microplate reader at 585 nm read the absorbance. The amount of NAG released by the product was proportional to the time of incubation with bovine hyaluronidase, reaching a plateau after 168 hours.

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

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Sylvia Moeckel

Full Text Available High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a to enrich specimens for brain tumor initiating cells and (b to confront cells with a therapeutic agent before expression profiling.As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.

A Tox21 Approach to Altered Epigenetic Landscapes: Assessing Epigenetic Toxicity Pathways Leading to Altered Gene Expression and Oncogenic Transformation In Vitro

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Craig L. Parfett

2017-06-01

Full Text Available An emerging vision for toxicity testing in the 21st century foresees in vitro assays assuming the leading role in testing for chemical hazards, including testing for carcinogenicity. Toxicity will be determined by monitoring key steps in functionally validated molecular pathways, using tests designed to reveal chemically-induced perturbations that lead to adverse phenotypic endpoints in cultured human cells. Risk assessments would subsequently be derived from the causal in vitro endpoints and concentration vs. effect data extrapolated to human in vivo concentrations. Much direct experimental evidence now shows that disruption of epigenetic processes by chemicals is a carcinogenic mode of action that leads to altered gene functions playing causal roles in cancer initiation and progression. In assessing chemical safety, it would therefore be advantageous to consider an emerging class of carcinogens, the epigenotoxicants, with the ability to change chromatin and/or DNA marks by direct or indirect effects on the activities of enzymes (writers, erasers/editors, remodelers and readers that convey the epigenetic information. Evidence is reviewed supporting a strategy for in vitro hazard identification of carcinogens that induce toxicity through disturbance of functional epigenetic pathways in human somatic cells, leading to inactivated tumour suppressor genes and carcinogenesis. In the context of human cell transformation models, these in vitro pathway measurements ensure high biological relevance to the apical endpoint of cancer. Four causal mechanisms participating in pathways to persistent epigenetic gene silencing were considered: covalent histone modification, nucleosome remodeling, non-coding RNA interaction and DNA methylation. Within these four interacting mechanisms, 25 epigenetic toxicity pathway components (SET1, MLL1, KDM5, G9A, SUV39H1, SETDB1, EZH2, JMJD3, CBX7, CBX8, BMI, SUZ12, HP1, MPP8, DNMT1, DNMT3A, DNMT3B, TET1, MeCP2, SETDB2, BAZ2

Home-Use Tests - Cholesterol

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... Medical Procedures In Vitro Diagnostics Home Use Tests Cholesterol Share Tweet Linkedin Pin it More sharing options ... a home-use test kit to measure total cholesterol. What cholesterol is: Cholesterol is a fat (lipid) ...

The 11th quality control survey for radioisotopes in vitro tests in Japan, 1989

Energy Technology Data Exchange (ETDEWEB)

1990-10-01

This report presents the results of the 11th quality control nationwide survey. Of 730 facilities performing radioisotopes in vitro tests in November 1989, 422 facilities (60.5%) participated in the present survey. The following 23 items were examined: adrenocorticotropic hormone (ACTH), albumin, carbohydrate antigen 125 (CA 125), carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA), calcitonin, cortisol, estradiol, ferritin, free thyroxine (FT{sub 4}), follicle stimulating hormone (FSH), gastrine, cholylglycine, glucagon, insulin, anti-DNA antibody, luteinizing hormone (LH), neuron specific enolase (NSE), parathyroid hormone (PTH), squamous cell carcinoma associated antigen (SCC), thyroxine (T{sub 4}), thyroxine binding globulin (TBG), and antithyroid stimulating hormone (TSH) receptor antibody. 'Within kit variation' between facilities showed large coefficient of variation for ACTH, CA125, CEA, estradiol, ferritin, FSH, glucagon, anti-DNA antibody, LH, PTH, and TSH receptor antibody. Both 'within kit variation' and 'between kit variation' showed small coefficient of variation for cortisol, free T{sub 4}, NSE, SCC, T{sub 4}, and TBG. The present survey was characterized by using immunoradiometric assay (IRMA) and non-isotope techniques, as well as radioimmunoassay. Kits for IRMA greatly varied from facility to facility. (N.K.).

In Vitro Propagation and Conservation of Bacopa monnieri L.

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Sharma, Neelam; Singh, Rakesh; Pandey, Ruchira

2016-01-01

Bacopa monnieri L. (common name brahmi) is a traditional and renowned Indian medicinal plant with high commercial value for its memory revitalizer potential. Demand for this herb has further escalated due to popularization of various brahmi-based drugs coupled with reported anticancer property. Insufficient seed availability and problems associated with seed propagation including short seed viability are the major constraints of seed conservation in the gene banks. In vitro clonal propagation, a prerequisite for in vitro conservation by enhanced axillary branching was standardized. We have developed a simple, single step protocol for in vitro establishment, propagation and medium-term conservation of B. monnieri. Single node explants, cultured on Murashige and Skoog's medium supplemented with BA (0.2 mg/L), exhibited shoot proliferation without callus formation. Rooting was achieved on the same medium. The in vitro raised plants were successfully transferred to soil with ~80 % survival. On the same medium, shoots could also be conserved for 12 months with high survival and genetic stability was maintained as revealed by molecular markers. The protocol optimized in the present study has been applied for culture establishment, shoot multiplication and medium-term conservation of several Bacopa germplasm, procured from different agro-ecological regions of India.

Strontium substituted hydroxyapatites: Synthesis and determination of their structural properties, in vitro and in vivo performance

Energy Technology Data Exchange (ETDEWEB)

Kaygili, Omer, E-mail: okaygili@firat.edu.tr [Department of Physics, Faculty of Science, Firat University, 23119 Elazig (Turkey); Keser, Serhat [Department of Chemistry, Faculty of Science, Firat University, 23119 Elazig (Turkey); Kom, Mustafa [Department of Surgery, Faculty of Veterinary Medicine, Firat University, 23119 Elazig (Turkey); Eroksuz, Yesari [Department of Pathology, Faculty of Veterinary Medicine, Firat University, 23119 Elazig (Turkey); Dorozhkin, Sergey V. [Kudrinskaja square 1-155, Moscow 123242 (Russian Federation); Ates, Tankut [Department of Physics, Faculty of Science, Firat University, 23119 Elazig (Turkey); Ozercan, Ibrahim H. [Department of Pathology, School of Medicine, Firat University, 23119 Elazig (Turkey); Tatar, Cengiz; Yakuphanoglu, Fahrettin [Department of Physics, Faculty of Science, Firat University, 23119 Elazig (Turkey)

2015-10-01

The objective of this study is to present a detailed report related to the synthesis and characterization of strontium substituted hydroxyapatites. Based on this purpose, hydroxyapatite (HAp) bioceramics with different amounts of strontium (e.g., 0, 0.45, 0.90, 1.35, 1.80 and 2.25 at.%) were prepared using a sol–gel method. The effects of Sr substitution on the structural properties and biocompatibility of the samples were studied by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) techniques, in vitro and in vivo tests. All the samples composed of the nanoparticles ranging from 21 to 27 nm. The presence of Sr at low levels influenced the crystal size, crystallinity degree, lattice parameters and volume of the unit cell of the HAp. Both in vitro conditions and soaking period in simulated body fluid (SBF) significantly affected these properties. Especially, the (Ca + Sr)/P molar ratio gradually decreases with increasing soaking period in SBF. Animal experiments revealed the bone formation and osseointegration for all samples, and as compared with other groups, more reasonable, were observed for the sample with the lowest Sr content. - Highlights: • Sr content affects the structural properties of hydroxyapatite. • Bone formation and osseointegration are observed for all the samples. • In vitro conditions cause a significant change in the (Ca + Sr)/P ratio.

Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements

International Nuclear Information System (INIS)

Liu, Hsiuchih; Chi, Chinwen; Liu, Tsungyun; Liu, Lianghui; Luh, Wenming; Hsieh, Changhuain; Wu, Wenguey

1991-01-01

Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations of in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue

10 CFR 32.71 - Manufacture and distribution of byproduct material for certain in vitro clinical or laboratory...

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2010-01-01

... certain in vitro clinical or laboratory testing under general license. 32.71 Section 32.71 Energy NUCLEAR... certain in vitro clinical or laboratory testing under general license. An application for a specific... only by physicians, veterinarians in the practice of veterinary medicine, clinical laboratories or...

COMPARISON OF BIOACTIVITY IN VITRO OF GLASS AND GLASS CERAMIC MATERIALS DURING SOAKING IN SBF AND DMEM MEDIUM

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GABRIELA LUTIŠANOVÁ

2011-09-01

Full Text Available This paper investigated the surface reactivity of two sets of glasses and glass ceramic materials belonging to the Li2O–SiO2–CaO–P2O5–CaF2 system. The in vitro bioactivity of coatings was evaluated using simulated body fluid (SBF and Dulbecco’s Modified Eagle’s Medium (DMEM soaking test in static regime for up to 28 days at 36.5°C in microincubator. The surface structure changes were examined by scanning electron microscopy (SEM and electron probe micro-analyzer (EPMA methods. The functional groups of the silicate and phosphates were identified by infrared spectroscopy (IR. The crystal phases of the glasses and glass ceramics were identified by X-ray diffraction analysis (XRD. The results suggest the bioactivity behavior for all compositions of glasses as well as glass ceramic samples after 28 days in the SBF and DMEM medium. The surface characterization and in vitro tests revealed a few variations in the reactivity of the different glasses and glass ceramic samples in their pristine form. The best results show the samples of glass and glass ceramic samples with higher content of fluorapatite (FA. The use of the acellular culture medium DMEM resulted in a delay at the start of precipitation.

In vitro bioactivity of glass-ceramic/fibroin composites

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Lachezar Radev

2017-06-01

Full Text Available Bioactive composite materials were prepared by mixing 20 wt.% of silk fibroin (SF and 80 wt.% of glassceramics from CaO-SiO2-P2O5-MgO system. In vitro bioactivity of the prepared composites was evaluated in 1.5 simulated body fluid (1.5 SBF in static conditions. The obtained samples before and after in vitro tests were characterized by X-ray diffraction (XRD analysis, Fourier transform infrared spectroscopy (FTIR, and X-ray photoelectron spectroscopy (XPS. The changes in 1.5 SBF solutions after soaking the samples were evaluated by inductively coupled plasma atomic emission spectroscopy (ICP-AES. MG63 osteosarcoma cells were used for the biological experiments. The obtained experimental data proved that the synthesized composites exhibit excellent in vitro bioactivity.

Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests

International Nuclear Information System (INIS)

Benavides, Tomas; Martinez, Veronica; Mitjans, Montserrat; Infante, Maria Rosa; Moran, Carmen; Clapes, Pere; Clothier, Richard; Vinardell, Maria Pilar

2004-01-01

The ultraviolet-A radiation damage effects on skin and eyes will be increased by phototoxic compounds which could be present in pharmaceutical or cosmetic formulations. Great efforts have been made in the last years to find surfactants to replace those with phototoxic potential in commercial use. Series of different in vitro models for phototoxicity, included to validated neutral red uptake (NRU) 3T3 phototoxicity assay are useful screening tools. The phototoxic effects of a novel family of glycerol amino acid-based surfactant compounds were examined via these assays. Human red blood cells and two immortalised cell lines, murine fibroblast cell line 3T3, and one human keratinocyte cell line, HaCaT, were the in vitro models employed to predict potential photoirritation. The phototoxic end-points assessed were hemolysis (human red blood cell test) and resazurin transformation to resorufin and NRU in cell culture methods. The results suggest that no phototoxic effects by any new amino acid derived-surfactants, could be identified

Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution

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María-Rocío Meini

2014-07-01

Full Text Available The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-β-lactamases.The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict

Test systems to identify reproductive toxicants.

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Riecke, K; Stahlmann, R

2000-09-01

Experience with drugs and other xenobiotics indicates that both animal testing and epidemiological studies are necessary to provide adequate data for an estimation of risks that might be associated with exposure to a chemical substance. In this review, the pros and cons of test systems for reproductive toxicity are discussed. Usually, several studies are performed to cover the different phases of the reproductive cycle. In the preclinical development of drugs, the three so-called 'segment testing protocols' have been used for several decades now. More recently, new testing concepts have been accepted internationally which include more flexibility in implementation. Several examples of compounds with the potential for reproductive toxicity are presented in more detail in a discussion of some pitfalls of the tests for fertility (phthalates and fluoroquinolones), teratogenicity (acyclovir and protease inhibitors) and postnatal developmental toxicity (fluoroquinolones). In addition, important aspects of kinetics and metabolism as a prerequisite for a rational interpretation of results from toxicological studies are briefly discussed. In vitro assays are useful for supplementing the routinely used in vivo approaches or for studying an expected or defined effect, but they are not suitable for revealing an unknown effect of a chemical on the complex reproductive process.

Density-dependence of functional spiking networks in vitro

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Ham, Michael I [Los Alamos National Laboratory; Gintautuas, Vadas [Los Alamos National Laboratory; Rodriguez, Marko A [Los Alamos National Laboratory; Bettencourt, Luis M A [Los Alamos National Laboratory; Bennett, Ryan [UNIV OF NORTH TEXAS; Santa Maria, Cara L [UNIV OF NORTH TEXAS

2008-01-01

During development, the mammalian brain differentiates into specialized regions with unique functional abilities. While many factors contribute to this functional specialization, we explore the effect neuronal density can have on neuronal interactions. Two types of networks, dense (50,000 neurons and glia support cells) and sparse (12,000 neurons and glia support cells), are studied. A competitive first response model is applied to construct activation graphs that represent pairwise neuronal interactions. By observing the evolution of these graphs during development in vitro we observe that dense networks form activation connections earlier than sparse networks, and that link-!llltropy analysis of the resulting dense activation graphs reveals that balanced directional connections dominate. Information theoretic measures reveal in addition that early functional information interactions (of order 3) are synergetic in both dense and sparse networks. However, during development in vitro, such interactions become redundant in dense, but not sparse networks. Large values of activation graph link-entropy correlate strongly with redundant ensembles observed in the dense networks. Results demonstrate differences between dense and sparse networks in terms of informational groups, pairwise relationships, and activation graphs. These differences suggest that variations in cell density may result in different functional specialization of nervous system tissue also in vivo.

Effect of combinations of marketed human anthelmintic drugs against Trichuris muris in vitro and in vivo

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Keiser Jennifer

2012-12-01

Full Text Available Abstract Background Soil-transmitted helminth (STH infections are responsible for a huge public health burden, however treatment options are limited. The discovery and development of novel efficacious drugs or drug combinations for the treatment of STH infections therefore has a high research priority. Methods We studied drug combination effects using the main standard anthelmintics, albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin in the Trichuris muris model. Drug combinations were first tested in vitro and additive and synergistic combinations investigated further in vivo in female mice using ratios based on the ED50 of the respective drugs. Results In vitro all 10 combinations of the standard anthelmintics tested against T. muris revealed synergistic behavior. We identified three drug combinations in vivo as strongly synergistic, namely mebendazole-ivermectin (Combination index (CI=0.16, mebendazole-levamisole (CI=0.17 and albendazole-mebendazole (CI=0.23. For albendazole-ivermectin, moderate synergism was observed (CI=0.81 and for albendazole-levamisole a nearly additive effect was documented (CI=0.93 in vivo. Five combinations (albendazole-pyrantel pamoate, mebendazole-pyrantel pamoate, levamisole-pyrantel pamoate, levamisole-ivermectin and pyrantel pamoate-ivermectin were antagonistic in vivo. Conclusion Our results strengthen the evidence that combination chemotherapy might play a role in the treatment of Trichuris infections. Albendazole-mebendazole should be studied in greater detail in preclinical studies.

In vitro study of manganese-doped bioactive glasses for bone regeneration.

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Miola, Marta; Brovarone, Chiara Vitale; Maina, Giovanni; Rossi, Federica; Bergandi, Loredana; Ghigo, Dario; Saracino, Silvia; Maggiora, Marina; Canuto, Rosa Angela; Muzio, Giuliana; Vernè, Enrica

2014-05-01

A glass belonging to the system SiO2-P2O5-CaO-MgO-Na2O-K2O was modified by introducing two different amounts of manganese oxide (MnO). Mn-doped glasses were prepared by melt and quenching technique and characterized by means of X-ray diffraction (XRD), scanning electron microscopy (SEM) observation and energy dispersion spectrometry (EDS) analysis. In vitro bioactivity test in simulated body fluid (SBF) showed a slight decrease in the reactivity kinetics of Mn-doped glasses compared to the glass used as control; however the glasses maintained a good degree of bioactivity. Mn-leaching test in SBF and minimum essential medium (MEM) revealed fluctuating trends probably due to a re-precipitation of Mn compounds during the bioactivity process. Cellular tests showed that all the Mn-doped glasses, up to a concentration of 50 μg/cm(2) (μg of glass powders/cm(2) of cell monolayer), did not produce cytotoxic effects on human MG-63 osteoblasts cultured for up to 5 days. Finally, biocompatibility tests demonstrated a good osteoblast proliferation and spreading on Mn-doped glasses and most of all that the Mn-doping can promote the expression of alkaline phosphatase (ALP) and some bone morphogenetic proteins (BMPs). Copyright © 2014 Elsevier B.V. All rights reserved.

In-vitro mutation breeding technology in maize

International Nuclear Information System (INIS)

Nesticky, M.

1988-08-01

Gamma-irradiation and in-vitro culture, separately or combined, as a tool for inducing mutation in maize were evaluated. This type of research has been hampered in maize because (i) maize is a cross pollinating crop and highly heterozygous and (ii) embryogenesis and plant regeneration of plants from in-vitro culture have been difficult. In the present study, carefully designed and elaborated experiments were conducted using an inbred line CH1 31 which is capable of somatic embryogenesis for the subject of mutagenesis and another line Bu 8Ro 2 for the test cross partner. Results showed: 1) Both the regeneration of plants from in-vitro culture and gamma-irradiation induced a similar spectrum of morphological variation. Although the variation with somaclones was more frequent that radiation induced mutations under the conditions used, combination of explant irradiation and in-vitro culture gave the highest frequencies of genetic variation. 2) Some of the mutations in quantitative characters can be recogned in heterozygous state. 3) Mutation can cause variation in combining ability (extent of heterosis). 4) Efficiency at embryogenesis differs with genotypes of maize. 3 refs, 11 figs, 4 tabs

An in vitro assay for compounds toxic to rumen protozoa

International Nuclear Information System (INIS)

Campbell, A.J.; Cumming, G.J.; Graham, C.A.; Leng, R.A.

1982-01-01

The viability of protozoa in whole rumen fluid was assessed by measuring the incorporation of Me- 14 C-choline in vitro. The use of the technique as an assay for testing antiprotozoal agents was evaluated with a variety of surfactant detergents which have previously been shown to have antiprotozoal activity in vivo. A good correlation was obtained between the potency of these compounds in vitro and in vivo. (auth)

The use and interpretation of in vitro data in regulatory toxicology: cosmetics, toiletries and household products.

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Indans, Ian

2002-02-28

There is currently a drive to eliminate animal testing for cosmetics, toiletries and household products; indeed, the European Union Cosmetics Directive aims to prohibit the use of experimental animals for the testing of finished cosmetic products after 2002. At present, national prohibitions are in place in the UK, Germany, Austria and the Netherlands, for the testing of finished cosmetic products and cosmetic ingredients. In the USA animal testing for certain types of finished products is mandatory. Against this background, the currently available regulatory in vitro tests comprise methods for eye irritation, skin corrosivity, genotoxicity, dermal penetration and photoirritation. The draft updates to the Organisation for Economic Co-operation and Development guidelines for eye and skin irritation advocate the use of in vitro or ex vivo methods prior to the commencement of animal studies. At present, testing for these endpoints cannot be completed in vitro, but potentially corrosive substances and products can be classified without the need for animal studies. Regulatory genotoxicity testing can be completed using only in vitro methods, provided that a clear negative outcome is obtained for each test. Data from dermal penetration studies may be used to refine risk assessments. Current developments in areas such as skin sensitisation and skin irritation promise that in the reasonably near future such information may be generated without the use of animals.

