The colloidal tool-box approach for fuel cell catalysts: Systematic study of perfluorosulfonate-ionomer impregnation and Pt loading

In this study, the correlation between the impregnation of proton exchange membrane fuel cell catalysts with perfluorosulfonate-ionomer (PFSI) and its electrochemical and electrocatalytic properties is investigated for different Pt loadings and carbon supports using a rotating-disk electrode (RDE) setup. We concentrate on its influence on the electrochemical surface area (ECSA) and the oxygen reduction reaction (ORR) activity. For this purpose, platinum (Pt) nanoparticles are prepared via a colloidal based preparation route and supported on three different carbon supports. Based on RDE experiments, we show that the ionomer has an influence both on the Pt utilization and the apparent kinetic current density of ORR. The experimental data reveal a strong interaction in the microstructure between the electrochemical properties and the surface properties of the carbon supports, metal loading and ionomer content. This study demonstrates that the colloidal synthesis approach offers interesting potential for systematic studies for the optimization of fuel cell catalysts.

Interference by the intracellular parasite Theileria parva with T-cell signal transduction pathways induces transformation and protection against apoptosis

The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.

Uncovering the dynamics of cardiac systems using stochastic pacing and frequency domain analyses

Alternans of cardiac action potential duration (APD) is a well-known arrhythmogenic mechanism which results from dynamical instabilities. The propensity to alternans is classically investigated by examining APD restitution and by deriving APD restitution slopes as predictive markers. However, experiments have shown that such markers are not always accurate for the prediction of alternans. Using a mathematical ventricular cell model known to exhibit unstable dynamics of both membrane potential and Ca2+ cycling, we demonstrate that an accurate marker can be obtained by pacing at cycle lengths (CLs) varying randomly around a basic CL (BCL) and by evaluating the transfer function between the time series of CLs and APDs using an autoregressive-moving-average (ARMA) model. The first pole of this transfer function corresponds to the eigenvalue (λalt) of the dominant eigenmode of the cardiac system, which predicts that alternans occurs when λalt≤−1. For different BCLs, control values of λalt were obtained using eigenmode analysis and compared to the first pole of the transfer function estimated using ARMA model fitting in simulations of random pacing protocols. In all versions of the cell model, this pole provided an accurate estimation of λalt. Furthermore, during slow ramp decreases of BCL or simulated drug application, this approach predicted the onset of alternans by extrapolating the time course of the estimated λalt. In conclusion, stochastic pacing and ARMA model identification represents a novel approach to predict alternans without making any assumptions about its ionic mechanisms. It should therefore be applicable experimentally for any type of myocardial cell.

Fossil and non-fossil source contributions to atmospheric carbonaceous aerosols during extreme spring grassland fires in Eastern Europe

In early spring the Baltic region is frequently affected by high-pollution events due to biomass burning in that area. Here we present a comprehensive study to investigate the impact of biomass/grass burning (BB) on the evolution and composition of aerosol in Preila, Lithuania, during springtime open fires. Non-refractory submicron particulate matter (NR-PM1) was measured by an Aerodyne aerosol chemical speciation monitor (ACSM) and a source apportionment with the multilinear engine (ME-2) running the positive matrix factorization (PMF) model was applied to the organic aerosol fraction to investigate the impact of biomass/grass burning. Satellite observations over regions of biomass burning activity supported the results and identification of air mass transport to the area of investigation. Sharp increases in biomass burning tracers, such as levoglucosan up to 683 ngm-3 and black carbon (BC) up to 17 μgm-3 were observed during this period. A further separation between fossil and non-fossil primary and secondary contributions was obtained by coupling ACSM PMF results and radiocarbon (14C) measurements of the elemental (EC) and organic (OC) carbon fractions. Non-fossil organic carbon (OCnf/ was the dominant fraction of PM1, with the primary (POCnf/ and secondary (SOCnf/ fractions contributing 26–44% and 13–23% to the total carbon (TC), respectively. 5–8% of the TC had a primary fossil origin (POCf/, whereas the contribution of fossil secondary organic carbon (SOCf/ was 4–13 %. Nonfossil EC (ECnf/ and fossil EC (ECf/ ranged from 13–24 and 7–13 %, respectively. Isotope ratios of stable carbon and nitrogen isotopes were used to distinguish aerosol particles associated with solid and liquid fossil fuel burning.

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

BACKGROUND: Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface. RESULTS: A novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 mug/cm(2). The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber. Dose-response measurements with ZnO nanoparticles (0.3-8.5 mug/cm(2)) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 mug/cm(2 )ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels. CONCLUSION: The ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Effects of TLR agonists on the hypoxia-regulated transcription factor HIF-1alpha and dendritic cell maturation under normoxic conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1alpha in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1alpha using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1alpha stabilization or activity through different mechanisms. Stabilization of HIF-1alpha protein by hypoxia or CoCl(2) did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1alpha controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1alpha in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1alpha under normoxic conditions with physiological roles that differ from those induced by hypoxia.

Absence of somatostatin SST(2) receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model

Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst(2)) internalization is a potent mechanism for tumor targeting with sst(2) affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst(2) internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst(2) receptors were injected intravenously with SOM230 or with the [Tyr(3), Thr(8)]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst(2) receptors. SOM230-induced sst(2) internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst(2) internalization triggered by intravenous [Tyr(3), Thr(8)]-octreotide, intravenous SOM230 did not elicit sst(2) internalization: immunohistochemically stained sst(2) in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5min, 10min, 1h, 6h, or 24h after SOM230 injection while sst(2) were found intracellularly after [Tyr(3), Thr(8)]-octreotide injection. The inability of stimulating sst(2) internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst(2) internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst(2) internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst(2) binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.