In vivo and in vitro effects of antioxidant compounds and PPAR γ modulators on rainbow trout oxidative stress and lipid metabolism.

The aquafeed use of raw plant materials, as protein and lipid sources, has been considered and approved as a sustainable alternative to fish products (fish meal and oils) because the current trend to use high-lipid diets has been shown to induce undesirable increase in fat depots or further physiological alterations, such as induction of oxidative stress. In the aquaculture perspective, the addition of natural substances with antioxidant properties is an emerging strategy for protecting biological systems and foodstuffs from oxidative damage. Among natural substances, hydroxytyrosol (HT) and caffeic acid (CA) have attracted considerable attention as food antioxidant additives and modulators of physiological and molecular pathways involved in energy metabolism and adiposity. The aim of this study was to evaluate the effects of CA and HT on lipid metabolism and oxidative stress of rainbow trout (Oncorhynchus mykiss). In vitro results showed the potential anti-obesogenic effects of the compounds CA and HT on the adipose tissue of the rainbow trout. To support these data, in vitro assays performed (MTT, ORO, immunofluorescence) resulted in accordance among them; only results from proliferating cell nuclear antigen (PCNA) assay were not significant. In vivo results showed a possible anti-obesogenic effect of CA in liver and HT in adipose tissue. Regarding oxidative stress, we could hypothesize a possible anti-oxidant role of CA in liver.

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.

Application of the specular and diffuse reflection analysis for in vitro diagnostics of dental erosion: correlation with enamel softening, roughness, and calcium release

We present assembly and application of an optical reflectometer for the analysis of dental erosion. The erosive procedure involved acid-induced softening and initial substance loss phases, which are considered to be difficult for visual diagnosis in a clinic. Change of the specular reflection signal showed the highest sensitivity for the detection of the early softening phase of erosion among tested methods. The exponential decrease of the specular reflection intensity with erosive duration was compared to the increase of enamel roughness. Surface roughness was measured by optical analysis, and the observed tendency was correlated with scanning electron microscopy images of eroded enamel. A high correlation between specular reflection intensity and measurement of enamel softening (r(2) ? -0.86) as well as calcium release (r(2) ? -0.86) was found during erosion progression. Measurement of diffuse reflection revealed higher tooth-to-tooth deviation in contrast to the analysis of specular reflection intensity and lower correlation with other applied methods (r(2) = 0.42-0.48). The proposed optical method allows simple and fast surface analysis and could be used for further optimization and construction of the first noncontact and cost-effective diagnostic tool for early erosion assessment in vivo.

In-vitro assessment of the forces generated by lingual fixed retainers

The aim of this article was to assess the effect of wire adaptation on the lingual surfaces of mandibular anterior teeth with 3 types of lingual retainers on the development of vertical and labiolingual forces.

In situ and in vitro comparison of laser fluorescence with visual inspection in detecting occlusal caries lesions

The aim of this study was to compare the in situ and in vitro performances of a laser fluorescence (LF) device (DIAGNOdent 2095) with visual inspection for the detection of occlusal caries in permanent teeth. Sixty-four sites were selected, and visual inspection and LF assessments were carried out, in vitro, three times by two independent examiners, with a 1-week interval between evaluations. Afterwards, the occlusal surfaces were mounted on the palatal portion of removable acrylic orthodontic appliances and placed in six volunteers. Assessments were repeated and validated by histological analysis of the tooth sections under a stereomicroscope. For both examiners, the highest intra-examiner values were observed for the visual inspection when in vitro and in situ evaluations were compared. The inter-examiner reproducibility varied from 0.61 to 0.64, except for the in vitro assessment using LF, which presented a lower value (0.43). The methods showed high specificity at the D(1) threshold (considering enamel and dentin caries as disease). In vitro evaluations showed the highest values of sensitivity for both methods when compared to the in situ evaluations at D(1) and D(2) (considering only dentinal caries as the disease) thresholds. For both methods, the results of sensitivity (at D(1) and D(2)) and accuracy (at D(1)) showed significant differences between in vitro and in situ conditions. However, the sensitivity (at D(1) and D(2)), specificity and accuracy (both at D(1)) of the methods were not significantly different when the same condition was considered. It can be concluded that visual inspection and LF showed better performance in vitro than in situ.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules – dexamethasone, triiodothyronine (T3) and insulin – on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

In Vitro Evaluation of Ventricular Cannulation for Rotodynamic Cardiac Assist Devices

The influence of positioning and geometry of ventricular cannulas for contemporary continuous flow Left Ventricular Assist Devices (LVADs) was evaluated in a non-beating isolated heart preparation with borescopic visualization. Preload and LVAD flow were varied to evaluate degrees of ventricular decompression up to the point of ventricular collapse. The performance of a flanged cannula was compared to a conventional bevel-tipped cannula: quantitatively by the maximal flow attainable, and qualitatively by visualization of fluid tracer particles within the ventricular chamber. Three forms of ventricular suck-down occurred: concentric collapse, gradual entrainment and instantaneous entrainment. In some circumstances, unstable oscillations of the ventricle were observed prior to complete collapse. Under conditions of low preload, the flanged cannula demonstrated less positional sensitivity, provided greater flow, and exhibited fewer areas of stagnation than the beveled cannula. These observations warrant further consideration of a flanged ventricular cannula to mitigate complications encountered with conventional cannulae.