Preparation, mechanical properties and in vitro cytocompatibility of multi-walled carbon nanotubes/poly(etheretherketone) nanocomposites.

Science.gov (United States)

Cao, Jianfei; Lu, Yue; Chen, Hechun; Zhang, Lifang; Xiong, Chengdong

2018-03-01

Desired bone repair material must have excellent biocompatibility and high bioactivity. Moreover, mechanical properties of biomaterial should be equivalent to those of human bones. For developing an alternative biocomposite for load-bearing orthopedic application, combination of bioactive fillers with polymer matrix is a feasible approach. In this study, a series of multi-walled carbon nanotubes (MWCNTs)/poly(etheretherketone) (PEEK) bioactive nanocomposites were prepared by a novel coprecipitation-compounding and injection-molding process. Scanning electron microscope (SEM) images revealed that MWCNTs were adsorbed on the surface of PEEK particles during the coprecipitation-compounding process and dispersed homogeneously in the nanocomposite because the conjugated PEEK polymers stabilized MWCNTs by forming strong π-π stack interactions. The mechanical testing revealed that mechanical performance of PEEK was significantly improved by adding MWCNTs (2-8 wt%) and the experimental values obtained were close to or higher than that of human cortical bone. In addition, incorporation of MWCNTs into PEEK matrix also enhanced the roughness and hydrophilicity of the nanocomposite surface. In vitro cytocompatibility tests demonstrated that the MWCNTs/PEEK nanocomposite was in favor of cell adhesion and proliferation of MC3T3-E1 osteoblast cells, exhibiting excellent cytocompatibility and biocompatibility. Thus, this MWCNTs/PEEK nanocomposite may be used as a promising bone repair material in orthopedic implants application.

How to assess the mutagenic potential of cosmetic products without animal tests?

Science.gov (United States)

Speit, Günter

2009-08-01

Animal experiments (in vivo tests) currently play a key role in genotoxicity testing. Results from in vivo tests are, in many cases, decisive for the assessment of a mutagenic potential of a test compound. The Seventh Amendment to the European Cosmetics Directive will, however, ban the European marketing of cosmetic/personal care products that contain ingredients that have been tested in animal experiments. If genotoxicity testing is solely based on the currently established in vitro tests, the attrition rate for chemicals used in cosmetic products will greatly increase due to irrelevant positive in vitro test results. There is urgent need for new and/or improved in vitro genotoxicity tests and for modified test strategies. Test strategies should consider all available information on chemistry of the test substance/the chemical class (e.g. SAR, metabolic activation and dermal adsorption). Test protocols for in vitro genotoxicity tests should be sensitive and robust enough to ensure that negative results can be accepted with confidence. It should be excluded that positive in vitro test results are due to high cytotoxicity or secondary genotoxic effects which may be thresholded and/or only occur under in vitro test conditions. Consequently, further research is needed to establish the nature of thresholds in in vitro assays and to determine the potential for incorporation of mode of action data into future risk assessments. New/improved tests have to be established and validated, considering the use of (metabolically competent) primary (skin) cells, 3D skin models and cells with defined capacity for metabolic activation (e.g. genetically engineered cell lines). The sensitivity and specificity of new and improved genotoxicity tests has to be determined by testing a battery of genotoxic and non-genotoxic chemicals. New or adapted international guidelines will be needed for these tests. The establishment of such a new genotoxicity testing strategy will take time and the

Radiation response in vitro of fibroblasts from a Fanconi anemia patient with marked clinical radiosensitivity

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Djuzenova, C.; Flentje, M. [Dept. of Radiotherapy, Univ. of Wuerzburg, Wuerzburg (Germany); Plowman, P.N. [Radiotherapy/Clinical Oncology, St. Bartholomew' s Hospital, London (United Kingdom)

2004-12-01

Background: fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay. Material and methods: in view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining. Results: the Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells. Conclusion: the increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA

Radiation response in vitro of fibroblasts from a Fanconi anemia patient with marked clinical radiosensitivity

International Nuclear Information System (INIS)

Djuzenova, C.; Flentje, M.; Plowman, P.N.

2004-01-01

Background: fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay. Material and methods: in view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining. Results: the Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells. Conclusion: the increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA

DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration.

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Sina Haas

Full Text Available Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

Science.gov (United States)

Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

1997-06-01

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

In Vitro Comparison of Dynesys, PEEK, and Titanium Constructs in the Lumbar Spine

Science.gov (United States)

Yeager, Matthew S.; Cook, Daniel J.; Cheng, Boyle C.

2015-01-01

Introduction. Pedicle based posterior dynamic stabilization systems aim to stabilize the pathologic spine while also allowing sufficient motion to mitigate adjacent level effects. Two flexible constructs that have been proposed to act in such a manner, the Dynesys Dynamic Stabilization System and PEEK rod, have yet to be directly compared in vitro to a rigid Titanium rod. Methods. Human lumbar specimens were tested in flexion extension, lateral bending, and axial torsion to evaluate the following conditions at L4-L5: Intact, Dynesys, PEEK rod, Titanium rod, and Destabilized. Intervertebral range of motion, interpedicular travel, and interpedicular displacement metrics were evaluated from 3rd-cycle data using an optoelectric tracking system. Results. Statistically significant decreases in ROM compared to Intact and Destabilized conditions were detected for the instrumented conditions during flexion extension and lateral bending. AT ROM was significantly less than Destabilized but not the Intact condition. Similar trends were found for interpedicular displacement in all modes of loading; however, interpedicular travel trends were less consistent. More importantly, no metrics under any mode of loading revealed significant differences between Dynesys, PEEK, and Titanium. Conclusion. The results of this study support previous findings that Dynesys and PEEK constructs behave similarly to a Titanium rod in vitro. PMID:26366303

Environmental Legionella spp. collected in urban test sites of South East Queensland, Australia, are virulent to human macrophages in vitro.

Science.gov (United States)

Lawrence, Amba; Eglezos, Sofroni; Huston, Wilhelmina

2016-01-01

Legionellae are frequent contaminants of potable water supplies, resulting in sporadic infections and occasional outbreaks. Isolates of Legionella were collected from urban test sites within South East Queensland and evaluated for their virulence potential in vitro. Two strains (from the species Legionella londiniensis and Legionella quinlivanii) were demonstrated to have the ability to infect human macrophages, while a strain from the species Legionella anisa did not maintain an infection over the same time course. This suggests that the spectrum of urban environmentally associated Legionella with potential to cause human disease might be greater than currently considered. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

In-vivo corneal pulsation in relation to in-vivo intraocular pressure and corneal biomechanics assessed in-vitro. An animal pilot study.

Science.gov (United States)

Rogala, Maja M; Danielewska, Monika E; Antończyk, Agnieszka; Kiełbowicz, Zdzisław; Rogowska, Marta E; Kozuń, Marta; Detyna, Jerzy; Iskander, D Robert

2017-09-01

The aim was to ascertain whether the characteristics of the corneal pulse (CP) measured in-vivo in a rabbit eye change after short-term artificial increase of intraocular pressure (IOP) and whether they correlate with corneal biomechanics assessed in-vitro. Eight New Zealand white rabbits were included in this study and were anesthetized. In-vivo experiments included simultaneous measurements of the CP signal, registered with a non-contact method, IOP, intra-arterial blood pressure, and blood pulse (BPL), at the baseline and short-term elevated IOP. Afterwards, thickness of post-mortem corneas was determined and then uniaxial tensile tests were conducted leading to estimates of their Young's modulus (E). At the baseline IOP, backward stepwise regression analyses were performed in which successively the ocular biomechanical, biometric and cardiovascular predictors were separately taken into account. Results of the analysis revealed that the 3rd CP harmonic can be statistically significantly predicted by E and central corneal thickness (Models: R 2  = 0.662, p biomechanics in-vitro was confirmed. In particular, spectral analysis revealed that higher amplitude and power of the 3rd CP harmonic indicates higher corneal stiffness, while the 1st CP harmonic correlates positively with the corresponding harmonic of the BPL signal. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antitumor Activity of Kielmeyera Coriacea Leaf Constituents in Experimental Melanoma, Tested in Vitro and in Vivo in Syngeneic Mice

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Carlos Rogério Figueiredo

2014-10-01

Full Text Available Purpose: The antitumor activity of Kielmeyera coriacea (Clusiaceae, a medicinal plant used in the treatment of parasitic, as well as fungal and bacterial infections by the Brazilian Cerrado population, was investigated. Methods: A chloroform extract (CE of K. coriacea was tested in the murine melanoma cell line (B16F10-Nex2 and a panel of human tumor cell lines. Tumor cell migration was determined by the wound-healing assay and the in vivo antitumor activity of CE was investigated in a melanoma cell metastatic model. 1H NMR and GC/MS were used to determine CE chemical composition. Results: We found that CE exhibited strong cytotoxic activity against murine melanoma cells and a panel of human tumor cell lines in vitro. CE also inhibited growth of B16F10-Nex2 cells at sub lethal concentrations, inducing cell cycle arrest at S phase, and inhibition of tumor cell migration. Most importantly, administration of CE significantly reduced the number of melanoma metastatic nodules in vivo. Chemical analysis of CE indicated the presence of the long chain fatty compounds, 1-eicosanol, 1-docosanol, and 2-nonadecanone as main constituents. Conclusion: These results indicate that K. coriacea is a promising medicinal plant in cancer therapy exhibiting antitumor activity both in vitro and in vivo against different tumor cell lines.

Genetic deletion of cdc42 reveals a crucial role for astrocyte recruitment to the injury site in vitro and in vivo

DEFF Research Database (Denmark)

Robel, Stefanie; Bardehle, Sophia; Lepier, Alexandra

2011-01-01

signals, the small RhoGTPase Cdc42, selectively in mouse astrocytes in vitro and in vivo. We used an in vitro scratch assay as a minimal wounding model and found that astrocytes lacking Cdc42 (Cdc42Δ) were still able to form protrusions, although in a nonoriented way. Consequently, they failed to migrate...... in a directed manner toward the scratch. When animals were injured in vivo through a stab wound, Cdc42Δ astrocytes developed protrusions properly oriented toward the lesion, but the number of astrocytes recruited to the lesion site was significantly reduced. Surprisingly, however, lesions in Cdc42Δ animals...

