The impact of DMARD and anti-TNF therapy on functional characterization of short-term T-cell activation in patients with rheumatoid arthritis - A follow-up study

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a systemic dysfunction of T-cells. In this study we tested the impact of DMARD and anti-TNF agents on short-term activation characteristics of T-cells. We enrolled 12 patients with newly diagnosed RA (naïve RA) who were treated with methothrexate (MTX) and glucocorticsteroid (GCS) and 22 patients with established RA non responding to conventional DMARD therapy who were treated with different anti-TNF agents. Nine healthy volunteers served as controls. Blood samples were taken at baseline, then at 4th and 8th week of therapy. The characteristics of several intracellular activation processes during short-term activation of T-cells including cytoplasmic Ca2+ level, mitochondrial Ca2+ level, reactive oxygen species (ROS) and nitric oxide (NO) generation were determined by a novel flow-cytometry technique. At baseline, the tested processes were comparable to controls in naïve RA. During GCS therapy, cytoplasmic Ca2+ level and ROS generation decreased. After the addition of MTX to GCS cytoplasmic Ca2+ level became comparable to controls, while ROS generation decreased further. In DMARD non responders, cytoplasmic Ca2+ level was higher than controls at baseline. The cytoplasmic Ca2+ level became comparable to controls and ROS generation decreased during each of the three anti-TNF-α agent therapies. Mitochondrial Ca2+ level and NO generation were unaltered in all of the patient groups. These results indicate that intracellular machinery is affected in T-cells of RA patients. This may alter the behavior of T-cells during activation. Different therapeutic approaches may modulate the abnormal T-cell functions. © 2014 Szalay et al

Measuring Fluorescent Dye in the Bubbly and Sediment-Laden Surfzone

Decisions about recreational beach closures would be enhanced if better estimates of surfzone contaminant transport and dilution were available. In situ methods for measuring fluorescent Rhodamine WT dye tracer in the surfzone are presented, increasing the temporal and spatial resolution over previous surfzone techniques. Bubbles and sand suspended by breaking waves in the surfzone interfere with in situ optical fluorometer dye measurements, increasing the lower bound for dye detection (≈ 1 ppb) and reducing (quenching) measured dye concentrations. Simultaneous turbidity measurements are used to estimate the level of bubble and sand interference and correct dye estimates. After correction, root-mean-square dye concentration errors are estimated to be < 5% of dye concentration magnitude, thus demonstrating the viability of in situ surfzone fluorescent dye measurements. The surfzone techniques developed here may be applicable to other environments with high bubble and sand concentrations (e.g., cascading rivers and streams)

New physical characterization of the Fontana Lapilli basaltic Plinian eruption, Nicaragua

The Fontana Lapilli deposit was erupted in the late Pleistocene from a vent, or multiple vents, located near Masaya volcano (Nicaragua) and is the product of one of the largest basaltic Plinian eruptions studied so far. This eruption evolved from an initial sequence of fluctuating fountain-like events and moderately explosive pulses to a sustained Plinian episode depositing fall beds of highly vesicular basaltic-andesite scoria (SiO2 > 53 wt%). Samples show unimodal grain size distribution and a moderate sorting that are uniform in time. The juvenile component predominates (> 96 wt%) and consists of vesicular clasts with both sub-angular and fluidal, elongated shapes. We obtain a maximum plume height of 32 km and an associated mass eruption rate of 1.4 × 108 kg s−1 for the Plinian phase. Estimates of erupted volume are strongly sensitive to the technique used for the calculation and to the distribution of field data. Our best estimate for the erupted volume of the majority of the climactic Plinian phase is between 2.9 and 3.8 km3 and was obtained by applying a power-law fitting technique with different integration limits. The estimated eruption duration varies between 4 and 6 h. Marine-core data confirm that the tephra thinning is better fitted by a power-law than by an exponential trend

A theoretical entropy score as a single value to express inhibitor selectivity

<p>Abstract</p> <p>Background</p> <p>Designing maximally selective ligands that act on individual targets is the dominant paradigm in drug discovery. Poor selectivity can underlie toxicity and side effects in the clinic, and for this reason compound selectivity is increasingly monitored from very early on in the drug discovery process. To make sense of large amounts of profiling data, and to determine when a compound is sufficiently selective, there is a need for a proper quantitative measure of selectivity.</p> <p>Results</p> <p>Here we propose a new theoretical entropy score that can be calculated from a set of IC₅₀data. In contrast to previous measures such as the 'selectivity score', Gini score, or partition index, the entropy score is non-arbitary, fully exploits IC₅₀data, and is not dependent on a reference enzyme. In addition, the entropy score gives the most robust values with data from different sources, because it is less sensitive to errors. We apply the new score to kinase and nuclear receptor profiling data, and to high-throughput screening data. In addition, through analyzing profiles of clinical compounds, we show quantitatively that a more selective kinase inhibitor is not necessarily more drug-like.</p> <p>Conclusions</p> <p>For quantifying selectivity from panel profiling, a theoretical entropy score is the best method. It is valuable for studying the molecular mechanisms of selectivity, and to steer compound progression in drug discovery programs.</p

Collagen reorganization at the tumor-stromal interface facilitates local invasion

BACKGROUND: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. METHODS: Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen. RESULTS: We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers. CONCLUSION: The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.National Institutes of Health (U.S.) (Grant HL052212)National Institutes of Health (U.S.) (Grant HL066105)National Institutes of Health (U.S.) (Grant ES015241)National Institutes of Health (U.S.) (Grant GM068762

The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

The maternal separation paradigm has been applied to C57BL/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology

Peripheral Nervous System Genes Expressed in Central Neurons Induce Growth on Inhibitory Substrates

Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons

Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1