A Systems-Level Approach to Human Epileptic Seizures

Epileptic seizures are due to the pathological collective activity of large cellular assemblies. A better understanding of this collective activity is integral to the development of novel diagnostic and therapeutic procedures. In contrast to reductionist analyses, which focus solely on small-scale characteristics of ictogenesis, here we follow a systems-level approach, which combines both small-scale and larger-scale analyses. Peri-ictal dynamics of epileptic networks are assessed by studying correlation within and between different spatial scales of intracranial electroencephalographic recordings (iEEG) of a heterogeneous group of patients suffering from pharmaco-resistant epilepsy. Epileptiform activity as recorded by a single iEEG electrode is determined objectively by the signal derivative and then subjected to a multivariate analysis of correlation between all iEEG channels. We find that during seizure, synchrony increases on the smallest and largest spatial scales probed by iEEG. In addition, a dynamic reorganization of spatial correlation is observed on intermediate scales, which persists after seizure termination. It is proposed that this reorganization may indicate a balancing mechanism that decreases high local correlation. Our findings are consistent with the hypothesis that during epileptic seizures hypercorrelated and therefore functionally segregated brain areas are re-integrated into more collective brain dynamics. In addition, except for a special sub-group, a highly significant association is found between the location of ictal iEEG activity and the location of areas of relative decrease of localised EEG correlation. The latter could serve as a clinically important quantitative marker of the seizure onset zone (SOZ).

Comparison of cartilage histopathology assessment systems on human knee joints at all stages of osteoarthritis development

To compare the MANKIN and OARSI cartilage histopathology assessment systems using human articular cartilage from a large number of donors across the adult age spectrum representing all levels of cartilage degradation.

CRF receptors in the rodent and human cardiovascular systems: Species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Protein buffering in model systems and in whole human saliva

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva.

Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation. Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation. Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Stress free configuration of the human eye

Numerical simulations of eye globes often rely on topographies that have been measured in vivo using devices such as the Pentacam or OCT. The topographies, which represent the form of the already stressed eye under the existing intraocular pressure, introduce approximations in the analysis. The accuracy of the simulations could be improved if either the stress state of the eye under the effect of intraocular pressure is determined, or the stress-free form of the eye estimated prior to conducting the analysis. This study reviews earlier attempts to address this problem and assesses the performance of an iterative technique proposed by Pandolfi and Holzapfel [1], which is both simple to implement and promises high accuracy in estimating the eye's stress-free form. A parametric study has been conducted and demonstrated reliance of the error level on the level of flexibility of the eye model, especially in the cornea region. However, in all cases considered 3-4 analysis iterations were sufficient to produce a stress-free form with average errors in node location <10(-6)mm and a maximal error <10(-4)mm. This error level, which is similar to what has been achieved with other methods and orders of magnitude lower than the accuracy of current clinical topography systems, justifies the use of the technique as a pre-processing step in ocular numerical simulations.

Effects of flame made zinc oxide particles in human lung cells - a comparison of aerosol and suspension exposures

Background Predominantly, studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach however, does not reflect particle inhalation. As a more realistic simulation of such a scenario, efforts were made towards direct delivery of aerosols to air-liquid-interface cultivated cell cultures by the use of aerosol exposure systems. This study aims to provide a direct comparison of the effects of zinc oxide (ZnO) NPs when delivered as either an aerosol, or in suspension to a triple cell co-culture model of the epithelial airway barrier. To ensure dose–equivalence, ZnO-deposition was determined in each exposure scenario by atomic absorption spectroscopy. Biological endpoints being investigated after 4 or 24h incubation include cytotoxicity, total reduced glutathione, induction of antioxidative genes such as heme-oxygenase 1 (HO–1) as well as the release of the (pro)-inflammatory cytokine TNFα. Results Off-gases released as by-product of flame ZnO synthesis caused a significant decrease of total reduced GSH and induced further the release of the cytokine TNFα, demonstrating the influence of the gas phase on aerosol toxicology. No direct effects could be attributed to ZnO particles. By performing suspension exposure to avoid the factor “flame-gases”, particle specific effects become apparent. Other parameters such as LDH and HO–1 were not influenced by gaseous compounds: Following aerosol exposure, LDH levels appeared elevated at both timepoints and the HO–1 transcript correlated positively with deposited ZnO-dose. Under submerged conditions, the HO–1 induction scheme deviated for 4 and 24h and increased extracellular LDH was found following 24h exposure. Conclusion In the current study, aerosol and suspension-exposure has been compared by exposing cell cultures to equivalent amounts of ZnO. Both exposure strategies differ fundamentally in their dose–response pattern. Additional differences can be found for the factor time: In the aerosol scenario, parameters tend to their maximum already after 4h of exposure, whereas under submerged conditions, effects appear most pronounced mainly after 24h. Aerosol exposure provides information about the synergistic interplay of gaseous and particulate phase of an aerosol in the context of inhalation toxicology. Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas–derived effects.