Design, Fabrication, and In Vitro Testing of an Anti-biofouling Glaucoma Micro-shunt.

Science.gov (United States)

Harake, Ryan S; Ding, Yuzhe; Brown, J David; Pan, Tingrui

2015-10-01

Glaucoma, one of the leading causes of irreversible blindness, is a progressive neurodegenerative disease. Chronic elevated intraocular pressure (IOP), a prime risk factor for glaucoma, can be treated by aqueous shunts, implantable devices, which reduce IOP in glaucoma patients by providing alternative aqueous outflow pathways. Although initially effective at delaying glaucoma progression, contemporary aqueous shunts often lead to numerous complications and only 50% of implanted devices remain functional after 5 years. In this work, we introduce a novel micro-device which provides an innovative platform for IOP reduction in glaucoma patients. The device design features an array of parallel micro-channels to provide precision aqueous outflow resistance control. Additionally, the device's microfluidic channels are composed of a unique combination of polyethylene glycol materials in order to provide enhanced biocompatibility and resistance to problematic channel clogging from biofouling of aqueous proteins. The microfabrication process employed to produce the devices results in additional advantages such as enhanced device uniformity and increased manufacturing throughput. Surface characterization experimental results show the device's surfaces exhibit significantly less non-specific protein adsorption compared to traditional implant materials. Results of in vitro flow experiments verify the device's ability to provide aqueous resistance control, continuous long-term stability through 10-day protein flow testing, and safety from risk of infection due to bacterial ingression.

Comparative In Vitro Efficacy of Doripenem and Imipenem Against Multi-Drug Resistant Pseudomonas aeruginosa.

Science.gov (United States)

Wali, Nadia; Mirza, Irfan Ali

2016-04-01

To compare the in vitro efficacy of doripenem and imipenem against multi-drug resistant (MDR) Pseudomonas aeruginosa from various clinical specimens. Descriptive cross-sectional study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from November 2012 to November 2013. MDR Pseudomonas aeruginosa isolates from various clinical samples were included in the study. Susceptibility of Pseudomonas aeruginosa against doripenem and imipenem was performed by E-test strip and agar dilution methods. The results were interpreted as recommended by Clinical Laboratory Standard Institute (CLSI) guidelines. The maximum number of Pseudomonas aeruginosa were isolated from pure pus and pus swabs. In vitro efficacy of doripenem was found to be more effective as compared to imipenem against MDR Pseudomonas aeruginosa with both E-test strip and agar dilution methods. Overall, p-values of 0.014 and 0.037 were observed when susceptibility patterns of doripenem and imipenem were evaluated with E-test strip and agar dilution methods. In vitro efficacy of doripenem was found to be better against MDR Pseudomonas aeruginosaas compared to imipenem when tested by both E-test and agar dilution methods.

Development of an in vitro laboratory manual for nuclear medicine technology students

International Nuclear Information System (INIS)

Meyers, A.

1989-01-01

This study evaluated existing in vitro education materials in qualitative and quantitative parameters that currently exist to educate potential clinicians of nationally accredited nuclear medicine programs. A review of over 300 articles, texts, and manuals pertaining to in vitro nuclear medicine procedures clearly demonstrated that no in vitro laboratory manual for undergraduate students presently exited. Every nuclear medicine program director in the United States was surveyed. They were asked for their overall philosophy in terms of developing an in vitro manual and requested to evaluate the significant of 22 general principles/concepts and 34 specific laboratory testing procedures. From the response to the survey, an in vitro nuclear medicine manual was created and appended to the study. The manual consists of lecture and study material, chapter reviews, and laboratory assignments and exercises

A simple way for targeted delivery of an antibiotic: In vitro evaluation of a nanoclay-based composite.

Directory of Open Access Journals (Sweden)

Leslie Valdés

Full Text Available The sodium-modified form of fluorohectorite nanoclay (NaFh is introduced as a potential drug carrier, demonstrating its ability for the controlled release of the broad-spectrum antibiotic Ciprofloxacin through in vitro tests. The new clay-drug composite is designed to target the local infections in the large intestine, where it delivers most of the incorporated drug thanks to its pH-sensitive behavior. The composite has been conceived to avoid the use of coating technology and to decrease the side-effects commonly associated to the burst-release of the ciprofloxacin at the stomach level. NaFh was obtained from lithium-fluorohectorite by ion exchange, and its lack of toxicity was demonstrated by in vivo studies. Ciprofloxacin hydrochloride (Cipro was encapsulated into the clay at different values of the pH, drug initial concentration, temperature and time. Systematic studies by X-ray diffraction (XRD, infrared and visible spectrophotometry (FT-IR and UV-vis, and thermal analysis (TGA indicated that the NaFh host exhibits a high encapsulation efficiency for Cipro, which reaches a 90% of the initial Cipro in solution at 65 oC, with initial concentration of drug in solution of 1.36 x 10-2 mol L-1 at acid pH. XRD revealed that a true intercalation of Cipro takes place between clay layers. TG showed an increased thermal stability of the drug when intercalated into the clay, as compared to the "free" Cipro. IR suggested a strong clay-Cipro interaction via ketone group, as well as the establishment of hydrogen bonds between the two materials. In vitro drug release tests revealed that NaFh is a potentially efficient carrier to deliver Cipro in the large intestine, where the release process is mediated by more than just one mechanism.

Assessment of flavonoids contents and in vitro antioxidant activity of Launaea procumbens

Directory of Open Access Journals (Sweden)

Khan Rahmat

2012-05-01

Full Text Available Abstract Background Launaea procumbens (LP has been used as a food supplement in Pakistan. In this study methanolic crude extract (LPME of the whole plant and its different fractions; n-hexane (LPHE; ethyl acetate (LPEE and chloroform (LPCE were studied for the determination of total flavonoid and phenolics contents along with multifaceted in vitro scavenging assays. Results Considerable amount of flavonoid and phenolics contents were found in all the fractions. Methanol and chloroform fraction exhibited efficient scavenging of DPPH·, ABTS·+, ·OH, superoxide, lipid peroxide and nitric oxide free radicals. Significant correlation was found between DPPH·, ABTS·+, superoxide radical, β-carotene bleaching restraint and phosphomolybdenum assay with total flavonoids and phenolics contents. High performance chromatography (HPLC of LPME revealed the presence of vitexin, orientin, rutin, hyperoside, catechin and myricetin. Conclusion These results reveal the presence of bioactive compounds in LPME, which might be contributed towards the various in vitro scavenging.

Study on the thyroid function of thoroughbred females in varying stages of pregnancy using 'in vitro' tests /sup 125/I-T/sub 3/ and /sup 125/I-T/sub 4/

Energy Technology Data Exchange (ETDEWEB)

de Martin, B W [Sao Paulo Univ. (Brazil). Faculdade de Medicina Veterinaria e Zootecnia

1975-01-01

A study is made on the thyroid function of thoroughbred female equines, aged between five through twelve years, in varying stages of pregnancy, using 'in vitro' tests /sup 125/I.T/sub 3/ and /sup 125/I-T/sub 4/.

Quantitative in vitro-to-in vivo extrapolation in a high-throughput environment

International Nuclear Information System (INIS)

Wetmore, Barbara A.

2015-01-01

High-throughput in vitro toxicity screening provides an efficient way to identify potential biological targets for environmental and industrial chemicals while conserving limited testing resources. However, reliance on the nominal chemical concentrations in these in vitro assays as an indicator of bioactivity may misrepresent potential in vivo effects of these chemicals due to differences in clearance, protein binding, bioavailability, and other pharmacokinetic factors. Development of high-throughput in vitro hepatic clearance and protein binding assays and refinement of quantitative in vitro-to-in vivo extrapolation (QIVIVE) methods have provided key tools to predict xenobiotic steady state pharmacokinetics. Using a process known as reverse dosimetry, knowledge of the chemical steady state behavior can be incorporated with HTS data to determine the external in vivo oral exposure needed to achieve internal blood concentrations equivalent to those eliciting bioactivity in the assays. These daily oral doses, known as oral equivalents, can be compared to chronic human exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. This review will describe the use of QIVIVE methods in a high-throughput environment and the promise they hold in shaping chemical testing priorities and, potentially, high-throughput risk assessment strategies

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

Science.gov (United States)

Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Beautiful Testing Leading Professionals Reveal How They Improve Software

CERN Document Server

Goucher, Adam

2009-01-01

Successful software depends as much on scrupulous testing as it does on solid architecture or elegant code. But testing is not a routine process, it's a constant exploration of methods and an evolution of good ideas. Beautiful Testing offers 23 essays from 27 leading testers and developers that illustrate the qualities and techniques that make testing an art. Through personal anecdotes, you'll learn how each of these professionals developed beautiful ways of testing a wide range of products -- valuable knowledge that you can apply to your own projects. Here's a sample of what you'll find i

In vitro culture of coconut (Cocos nucifera L.) zygotic embryos.

Science.gov (United States)

Engelmann, Florent; Malaurie, Bernard; N'Nan, Oulo

2011-01-01

Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Côte d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells.

Science.gov (United States)

Saito, Kazutoshi; Miyazawa, Masaaki; Nukada, Yuko; Sakaguchi, Hitoshi; Nishiyama, Naohiro

2013-03-01

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro and in vivo models for testing arrhythmogenesis in drugs.

Science.gov (United States)

Carlsson, L

2006-01-01

The steadily increasing list of drugs associated with prolongation of the QT interval and torsades de pointes (TdP) constitute a medical problem of major concern. Hence, there is a need at an early stage to identify drug candidates with an inherent capacity to induce repolarization-related proarrhythmias, avoiding exposure of large populations to potentially harmful drugs. Furthermore, the availability of clinically relevant and predictive animal models should reduce the risk that effective and potentially life-saving drugs never reach the market. This review will discuss the pros and cons of some in vivo and in vitro animal models for assessing proarrhythmia liability.

