A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

In vitro fabrication of autologous living tissue-engineered vascular grafts based on prenatally harvested ovine amniotic fluid-derived stem cells

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications. Copyright © 2013 John Wiley & Sons, Ltd.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Nonlinear quasi-static finite element simulations predict in vitro strength of human proximal femora assessed in a dynamic sideways fall setup

Osteoporotic proximal femur fractures are caused by low energy trauma, typically when falling on the hip from standing height. Finite element simulations, widely used to predict the fracture load of femora in fall, usually include neither mass-related inertial effects, nor the viscous part of bone's material behavior. The aim of this study was to elucidate if quasi-static non-linear homogenized finite element analyses can predict in vitro mechanical properties of proximal femora assessed in dynamic drop tower experiments. The case-specific numerical models of thirteen femora predicted the strength (R2=0.84, SEE=540 N, 16.2%), stiffness (R2=0.82, SEE=233 N/mm, 18.0%) and fracture energy (R2=0.72, SEE=3.85 J, 39.6%); and provided fair qualitative matches with the fracture patterns. The influence of material anisotropy was negligible for all predictions. These results suggest that quasi-static homogenized finite element analysis may be used to predict mechanical properties of proximal femora in the dynamic sideways fall situation.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug-induced skin diseases

BACKGROUND: Cytotoxic cells are involved in most forms of drug-induced skin diseases. Till now, no in vitro test addressed this aspect of drug-allergic responses. Our report evaluates whether drug-induced cytotoxic cells can be detected in peripheral blood of nonacute patients with different forms of drug hypersensitivity, and also whether in vitro detection of these cells could be helpful in drug-allergy diagnosis. METHODS: GranzymeB enzyme-linked immunosorbent spot-forming (ELISPOT) and cell surface expression of the degranulation marker CD107a were evaluated on peripheral blood mononuclear cells from 12 drug-allergic patients in remission state and 16 drug-exposed healthy controls. RESULTS: In 10/12 allergic patients culprit but not irrelevant drug elicited granzymeB release after 48-72 h stimulation. It was clearly positive in patients with high proliferative response to the drug, measured in lymphocyte transformation tests. In patients, who showed moderate or low proliferation and low drug-response in granzymeB ELISPOT, overnight preincubation with interleukin (IL)-7/IL-15 enhanced drug-specific granzymeB release and allowed to clearly identify the offending agent. CD107a staining was positive on CD4+/CD3+, CD8+/CD3+ T cells as well as CD56+/CD3- natural killer cells. None of the drug-exposed healthy donors reacted to the tested drugs and allergic patients reacted only to the offending, but not to tolerated drugs. CONCLUSION: GranzymeB ELISPOT is a highly specific in vitro method to detect drug-reacting cytotoxic cells in peripheral blood of drug-allergic patients even several years after disease manifestation. Together with IL-7/IL-15 preincubation, it may be helpful in indentifying the offending drug even in some patients with weak proliferative drug-response.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

In vitro detection of secondary caries associated with composite restorations on approximal surfaces using laser fluorescence

This study evaluated the performance of the DIAGNOdent pen laser fluorescence device (LFpen) in comparison with visual examination (VE), bitewing radiographs (BW) and visual examination combined with bitewing radiographs (VEBW) in detecting secondary approximal caries associated with composite restorations. In total, 60 approximal surfaces from 43 permanent molars with composite restorations were assessed twice by two examiners using the LFpen, VE, BW and VEBW. After histological preparation and hardness measurements, the sample was assigned to either a crown or root caries group, depending on the location of the lesions as the gold standard. For crown caries at D1, the highest values of specificity and sensitivity were observed for the LFpen at a cutoff value of 18 (1.00) and for the VEBW (0.89). At D3 (cutoff of 30), the LFpen showed the highest values of sensitivity and specificity. For root caries, the LFpen and VEBW showed the highest values of specificity (0.54), sensitivity (0.81) and accuracy (0.69). The Spearman rank correlation coefficients for crown/root caries with histology were 0.54/0.37 (LFpen), 0.29/0.10 (BW), 0.29/0.18 (VE) and 0.23/0.37 (VEBW). For the LFpen, the ICC varied from 0.80 (interexaminer) to 0.97 (intraexaminer B); the kappa value was 0.19 for BW and 0.35 for VE (interexaminer). Intraexaminer kappa values for BW were 0.25 (A) and 0.29 (B), and those for VE were 0.31 (A) and 0.32 (B). The LFpen device exhibited a performance comparable to that of conventional methods but with higher interexaminer reproducibility. Therefore, the LFpen should be considered an auxiliary method for the detection of secondary approximal caries associated with composite restorations.

Development and in-vitro characterization of an implantable flow sensing transducer for hydrocephalus

An implantable transducer for monitoring the flow of Cerebrospinal fluid (CSF) for the treatment of hydrocephalus has been developed which is based on measuring the heat dissipation of a local thermal source. The transducer uses passive telemetry at 13.56 MHz for power supply and read out of the measured flow rate. The in vitro performance of the transducer has been characterized using artificial Cerebrospinal Fluid (CSF) with increased protein concentration and artificial CSF with 10\% fresh blood. After fresh blood was added to the artificial CSF a reduction of flow rate has been observed in case that the sensitive surface of the flow sensor is close to the sedimented erythrocytes. An increase of flow rate has been observed in case that the sensitive surface is in contact with the remaining plasma/artificial CSF mix above the sediment which can be explained by an asymmetric flow profile caused by the sedimentation of erythrocythes having increased viscosity compared to artificial CSF. After removal of blood from artificial CSF, no drift could be observed in the transducer measurement which could be associated to a deposition of proteins at the sensitive surface walls of the packaged flow transducer. The flow sensor specification requirement of +-10\% for a flow range between 2 ml/h and 40 ml/h. could be confirmed at test conditions of 37 degrees C.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Cartilage, bone and synovial histomorphometry in animal models of osteoarthritis

http://www.ncbi.nlm.nih.gov/pubmed/20864016

Dose-response effect of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and interferon-y on the in vitro production of epithelial neutrophil activating peptide-78 (ENA-78), IL-8, and IL-6 by human endometrial stromal cells

The production of epithelial neutrophil activating peptide-78 (NA-78) and the interleukins IL-8 and IL-6 by endometrial stromal cells is stimulated by pro-inflammatory interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α). IL-8 is suggested to play a role in the pathogenesis of endometriosis, and in these women the peritoneal fluid concentrations of ENA-78 and IL-8 are increased. TNF-α has been tested together with interferon-γ because of their cooperative stimulation of IL-6. The release of IL-8, however, is inhibited with increasing interferon levels. The aim of the study was the analysis of the production of ENA-78, IL-6 and IL-8 by cultured human endometrial stromal cells in the presence of varying concentrations of IL-1β, TNF-α, and interferon-γ.