In Vitro and In Vivo Imaging Characteristics Assessment of Polymeric Coils Compared with Standard Platinum Coils for the Treatment of Intracranial Aneurysms

BACKGROUND AND PURPOSE:Conventional platinum coils cause imaging artifacts that reduce imaging quality and therefore impair imaging interpretation on intraprocedural or noninvasive follow-up imaging. The purpose of this study was to evaluate imaging characteristics and artifact production of polymeric coils compared with standard platinum coils in vitro and in vivo.MATERIALS AND METHODS:Polymeric coils and standard platinum coils were evaluated in vitro with the use of 2 identical silicon aneurysm models coiled with a packing attenuation of 20% each. DSA, flat panel CT, CT, and MR imaging were performed. In vivo evaluation of imaging characteristics of polymeric coils was performed in experimentally created rabbit carotid bifurcation aneurysms. DSA, CT/CTA, and MR imaging were performed after endovascular treatment of the aneurysms. Images were evaluated regarding visibility of individual coils, coil mass, artifact production, and visibility of residual flow within the aneurysm.RESULTS:Overall, in vitro and in vivo imaging showed relevantly reduced artifact production of polymeric coils in all imaging modalities compared with standard platinum coils. Image quality of CT and MR imaging was improved with the use of polymeric coils, which permitted enhanced depiction of individual coil loops and residual aneurysm lumen as well as the peri-aneurysmal area. Remarkably, CT images demonstrated considerably improved image quality with only minor artifacts compared with standard coils. On DSA, polymeric coils showed transparency and allowed visualization of superimposed vessel structures.CONCLUSIONS:This initial experimental study showed improved imaging quality with the use of polymeric coils compared with standard platinum coils in all imaging modalities. This might be advantageous for improved intraprocedural imaging for the detection of complications and posttreatment noninvasive follow-up imaging.

In vitro hemodynamic evaluation of ventricular suction conditions of the EVAHEART ventricular assist pump

Purpose: Mismatches between pump output and venous return in a continuous-flow ventricular assist device may elicit episodes of ventricular suction. This research describes a series of in vitro experiments to characterize the operating conditions under which the EVAHEART centrifugal blood pump (Sun Medical Technology Research Corp., Nagano, Japan) can be operated with minimal concern regarding left ventricular (LV) suction. Methods: The pump was interposed into a pneumatically driven pulsatile mock circulatory system (MCS) in the ventricular apex to aorta configuration. Under varying conditions of preload, afterload, and systolic pressure, the speed of the pump was increased step-wise until suction was observed. Identification of suction was based on pump inlet pressure. Results: In the case of reduced LV systolic pressure, reduced preload (=10 mmHg), and afterload (=60 mmHg), suction was observed for speeds =2,200 rpm. However, suction did not occur at any speed (up to a maximum speed of 2,400 rpm) when preload was kept within 10-14 mmHg and afterload =80 mmHg. Although in vitro experiments cannot replace in vivo models, the results indicated that ventricular suction can be avoided if sufficient preload and afterload are maintained. Conclusion: Conditions of hypovolemia and/or hypotension may increase the risk of suction at the highest speeds, irrespective of the native ventricular systolic pressure. However, in vitro guidelines are not directly transferrable to the clinical situation; therefore, patient-specific evaluation is recommended, which can be aided by ultrasonography at various points in the course of support.

An in vitro study on spontaneous myometrial contractility in the mare during estrus and diestrus

Uterine smooth muscle specimens were collected from euthanatized mares in estrus and diestrus. Longitudinal and circular specimens were mounted in organ baths and the signals transcribed to a Grass polygraph. After equilibration time and 2 g preload, their physiologic isometric contractility was recorded for a continuous 2.0 h. Area under the curve, frequency and time occupied by contractions were studied. Differences between cycle phases, between muscle layers, and over the recorded time periods were statistically evaluated using linear mixed-effect models. In the mare, physiologic contractility of the uterus decreased significantly over time for all variables evaluated (time as covariate on a continuous scale). For area under the curve, there was a significant effect of muscle layer (longitudinal > circular). For frequency, higher values were recorded in estrus for circular smooth muscle layer, whereas higher values were seen in longitudinal smooth muscle layers during diestrus. In longitudinal layer and in diestrus, more time was occupied by contractions than in circular layer, and in estrus. This study is describing physiologic myometrial motility in the organ bath depending on cycle phase.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system