The in vitro biokinetics of chlorpromazine and diazepam in aggregating rat brain cell cultures after repeated exposure

NARCIS (Netherlands)

Broeders, Jessica J W; Hermens, Joop L M; Blaauboer, Bas J; Zurich, Marie-Gabrielle

2015-01-01

Neurotoxic effects of compounds can be tested in vitro using cell systems. One example is aggregating rat brain cell cultures. For the extrapolation of in vitro data to the in vivo situation, it is important to take the biokinetics of the test compound into account. In addition, the exposure in vivo

In vitro Evaluation of Trimethoprim and Sulfamethoxazole from Fixed ...

African Journals Online (AJOL)

spectrophotometry, Fixed-dose combination generic drugs. Tropical Journal ... without testing their in-vivo performance [4]. In ..... pharmacokinetic parameter such as AUC, Cmax or Tmax. .... granules: factors affecting drug release in vitro. Int J.

Peri-Implant Strain in an In Vitro Model.

Science.gov (United States)

Hussaini, Souheil; Vaidyanathan, Tritala K; Wadkar, Abhinav P; Quran, Firas A Al; Ehrenberg, David; Weiner, Saul

2015-10-01

An in vitro experimental model was designed and tested to determine the influence that peri-implant strain may have on the overall crestal bone. Strain gages were attached to polymethylmethacrylate (PMMA) models containing a screw-type root form implant at sites 1 mm from the resin-implant interface. Three different types of crown superstructures (cemented, 1-screw [UCLA] and 2-screw abutment types) were tested. Loading (1 Hz, 200 N load) was performed using a MTS Mechanical Test System. The strain gage data were stored and organized in a computer for statistical treatment. Strains for all abutment types did not exceed the physiological range for modeling and remodeling of cancellous bone, 200-2500 με (microstrain). For approximately one-quarter of the trials, the strain values were less than 200 με the zone for bone atrophy. The mean microstrain obtained was 517.7 με. In conclusion, the peri-implant strain in this in vitro model did not exceed the physiologic range of bone remodeling under axial occlusal loading.

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

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Chatterjee, Sagnik, E-mail: Sagnik.Chatterjee@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium); Richert, Lysiane, E-mail: l.richert@kaly-cell.com [KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim (France); Augustijns, Patrick, E-mail: Patrick.Augustijns@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium); Annaert, Pieter, E-mail: Pieter.Annaert@pharm.kuleuven.be [Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O and N2, Herestraat 49 — bus 921, 3000 Leuven (Belgium)

2014-01-01

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. - Highlights: • Novel in vitro assay to detect drug-induced cholestasis • Rat and human sandwich-cultured hepatocytes (SCH) as in vitro models • Cholestatic compounds sensitize SCH to toxic effects of accumulating bile acids • Drug

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

International Nuclear Information System (INIS)

Chatterjee, Sagnik; Richert, Lysiane; Augustijns, Patrick; Annaert, Pieter

2014-01-01

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. - Highlights: • Novel in vitro assay to detect drug-induced cholestasis • Rat and human sandwich-cultured hepatocytes (SCH) as in vitro models • Cholestatic compounds sensitize SCH to toxic effects of accumulating bile acids • Drug

The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer

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Tarig Magdeldin

2014-07-01

Full Text Available The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti–epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01. Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

Adverse reactions to cosmetics and methods of testing.

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Nigam, P K

2009-01-01

Untoward reactions to cosmetics, toiletries, and topical applications are the commonest single reason for hospital referrals with allergic contact dermatitis. In most cases, these are only mild or transient and most reactions being irritant rather than allergic in nature. Various adverse effects may occur in the form of acute toxicity, percutaneous absorption, skin irritation, eye irritation, skin sensitization and photosensitization, subchronic toxicity, mutagenicity/genotoxicity, and phototoxicity/photoirritation. The safety assessment of a cosmetic product clearly depends upon how it is used, since it determines the amount of substance which may be ingested, inhaled, or absorbed through the skin or mucous membranes. Concentration of ingredients used in the different products is also important. Various test procedures include in vivo animal models and in vitro models, such as open or closed patch test, in vivo skin irritation test, skin corrosivity potential tests (rat skin transcutaneous electrical resistance test, Episkin test), eye irritation tests (in vivo eye irritancy test and Draize eye irritancy test), mutagenicity/genotoxicity tests (in vitro bacterial reverse mutation test and in vitro mammalian cell chromosome aberration test), and phototoxicity/photoirritation test (3T3 neutral red uptake phototoxicity test). Finished cosmetic products are usually tested in small populations to confirm the skin and mucous membrane compatibility, and to assess their cosmetic acceptability.

Endometrial carcinoma in vitro chemosensitivity testing of single and combination chemotherapy regimens using the novel microculture kinetic apoptosis assay: implications for endometrial cancer treatment.

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Ballard, Karen S; Homesley, Howard D; Hodson, Charles; Presant, Cary A; Rutledge, James; Hallquist, Allan; Perree, Mathieu

2010-03-01

The in vitro microculture kinetic (MiCK) apoptosis assay has been used to predict single or combination chemotherapy response in leukemia patients. This feasibility study addressed MiCK in endometrial cancer specimens. Endometrial cancer specimens from total abdominal hysterectomies were processed at a central laboratory. Single cell suspensions of viable endometrial cancer cells were plated in individual wells. Single and combination regimens were tested: combinations of doxorubicin, cisplatin, and paclitaxel and carboplatin and paclitaxel (Gynecologic Oncology Group [GOG] 209 endometrial cancer phase III trial arms) as well as single agent testing with paclitaxel, carboplatin, doxorubicin, cisplatin, ifosfamide, and vincristine (active agents in GOG trials). Apoptosis was measured continuously over 48 hours. Fifteen of nineteen patients had successful assays. The highest mean chemo sensitivity was noted in the combination of cisplatin, doxorubicin, and paclitaxel with lower mean chemosensitivity for carboplatin and paclitaxel. Combination chemotherapy had higher chemosensitivity than single drug chemotherapy. However, in 25% of patients a single drug had higher chemosensitivity than combination chemotherapy. As single agents, ifosfamide, cisplatin, and paclitaxel had the highest kinetic unit values. Using a panel of agents simulating clinical dose regimens, the MiCK assay was feasible in evaluating in vitro chemosensitivity of endometrial cancer. MiCK assay results correlated with GOG clinical trial results. However, 25% of patients might be best treated with single agent chemotherapy selected by MiCK. Ifosfamide, cisplatin, and paclitaxel appear to have high activity as single agents. MiCK may be useful in future new drug testing and individualizing endometrial cancer patient's chemotherapy management.

Comparação de métodos para testar a citotoxicidade "in vitro" de materiais biocompatíveis Comparison of methods to test an "in vitro" test of cytotoxicity of biocompatible hospital materials

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Aurea Silveira Cruz

1998-04-01

Full Text Available OBJETIVO: Comparar a sensibilidade do método de difusão em ágar e do método de extração utilizando as linhagens celulares RC-IAL (células fibroblásticas de rim de coelho e HeLa (células epiteliais de carcinoma do colo do útero humano, na avaliação da citotoxicidade "in vitro" de materiais de uso médico-hospitalar. MATERIAL E MÉTODO: Foram testadas 50 amostras escolhidas por sorteio, entre as já conhecidamente positivas e negativas e identificadas como: algodão, espuma, borracha, látex, celulose e acrílico. Além, das amostras citadas foram testadas experimentalmente várias concentrações de SDS (duodecil sulfato de sódio nas culturas celulares RC-IAL e HeLa. RESULTADOS: Das 50 amostras testadas , 44 (88% foram positivas para os dois métodos. Mas quando comparado o SDS nos dois métodos foram observados resultados positivos nas concentrações de 0,5 a 0,05 µg/ml no método de difusão em ágar e no método de extração somente foi observado efeito citotóxico até a concentração de 0,25 µg/ml. CONCLUSÃO: Os resultados encontrados são similares aos observados por outros autores que testaram materiais como, por exemplo, ligas metálicas. Quando foi usado o SDS observou-se, nas duas linhagens celulares, diferenças favoráveis ao método de difusão em ágar em duas concentrações, isto é, a sensibilidade deste método foi significantemente maior, por inspecção, em relação ao método de extração, além de se constituir em método mais simples de ser realizado.OBJECTIVE: A comparison of the sensitivity of the agar diffusion method with that of extraction using cell-lines RC-IAL (fibroblastic of rabbit kidney and HeLa (epithelial carcionoma cells from the cervix uteri of the humam uterus, in the in vitro evaluation of materials of medical and hospital. MATERIAL AND METHODS: Fifteen samples chosen at random, from among the already known positives and negatives in our stock, were tested and identified as cotton

The effectiveness of adhesives on the retention of mandibular free end saddle partial dentures: An in vitro study.

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Quiney, Daniel; Nishio Ayre, Wayne; Milward, Paul

2017-07-01

Existing in vitro methods for testing denture adhesives do not fully replicate the complex oral geometries and environment; and in vivo methods are qualitative, prone to bias and not easily reproducible. The purpose of this study was to develop a novel, quantitative and more accurate model to test the effect of adhesives on the retentive force of mandibular free end saddle partial dentures. An in vitro model was developed based on an anatomically accurate cast of a clinical case. Experimentally, the amount of adhesive was varied (0.2g-1g) and the tensile force required for displacement was measured. Different commercially available adhesives were then tested at the optimum volume using the in vitro model. A 3D finite element model of the denture was used to assess how the forces to induce denture displacement varied according to the position of the force along the saddle length. The mass of adhesive was found to significantly alter retention forces, with 0.4-0.7g being the optimum range for this particular scenario. Use of adhesives significantly improved mandibular free end saddle partial denture retention with the worst performing adhesive increasing retention nine-fold whilst the best performing adhesive increased retention twenty three-fold. The finite element model revealed that 77% more force was required to displace the denture by positioning forces towards the mesial end of the saddle compared to the distal end. An in vitro denture adhesive model was developed, which demonstrated that mass of adhesive plays a significant role in enhancing denture retention and supported the design principle of placing as few teeth as clinically necessary on the distal end of the free end saddles. Limiting the position of teeth on free end saddles to the mesial and mid portion of the saddle will reduce displacements caused by mastication. The movement of mandibular free end saddle partial dentures can be restricted with the use of denture adhesives. Altering the mass of

Toxicity of polymeric nanoparticles in vivo and in vitro

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Voigt, Nadine; Henrich-Noack, Petra; Kockentiedt, Sarah; Hintz, Werner; Tomas, Jürgen; Sabel, Bernhard A.