Evaluation of two fully automated novel enzyme-linked immunosorbent assays for the determination of human adiponectin in serum

BACKGROUND: In contrast to RIA, recently available ELISAs provide the potential for fully automated analysis of adiponectin. To date, studies reporting on the diagnostic characteristics of ELISAs and investigating on the relationship between ELISA- and RIA-based methods are rare. METHODS: Thus, we established and evaluated a fully automated platform (BEP 2000; Dade-Behring, Switzerland) for determination of adiponectin levels in serum by two different ELISA methods (competitive human adiponectin ELISA; high sensitivity human adiponectin sandwich ELISA; both Biovendor, Czech Republic). Further, as a reference method, we also employed a human adiponectin RIA (Linco Research, USA). Samples from 150 patients routinely presenting to our cardiology unit were tested. RESULTS: ELISA measurements could be accomplished in less than 3 h, measurement of RIA had a duration of 24 h. The ELISAs were evaluated for precision, analytical sensitivity and specificity, linearity on dilution and spiking recovery. In the investigated patients, type 2 diabetes, higher age and male gender were significantly associated with lower serum adiponectin concentrations. Correlations between the ELISA methods and the RIA were strong (competitive ELISA, r=0.82; sandwich ELISA, r=0.92; both p<0.001). However, Deming regression and Bland-Altman analysis indicated lack of agreement of the 3 methods preventing direct comparison of results. The equations of the regression lines are: Competitive ELISA=1.48 x RIA-0.88; High sensitivity sandwich ELISA=0.77 x RIA+1.01. CONCLUSIONS: Fully automated measurement of adiponectin by ELISA is feasible and substantially more rapid than RIA. The investigated ELISA test systems seem to exhibit analytical characteristics allowing for clinical application. In addition, there is a strong correlation between the ELISA methods and RIA. These findings might promote a more widespread use of adiponectin measurements in clinical research.

TLR2 and TLR4 agonists induce production of the vasoactive peptide endothelin-1 by human dendritic cells

Endothelin-1 (ET-1) is mainly secreted by endothelial cells and acts as a potent vasoconstrictor. In addition ET-1 has also been shown to have pleiotropic effects on a variety of other systems including adaptive immunity. There are two main ET-1 receptors, ET(A) and ET(B), which have different tissue and functional distributions. Dendritic cells (DC) are pivotal antigen-presenting cells linking the innate with the adaptive immune system. DC are sentinels expressing pattern-recognition receptors, e.g. the toll-like receptors (TLR) for detecting danger signals released from pathogens or tissue injury. Here we show for the first time that stimulation of human monocyte-derived DC with exogenous as well as endogenous selective TLR4 and TLR2 agonists induces the production of ET-1 in a dose- and time-dependent manner. 'Alternative' activation of DC in the presence of 1alpha,25-dihydroxyvitamin D(3) results in a marked potentiation of the endothelin response, whereas prostaglandin E(2) or dexamethasone do not increase ET-1 production. Furthermore, chetomin, an inhibitor of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), prevents TLR-mediated secretion of ET-1. Surprisingly, stimulation of human monocytes with LPS does not lead to secretion of detectable amounts of ET-1. These results suggest a role of ET-1 as an important player in human DC biology and innate immunity in general.