STUDY DESIGN: Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. OBJECTIVES: To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. SUMMARY OF BACKGROUND DATA: With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. METHODS: Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. RESULTS: Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. CONCLUSIONS: Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

The in vitro evidence for an effect of high homeopathic potencies--a systematic review of the literature

OBJECTIVE: Systematic assessment of the in vitro research on high potency effects. METHOD: Publications of experiments were collected through databases, experts, previous reviews, citation tracking. Inclusion criteria: stepwise agitated dilutions <10(-23); cells or molecules from human or animal. Experiments were assessed with the modified SAPEH score. RESULTS: From 75 publications, 67 experiments (1/3 of them replications) were evaluated. Nearly 3/4 of them found a high potency effect, and 2/3 of those 18 that scored 6 points or more and controlled contamination. Nearly 3/4 of all replications were positive. Design and experimental models of the reviewed experiments were inhomogenous, most were performed on basophiles. CONCLUSIONS: Even experiments with a high methodological standard could demonstrate an effect of high potencies. No positive result was stable enough to be reproduced by all investigators. A general adoption of succussed controls, randomization and blinding would strengthen the evidence of future experiments.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Multimeric tyrosine sulfate acts as an endothelial cell protectant and prevents complement activation in xenotransplantation models

BACKGROUND: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. METHOD: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. RESULTS: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. CONCLUSIONS: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application.

Use of homeopathic preparations in phytopathological models and in field trails: a critical review

Background: The literature on the applications of homeopathy for controlling plant diseases in both plant pathological models and field trials was first reviewed by Scofield in 1984. No other review on homeopathy in plant pathology has been published since, though much new research has subsequently been carried out using more advanced methods. Objectives: To conduct an up-to-date review of the existing literature on basic research in homeopathy using phytopathological models and experiments in the field. Methods: A literature search was carried out on publications from 1969 to 2009, for papers that reported experiments on homeopathy using phytopathological models (in vitro and in planta) and field trials. The selected papers were summarized and analysed on the basis of a Manuscript Information Score (MIS) to identify those that provided sufficient information for proper interpretation (MIS ≥ 5). These were then evaluated using a Study Methods Evaluation Procedure (SMEP). Results: A total of 44 publications on phytopathological models were identified: 19 papers with statistics, 6 studies with MIS ≥ 5. Publications on field were 9, 6 with MIS ≥ 5. In general, significant and reproducible effects with decimal and centesimal potencies were found, including dilution levels beyond the Avogadro's number. Conclusions: The prospects for homeopathic treatments in agriculture are promising, but much more experimentation is needed, especially at a field level, and on potentisation techniques, effective potency levels and conditions for reproducibility. Phytopathological models may also develop into useful tools to answer pharmaceutical questions.

The state of in vitro science for use in bioaccumulation assessments for fish

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Program's Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

A nonlinear QCT-based finite element model validation study for the human femur tested in two configurations in vitro

Purpose Femoral fracture is a common medical problem in osteoporotic individuals. Bone mineral density (BMD) is the gold standard measure to evaluate fracture risk in vivo. Quantitative computed tomography (QCT)-based homogenized voxel finite element (hvFE) models have been proved to be more accurate predictors of femoral strength than BMD by adding geometrical and material properties. The aim of this study was to evaluate the ability of hvFE models in predicting femoral stiffness, strength and failure location for a large number of pairs of human femora tested in two different loading scenarios. Methods Thirty-six pairs of femora were scanned with QCT and total proximal BMD and BMC were evaluated. For each pair, one femur was positioned in one-legged stance configuration (STANCE) and the other in a sideways configuration (SIDE). Nonlinear hvFE models were generated from QCT images by reproducing the same loading configurations imposed in the experiments. For experiments and models, the structural properties (stiffness and ultimate load), the failure location and the motion of the femoral head were computed and compared. Results In both configurations, hvFE models predicted both stiffness (R2=0.82 for STANCE and R2=0.74 for SIDE) and femoral ultimate load (R2=0.80 for STANCE and R2=0.85 for SIDE) better than BMD and BMC. Moreover, the models predicted qualitatively well the failure location (66% of cases) and the motion of the femoral head. Conclusions The subject specific QCT-based nonlinear hvFE model cannot only predict femoral apparent mechanical properties better than densitometric measures, but can additionally provide useful qualitative information about failure location.

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

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

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.