2014-06-01

Polybutylcyanoacrylate nanoparticles (PBCA NPs) are candidates for a drug delivery system, which can cross the blood-brain barrier (BBB). Because little is known about their toxicity, we exposed cells to PBCA NPs in vitro and in vivo and monitored their life and death assays. PBCA NPs were fabricated with different surfactants according to the mini-emulsion technique. Viabilities of HeLa and HEK293 cells after NP incubation were quantified by analysing cellular metabolic activity (MTT-test). We then repetitively injected i.v. rhodamine-labelled PBCA NP variations into rats and monitored the survival and morphology of retrogradely labelled neurons by in vivo confocal neuroimaging (ICON) for five weeks. To test for carrier-efficacy and safety, PBCA NPs loaded with Kyotorphin were injected in rats, and a hot plate test was used to quantify analgesic effects. In vitro, we found dose-dependent cell death which was, however, only detectable at very high doses and mainly seen in the cultures incubated with NPs fabricated with the tensids SDS and Tween. However, the in vivo experiments did not show any NP-induced neuronal death, even with particles which were toxic at high dose in vitro, i.e. NPs with Tween and SDS. The increased pain threshold at the hot plate test demonstrated that PBCA NPs are able to cross the BBB and thus comprise a useful tool for drug delivery into the central nervous system (CNS). Our findings showing that different nanoparticle formulations are non-toxic have important implications for the value of NP engineering approaches in medicine.

Carboxylated nanodiamonds can be used as negative reference in in vitro nanogenotoxicity studies.

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Moche, H; Paget, V; Chevalier, D; Lorge, E; Claude, N; Girard, H A; Arnault, J C; Chevillard, S; Nesslany, F

2017-08-01

Nanodiamonds (NDs) are promising nanomaterials for biomedical applications. However, a few studies highlighted an in vitro genotoxic activity for detonation NDs, which was not evidenced in one of our previous work quantifying γ-H2Ax after 20 and 100 nm high-pressure high-temperature ND exposures of several cell lines. To confirm these results, in the present work, we investigated the genotoxicity of the same 20 and 100 nm NDs and added intermediate-sized NDs of 50 nm. Conventional in vitro genotoxicity tests were used, i.e., the in vitro micronucleus and comet assays that are recommended by the French National Agency for Medicines and Health Products Safety for the toxicological evaluation of nanomedicines. In vitro micronucleus and in vitro comet assays (standard and hOGG1-modified) were therefore performed in two human cell lines, the bronchial epithelial 16HBE14o- cells and the colon carcinoma T84 cells. Our results did not show any genotoxic activity, whatever the test, the cell line or the size of carboxylated NDs. Even though these in vitro results should be confirmed in vivo, they reinforce the potential interest of carboxylated NDs for biomedical applications or even as a negative reference nanoparticle in nanotoxicology. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients.

Science.gov (United States)

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.

Pituitary tumour causing gigantism. Morphology and in vitro hormone secretion.

Science.gov (United States)

Anniko, M; Ritzén, E M

1986-01-01

True gigantism with overproduction of growth hormone (GH) and prolactin (PRL) was diagnosed in a 13-year-old boy. The clinical history indicated that the tumour had caused an oversecretion of GH since the age of 4-5 years. At diagnosis, the sella turcica was markedly enlarged. No infiltrative growth was noted at surgery. Endocrine investigations showed elevated GH and PRL secretion. Light and electron microscopy of tumour tissue revealed densely packed pleomorphic cells of both GH and PRL type. In addition, oncocyte-like cells were observed. Organ culture of pieces of tumour tissue demonstrated continued secretion of GH and PRL into the medium for more than 5 days in vitro. Addition of bromocriptine to the medium caused a rapid decline in PRL secretion while GH secretion remained the same. X-ray irradiation in vitro also caused a decrease in PRL secretion. These effects of bromocriptine and X-ray on hormone secretion in vitro mirrored the corresponding effect of treatment, when the patient showed signs of tumour recurrence after pituitary surgery. It is concluded that also in childhood, the in vitro response of tumour tissue to various treatments may be explored as a possible way to predict the efficacy of pharmacological or irradiation treatment of pituitary tumours.

Acanthopanax trifoliatus inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo

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Tzu-Mei Chien

2015-10-01

Full Text Available Acanthopanax trifoliatus is a well-known herb that is used for the treatment of bruising, neuralgia, impotence, and gout in Taiwan. This herb exhibits multifunctional activities, including anticancer, anti-inflammation, and antioxidant effects. This paper investigated the in vitro and in vivo anti-inflammatory effect of A. trifoliatus. High-performance liquid chromatography analysis established the fingerprint chromatogram of the ethyl acetate fraction of A. trifoliatus (EAAT. The anti-inflammatory effect of EAAT was detected using lipopolysaccharide (LPS stimulation of the mouse macrophage cell line RAW264.7 in vitro and LPS-induced lung injury in vivo. The effects of EAAT on LPS-induced production of inflammatory mediators in RAW264.7 murine macrophages and the mouse model were measured using enzyme-linked immunosorbent assay and Western blot. EAAT attenuated the production of LPS-induced nitric oxide (NO, tumor necrosis factor-alpha, interleukin-1β (IL-1β, and IL-6 in vitro and in vivo. Pretreatment with EAAT markedly reduced LPS-induced histological alterations in lung tissues. Furthermore, EAAT significantly reduced the number of total cells and protein concentration levels in the bronchoalveolar lavage fluid. Western blotting test results revealed that EAAT blocked protein expression of inducible NO synthase, cyclooxygenase-2, phosphorylation of Nuclear factor-kappa-B Inhibitor alpha (IκB-α protein, and mitogen-activated protein kinases in LPS-stimulated RAW264.7 cells as well as LPS-induced lung injury. This study suggests that A. trifoliatus may be a potential therapeutic candidate for the treatment of inflammatory diseases.

Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

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Middendorf, Jill M; Griffin, Darvin J; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Cohen, Itai; Bonassar, Lawrence J

2017-10-01

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Development of an in vitro Assay, based on the BioFilm Ring Test®, for Rapid Profiling of Biofilm-Growing Bacteria

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Enea Gino Di Domenico

2016-09-01

Full Text Available Microbial biofilm represents a major virulence factor associated with chronic and recurrent infections. Pathogenic bacteria embedded in biofilms are highly resistant to environmental and chemical agents, including antibiotics and therefore difficult to eradicate. Thus, reliable tests to assess biofilm formation by bacterial strains as well as the impact of chemicals or antibiotics on biofilm formation represent desirable tools for a most effective therapeutic management and microbiological risk control. Current methods to evaluate biofilm formation are usually time-consuming, costly, and hardly applicable in the clinical setting.The aim of the present study was to develop and assess a simple and reliable in vitro procedure for the characterization of biofilm-producing bacterial strains for future clinical applications based on the BioFilm Ring Test® (BRT technology. The procedure developed for clinical testing (cBRT can provide an accurate and timely (5 hours measurement of biofilm formation for the most common pathogenic bacteria seen in clinical practice. The results gathered by the cBRT assay were in agreement with the traditional crystal violet (CV staining test, according to the kappa coefficient test (kappa = 0.623. However, the cBRT assay showed higher levels of specificity (92.2% and accuracy (88.1% as compared to CV. The results indicate that this procedure offers an easy, rapid and robust assay to test microbial biofilm and a promising tool for clinical microbiology.

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo

DEFF Research Database (Denmark)

Verhagen, H.; Aruoma, O.I.; van Delft, J.H.M.

2003-01-01

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near......, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2......) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9...

REVEAL: Software Documentation and Platform Migration

Science.gov (United States)

Wilson, Michael A.; Veibell, Victoir T.; Freudinger, Lawrence C.

2008-01-01

The Research Environment for Vehicle Embedded Analysis on Linux (REVEAL) is reconfigurable data acquisition software designed for network-distributed test and measurement applications. In development since 2001, it has been successfully demonstrated in support of a number of actual missions within NASA s Suborbital Science Program. Improvements to software configuration control were needed to properly support both an ongoing transition to operational status and continued evolution of REVEAL capabilities. For this reason the project described in this report targets REVEAL software source documentation and deployment of the software on a small set of hardware platforms different from what is currently used in the baseline system implementation. This report specifically describes the actions taken over a ten week period by two undergraduate student interns and serves as a final report for that internship. The topics discussed include: the documentation of REVEAL source code; the migration of REVEAL to other platforms; and an end-to-end field test that successfully validates the efforts.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

Science.gov (United States)

Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC as a cellular alternative for in vitro ocular toxicity testing.

Directory of Open Access Journals (Sweden)

Edith Aberdam

Full Text Available Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Revealed preference with limited consideration

NARCIS (Netherlands)

Demuynck, T.; Seel, C.

2014-01-01

We derive revealed preference tests for models where individuals use consideration sets to simplify their consumption problem. Our basic test provides necessary and sufficient conditions for consistency of observed choices with the existence of consideration set restrictions. The same conditions can

Comparative study for antibacterial potential of in vitro and in vivo grown Ocimum basilicum L. plant extracts

Energy Technology Data Exchange (ETDEWEB)

Shafique, M; Khan, S J [Pakistan Councile of Scientific and Industrial Research, Lahore (Pakistan). Dept. of Food and Biotechnology

2011-09-15

The antimicrobial activities of in vitro grown callus extract and in vivo grown Ocimum basilicum L. plant leaves extracts were studied and compared. Effect of extraction solvent was also assessed. These extracts were tested in vitro against eight bacterial strains following disc diffusion method. The results indicated that in vitro grown callus extracts of O. basilicum exhibited higher antimicrobial activity against tested Gram positive microorganisms as compared to in vivo grown plant material extract. These findings indicate towards potential use of biotechnology for natural therapeutic agent production. (author)

Comparative study for antibacterial potential of in vitro and in vivo grown Ocimum basilicum L. plant extracts

International Nuclear Information System (INIS)

Shafique, M.; Khan, S.J.

2011-01-01

The antimicrobial activities of in vitro grown callus extract and in vivo grown Ocimum basilicum L. plant leaves extracts were studied and compared. Effect of extraction solvent was also assessed. These extracts were tested in vitro against eight bacterial strains following disc diffusion method. The results indicated that in vitro grown callus extracts of O. basilicum exhibited higher antimicrobial activity against tested Gram positive microorganisms as compared to in vivo grown plant material extract. These findings indicate towards potential use of biotechnology for natural therapeutic agent production. (author)

In Vitro Wound Healing Potential and Identification of Bioactive Compounds from Moringa oleifera Lam

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Abubakar Amali Muhammad

2013-01-01

Full Text Available Moringa oleifera Lam. (M. oleifera from the monogeneric family Moringaceae is found in tropical and subtropical countries. The present study was aimed at exploring the in vitro wound healing potential of M. oleifera and identification of active compounds that may be responsible for its wound healing action. The study included cell viability, proliferation, and wound scratch test assays. Different solvent crude extracts were screened, and the most active crude extract was further subjected to differential bioguided fractionation. Fractions were also screened and most active aqueous fraction was finally obtained for further investigation. HPLC and LC-MS/MS analysis were used for identification and confirmation of bioactive compounds. The results of our study demonstrated that aqueous fraction of M. oleifera significantly enhanced proliferation and viability as well as migration of human dermal fibroblast (HDF cells compared to the untreated control and other fractions. The HPLC and LC-MS/MS studies revealed kaempferol and quercetin compounds in the crude methanolic extract and a major bioactive compound Vicenin-2 was identified in the bioactive aqueous fraction which was confirmed with standard Vicenin-2 using HPLC and UV spectroscopic methods. These findings suggest that bioactive fraction of M. oleifera containing Vicenin-2 compound may enhance faster wound healing in vitro.

Strategy for the hemocompatibility testing of microparticles.

Science.gov (United States)

Braune, S; Basu, S; Kratz, K; Johansson, J Bäckemo; Reinthaler, M; Lendlein, A; Jung, F

2016-01-01

microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis.Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells.The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood.

Extracellular matrices for gastrointestinal surgery: Ex vivo testing and current applications

Science.gov (United States)

Hoeppner, Jens; Marjanovic, Goran; Helwig, Peter; Hopt, Ulrich Theodor; Keck, Tobias

2010-01-01

AIM: To assess the effects of bile and pancreatic juice on structural and mechanical resistance of extracellular matrices (ECMs) in vitro. METHODS: Small-intestinal submucosa (SIS), porcine dermal matrix (PDM), porcine pericardial matrix (PPM) and bovine pericardial matrix (BPM) were incubated in human bile and pancreatic juice in vitro. ECMs were examined by macroscopic observation, scanning electron microscopy (SEM) and testing of mechanical resistance. RESULTS: PDM dissolved within 4 d after exposure to bile or pancreatic juice. SIS, PPM and PDM retained their integrity for > 60 d when incubated in either digestive juice. The effect of bile was found to be far more detrimental to mechanical stability than pancreatic juice in all tested materials. In SIS, the loss of mechanical stability after incubation in either of the digestive secretions was less distinct than in PPM and BPM [mFmax 4.01/14.27 N (SIS) vs 2.08/5.23 N (PPM) vs 1.48/7.89 N (BPM)]. In SIS, the extent of structural damage revealed by SEM was more evident in bile than in pancreatic juice. In PPM and BPM, structural damage was comparable in both media. CONCLUSION: PDM is less suitable for support of gastrointestinal healing. Besides SIS, PPM and BPM should also be evaluated experimentally for gastrointestinal indications. PMID:20731016

PLGA/DPPC/trimethylchitosan spray-dried microparticles for the nasal delivery of ropinirole hydrochloride: in vitro, ex vivo and cytocompatibility assessment

Energy Technology Data Exchange (ETDEWEB)

Karavasili, Christina [Laboratory of Pharmaceutical Technology, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 (Greece); Bouropoulos, Nikolaos [Department of Materials Science, University of Patras, 26504 Rio, Patras (Greece); Foundation for Research and Technology, Hellas-Institute of Chemical Engineering and High Temperature, P.O. Box 1414, 26504 Patras (Greece); Sygellou, Lamprini [Foundation for Research and Technology, Hellas-Institute of Chemical Engineering and High Temperature, P.O. Box 1414, 26504 Patras (Greece); Amanatiadou, Elsa P.; Vizirianakis, Ioannis S. [Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-54124 Thessaloniki (Greece); Fatouros, Dimitrios G., E-mail: dfatouro@pharm.auth.gr [Laboratory of Pharmaceutical Technology, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 (Greece)

2016-02-01

In the present study we investigated polymer-lipid microparticles loaded with ropinirole hydrochloride (RH) for nasal delivery. RH microparticles were further evaluated by means of scanning electron microscopy (SEM), ζ-potential measurements, Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). In vitro release studies were performed in simulated nasal electrolyte solution (SNES) pH 5.5 at 35 °C. Ex vivo permeation studies were conducted across sheep nasal mucosa. Cytocompatibility was tested in cultured human airway epithelial cells (Calu-3). SEM studies revealed spheroid microparticles in the range of 2.09 μm to 2.41 μm. The presence of trimethylchitosan (TMC) induced a slight shift towards less negative ζ-potential values. Surface chemistry (XPS) revealed the presence of dipalmitoylphospatidylcholine (DPPC) and poly(lactic-co-glycolic acid) (PLGA) onto microparticles' surface, further corroborating the FT-IR and XRD findings. In vitro release studies showed that the microparticle composition can partly modulate the release of RH. Ex vivo studies demonstrated a 2.35-folded enhancement of RH permeation when RH was co-formulated with TMC of low molecular weight, compared to the control. All formulations tested were found to be non-toxic to cells. The results suggest that polymer-lipid microparticles may be a promising carrier for the nasal delivery of RH. - Highlights: • Development of microparticles comprising PLGA/DPPC/TMC for nasal drug delivery. • Physicochemical characterization showed that DPPC dominated microparticles' surface. • Microparticles enhanced permeation of ropinirole across sheep nasal epithelium. • The cytotoxicity assay with Calu-3 cells demonstrated satisfactory cell viability.

PLGA/DPPC/trimethylchitosan spray-dried microparticles for the nasal delivery of ropinirole hydrochloride: in vitro, ex vivo and cytocompatibility assessment

International Nuclear Information System (INIS)

Karavasili, Christina; Bouropoulos, Nikolaos; Sygellou, Lamprini; Amanatiadou, Elsa P.; Vizirianakis, Ioannis S.; Fatouros, Dimitrios G.

2016-01-01

In the present study we investigated polymer-lipid microparticles loaded with ropinirole hydrochloride (RH) for nasal delivery. RH microparticles were further evaluated by means of scanning electron microscopy (SEM), ζ-potential measurements, Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). In vitro release studies were performed in simulated nasal electrolyte solution (SNES) pH 5.5 at 35 °C. Ex vivo permeation studies were conducted across sheep nasal mucosa. Cytocompatibility was tested in cultured human airway epithelial cells (Calu-3). SEM studies revealed spheroid microparticles in the range of 2.09 μm to 2.41 μm. The presence of trimethylchitosan (TMC) induced a slight shift towards less negative ζ-potential values. Surface chemistry (XPS) revealed the presence of dipalmitoylphospatidylcholine (DPPC) and poly(lactic-co-glycolic acid) (PLGA) onto microparticles' surface, further corroborating the FT-IR and XRD findings. In vitro release studies showed that the microparticle composition can partly modulate the release of RH. Ex vivo studies demonstrated a 2.35-folded enhancement of RH permeation when RH was co-formulated with TMC of low molecular weight, compared to the control. All formulations tested were found to be non-toxic to cells. The results suggest that polymer-lipid microparticles may be a promising carrier for the nasal delivery of RH. - Highlights: • Development of microparticles comprising PLGA/DPPC/TMC for nasal drug delivery. • Physicochemical characterization showed that DPPC dominated microparticles' surface. • Microparticles enhanced permeation of ropinirole across sheep nasal epithelium. • The cytotoxicity assay with Calu-3 cells demonstrated satisfactory cell viability.

Comparison of in vitro and in vivo phototoxicity tests with S-(-)-10,11-dihydroxyfarnesic acid methyl ester produced by Beauveria bassiana KACC46831.

Science.gov (United States)

Kim, Min-A; Son, Hyeong-U; Yoon, Cheol-Sik; Nam, Sung-Hee; Choi, Young-Cheol; Lee, Sang-Han

2014-09-01

Beauveria bassiana is a fungi that is well-known for demonstrating a resistance to environmental change. To confirm whether S-(-)-10,11-dihydroxyfarnesic acid methyl ester (DHFAME) produced by Beauveria bassiana KACC46831 causes phototoxicity when used for cosmetic purposes due to its anti-tyrosinase activity, we conducted in vitro and in vivo phototoxicity tests. There were no significant changes or damage observed in the compound-treated group with regards to skin phototoxicity, while 8-methoxypsoralen, which served as a positive control, induced toxic effects. The in vitro 3T3 neutral red uptake assay, an alternative assessment, was used for further confirmation of the phototoxicity. The results showed that DHFAME did not exhibit phototoxicity at the designated concentrations, with or without UV irradiation in the 3T3 cells. These results indicated that the methyl ester produced by Beauveria bassiana KACC46831 does not induce phototoxicity in the skin. Therefore, the results of the present study indicate that DHFAME shows potential for use as a cosmetic ingredient that does not cause skin phototoxicity.

Evaluation of a three compartment in vitro gastrointestinal simulator dissolution apparatus to predict in vivo dissolution.

Science.gov (United States)

Takeuchi, Susumu; Tsume, Yasuhiro; Amidon, Gregory E; Amidon, Gordon L

2014-11-01

In vitro dissolution tests are performed for new formulations to evaluate in vivo performance, which is affected by the change of gastrointestinal (GI) physiology, in the GI tract. Thus, those environmental changes should be introduced to an in vitro dissolution test. Many studies have successfully shown the improvement of in vitro-in vivo correlations (IVIVC) by introducing those physiological changes into dissolution tests. The gastrointestinal simulator (GIS), a multicompartment in vitro dissolution apparatus, was developed to evaluate in vivo drug dissolution. A gastric-emptying rate along with transit rate are key factors to evaluate in vivo drug dissolution and, hence, drug absorption. Dissolution tests with the GIS were performed with Biopharmaceutical Classification System class I drugs at five different gastric-emptying rates in the fasted state. Computational models were used to determine in vivo gastric-emptying time for propranolol and metoprolol based on the GIS dissolution results. Those were compared with published clinical data to determine the gastric half-emptying time. In conclusion, the GIS is a practical tool to assess dissolution properties and can improve IVIVC. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Medical chilling device designed for hypothermic hydration graft storage system: Design, thermohydrodynamic modeling, and preliminary testing

Energy Technology Data Exchange (ETDEWEB)

Seo, Jung Hwan [Hongik University, Seoul (Korea, Republic of)

2015-02-15

Hypothermic hydration graft storage is essential to reduce the metabolic demand of cells in vitro. The alleviated metabolic demands reduce the emergence rate of anaerobic metabolism generating adenosine triphosphate (ATP) energy that creates free radicals. The cessive free radicals can damage cells and tissues due to their highly oxidative power with molecules. Current cooling systems such as a conventional air cooling system and an ice pack system are inappropriate for chilling cell tissues in vitro because of inconvenience in use and inconsistent temperature sustainability caused by large size and progressive melting, respectively. Here, we develop a medical chilling device (MCD) for hypothermic hydration graft storage based on thermo-hydrodynamic modeling and thermal electric cooling technology. Our analysis of obtained hydrodynamic thermal behavior of the MCD revealed that the hypothermic condition of 4 .deg. C was continuously maintained, which increased the survival rates of cells in vitro test by reduced free radicals. The validated performance of the MCD promises future development of an optimal hypothermic hydration graft storage system designed for clinical use.

State of the art in non-animal approaches for skin sensitization testing: from individual test methods towards testing strategies.

Science.gov (United States)

Ezendam, Janine; Braakhuis, Hedwig M; Vandebriel, Rob J

2016-12-01

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)-protein binding, KE2-keratinocyte activation, KE3-dendritic cell activation and KE4-T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an

Elucidation of Formulation and Delivery Device-Related Effects on In Vitro Performance of Nasal Spray with Implication to Rational Product Specification Identification.

Science.gov (United States)

Grmaš, Jernej; Stare, Katarina; Božič, Dane; Injac, Rade; Dreu, Rok

2017-08-01

The aim of this work is to use an experimental design approach to identify and study influential formulation and delivery device properties, which can be controlled by final product manufacturer, to establish design space, within which desired in vitro performance can be reached. Combining three factors, viscosity of suspension, nozzle orifice diameter (OD), and shot weight (SW), at three levels resulted in D-optimal experimental design with 20 runs. Responses within this study were droplet size distribution (DSD) and spray pattern (SP) in vitro tests. In addition, the amount of mechanical work needed for actuation was integrated from force profiles and used as a response. Results were fit to quadratic model by regression, which allowed also for determination of second-order and interaction effects between factors. Models were further optimized by keeping significant terms only. Optimized models were used to create response surfaces and design space with confidence levels. Viscosity has a dominant effect on DSD and modest effect on SP, with lower viscosities related to generation of smaller DSD and larger SP. Orifice diameter was found to have the highest impact on SP, with larger diameter resulting in larger SP. This effect was additionally confirmed by results of Plume Geometry in vitro test. Shot weight factor exerts significant influence on all tested metrics. Work, however, did not vary greatly with suspension viscosity or orifice diameter. Shot weight is the most dominant factor for work and important for DSD having a positive effect on both responses. In the case of SP, its relationship with shot weight is described by second-order polynomial fit. Inspection of raw data revealed that density of droplets within SP area is different for different shot weights. Presented study elucidated an inherent relationship between factors and responses and established mathematical models (response surfaces) for predictive purposes to target specific in vitro performance

In vitro propagation of avocado (Persea drymifolia Ness.)

International Nuclear Information System (INIS)

Barrera-Guerra, J.L.; Ramirez-Malagon, R.; Martinez-Jaime, O.A.

2001-01-01

In the past 20 years, reports on micropropagation and rooting in vitro of avocado shoots, with diverse origins and treatments, have been published. However, none of them reached the level required for large scale propagation of the species. It is considered that, in the first place, the micropropagation of avocado requires an efficient system of rooting. Therefore, a system to induce the rooting in vitro of avocado shoots, based on indole-3-butyric acid (IBA) pulses and some treatments based on thidiazuron (TDZ) was tested, using 40 explants per treatment. The treatments with TDZ did not succeed in rooting shoots. Some treatments with pulses of IBA induced the following rooting results: without growth regulators, 16%; 4,000 mg L -1 of IBA for 5 seconds, 8.3% rooted; 100 mg L -1 for 72 hours, 20%; 50 mg L -1 for 72 hours, 15.4%; 150 mg L -1 for 24 hours, 5% rooted. It is considered possible to improve these results by adjusting the range of IBA concentrations as well as in the time range of pulse applications. Finally, it is easier to establish in vitro explants derived from mature seeds or embryos germinated in vitro. (author)

Comparative In Vitro Efficacy of Doripenem and Imipenem Against Multi-Drug Resistant Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Wali, N.; Mirza, I. A.

2016-01-01

Objective: To compare the in vitro efficacy of doripenem and imipenem against multi-drug resistant (MDR) Pseudomonas aeruginosa from various clinical specimens. Study Design: Descriptive cross-sectional study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from November 2012 to November 2013. Methodology: MDR Pseudomonas aeruginosa isolates from various clinical samples were included in the study. Susceptibility of Pseudomonas aeruginosa against doripenem and imipenem was performed by E-test strip and agar dilution methods. The results were interpreted as recommended by Clinical Laboratory Standard Institute (CLSI) guidelines. Results: The maximum number of Pseudomonas aeruginosa were isolated from pure pus and pus swabs. In vitro efficacy of doripenem was found to be more effective as compared to imipenem against MDR Pseudomonas aeruginosa with both E-test strip and agar dilution methods. Overall, p-values of 0.014 and 0.037 were observed when susceptibility patterns of doripenem and imipenem were evaluated with E-test strip and agar dilution methods. Conclusion: In vitro efficacy of doripenem was found to be better against MDR Pseudomonas aeruginosa as compared to imipenem when tested by both E-test and agar dilution methods. (author)

Developing a quick and inexpensive in vitro (non-animal) bioassay for mascara irritation.

Science.gov (United States)

Thomason, H; Montagnes, D J S

2014-04-01

Mascara is a mild irritant that causes a range of medical problems. Animal models to predict ocular irritation have, however, been questioned at a number of levels, and there is a continued need to develop in vitro testing methods. We assess changes in an easily quantifiable attribute, ciliated protozoan growth rate, as a sensitive, sublethal measure. Specifically, we test six, randomly chosen, commercial mascara products against a control (as treatments) and reveal through ANOVA (n = 6, α = 0.05) significant differences in the specific growth rate to treatments (for both protozoa). We provide evidence that two easily cultured protozoa (Paramecium caudatum, Blepharisma japonicum) should be considered as models to assess ocular irritancy (and possibly cosmetics in general) and establish the groundwork for such studies to be applied at a more commercial level. We do this by developing a bioassay for mascara toxicity and indicate the low cost (after equipment is purchased, on the order of $100s) and the ease of performing such tests (able to be conducted by undergraduate students), as a consideration for their future commercial application. We first examined dose dependence of responses, revealing that there was a need to conduct preliminary work to determine appropriate levels for sublethal responses. We then show that some products resulted in mortality at high concentrations, others decreased growth rate by >50% (compared with the control), whereas others had no significant effect, compared with the control. We have provided a novel, quick and inexpensive means to assess mascara; the next step is to validate these ciliate bioassays by comparison with animal testing and epidemiological studies, which is beyond the scope of this fundamental 'proof-of-concept' study. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Equating spatial summation in visual field testing reveals greater loss in optic nerve disease.

Science.gov (United States)

Kalloniatis, Michael; Khuu, Sieu K

2016-07-01

To test the hypothesis that visual field assessment in ocular disease measured with target stimuli within or close to complete spatial summation results in larger threshold elevation compared to when measured with the standard Goldmann III target size. The hypothesis predicts a greater loss will be identified in ocular disease. Additionally, we sought to develop a theoretical framework that would allow comparisons of thresholds with disease progression when using different Goldmann targets. The Humphrey Field Analyser (HFA) 30-2 grid was used in 13 patients with early/established optic nerve disease using the current Goldmann III target size or a combination of the three smallest stimuli (target size I, II and III). We used data from control subjects at each of the visual field locations for the different target sizes to establish the number of failed points (events) for the patients with optic nerve disease, as well as global indices for mean deviation (MD) and pattern standard deviation (PSD). The 30-2 visual field testing using alternate target size stimuli showed that all 13 patients displayed more defects (events) compared to the standard Goldmann III target size. The median increase for events was seven additional failed points: (range 1-26). The global indices also increased when the new testing approach was used (MD -3.47 to -6.25 dB and PSD 4.32 to 6.63 dB). Spatial summation mapping showed an increase in critical area (Ac) in disease and overall increase in thresholds when smaller target stimuli were used. When compared to the current Goldmann III paradigm, the use of alternate sized targets within the 30-2 testing protocol revealed a greater loss in patients with optic nerve disease for both event analysis and global indices (MD and PSD). We therefore provide evidence in a clinical setting that target size is important in visual field testing. © 2016 The Authors Ophthalmic & Physiological Optics © 2016 The College of Optometrists.

In vitro evaluation of fluorescence glucose biosensor response.

Science.gov (United States)

Aloraefy, Mamdouh; Pfefer, T Joshua; Ramella-Roman, Jessica C; Sapsford, Kim E

2014-07-08

Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

In Vitro Evaluation of Fluorescence Glucose Biosensor Response

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Mamdouh Aloraefy