Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Single-pulse transcranial magnetic stimulation over the frontal eye field can facilitate and inhibit saccade triggering

The aim of this study was to investigate the effect of single-pulse transcranial magnetic stimulation on the triggering of saccades. The right frontal eye field was stimulated during modified gap and overlap paradigms with flashed presentation of the lateral visual target of 80 ms. In order to examine possible facilitating or inhibitory effects on saccade triggering, three different time intervals of stimulation were chosen, i.e. simultaneously with onset of the target, during the presentation and after target end. Stimulation applied simultaneously with target onset significantly decreased the latency of contralateral saccades in the gap but not in the overlap paradigm. Stimulation after target end significantly increased saccade latency for both sides in the gap paradigm and for the contralateral side in the overlap paradigm. Stimulation during presentation had no effect in either paradigm. The results show that, depending on the time interval and the paradigm tested, a facilitation or inhibition of saccade triggering can be achieved. The results are discussed in a context of two probable transcranial magnetic stimulation effects, a direct interference with the frontal eye field on the one hand and a remote interference with the superior colliculus on the other hand.

Novel weapons: Invasive plant suppresses fungal mutualists in America but not in its native Europe

Why some invasive plant species transmogrify from weak competitors at home to strong competitors abroad remains one of the most elusive questions in ecology. Some evidence suggests that disproportionately high densities of some invaders are due to the release of biochemicals that are novel, and therefore harmful, to naive organisms in their new range. So far, such evidence has been restricted to the direct phytotoxic effects of plants on other plants. Here we found that one of North America's most aggressive invaders of undisturbed forest understories, Alliaria petiolata (garlic mustard) and a plant that inhibits mycorrhizal fungal mutualists of North American native plants, has far stronger inhibitory effects on mycorrhizas in invaded North American soils than on mycorrhizas in European soils where A. petiolata is native. This antifungal effect appears to be due to specific flavonoid fractions in A. petiolata extracts. Furthermore, we found that suppression of North American mycorrhizal fungi by A. petiolata corresponds with severe inhibition of North American plant species that rely on these fungi, whereas congeneric European plants are weakly affected. These results indicate that phytochemicals, benign to resistant mycorrhizal symbionts in the home range, may be lethal to naive native mutualists in the introduced range and indirectly suppress the plants that rely on them.

Annexin-1 regulates macrophage IL-6 and TNF via glucocorticoid-induced leucine zipper

Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In response to LPS administration, ANXA1(-/-) mice exhibited significantly increased serum IL-6 and TNF compared with wild-type (WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1(-/-) than in WT peritoneal macrophages in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accompanied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1(-/-) cells. In contrast, GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1(-/-) cells by silencing of ANXA1 in WT cells and in macrophages of ANXA1(-/-) mice in vivo. GC-induced GILZ expression and GC inhibition of NF-kappaB activation were restored by expression of ANXA1 in ANXA1(-/-) cells, and GILZ overexpression in ANXA1(-/-) macrophages reduced ERK MAPK phosphorylation and restored sensitivity of cytokine expression and NF-kappaB activation to GC. These data confirm ANXA1 as a key inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory effects of ANXA1.

Late cardiac sodium current can be assessed using automated patch-clamp

The cardiac late Na (+) current is generated by a small fraction of voltage-dependent Na (+) channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na (+) component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

ABCB5 Identifies Immunoregulatory Dermal Cells

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis

OBJECTIVE The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Does acupuncture needling induce analgesic effects comparable to diffuse noxious inhibitory controls?

Diffuse noxious inhibitory control (DNIC) is described as one possible mechanism of acupuncture analgesia. This study investigated the analgesic effect of acupuncture without stimulation compared to nonpenetrating sham acupuncture (NPSA) and cold-pressor-induced DNIC. Forty-five subjects received each of the three interventions in a randomized order. The analgesic effect was measured using pressure algometry at the second toe before and after each of the interventions. Pressure pain detection threshold (PPDT) rose from 299 kPa (SD 112 kPa) to 364 kPa (SD 144), 353 kPa (SD 135), and 467 kPa (SD 168) after acupuncture, NPSA, and DNIC test, respectively. There was no statistically significant difference between acupuncture and NPSA at any time, but a significantly higher increase of PPDT in the DNIC test compared to acupuncture and NPSA. PPDT decreased after the DNIC test, whereas it remained stable after acupuncture and NPSA. Acupuncture needling at low pain stimulus intensity showed a small analgesic effect which did not significantly differ from placebo response and was significantly less than a DNIC-like effect of a painful noninvasive stimulus.

Antifibrotic effects of tocotrienols on human Tenon's fibroblasts

Antifibrotic effects of α- (40, 60, 80, 100, and 120 μM), γ- (10, 20, 30, and 40 μM) and δ-tocotrienol (10, 20, 30, and 40 μM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7 days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100 μg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. Results All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for α-tocotrienol 80 μM with 36.7% and at day 7 for α-tocotrienol 80 μM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80 μM for α- and above 30 μM for γ- and δ-tocotrienol. The highest collagen synthesis inhibition has been found with 80 µM α-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 µM α- and 30 µM γ-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. Conclusion In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon’s fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surger

Inhibitory regulation of dendritic activity in vivo

The spatiotemporal control of neuronal excitability is fundamental to the inhibitory process. We now have a wealth of information about the active dendritic properties of cortical neurons including axonally generated sodium action potentials as well as local sodium spikelets generated in the dendrites, calcium plateau spikes, and NMDA spikes. All of these events have been shown to be highly modified by the spatiotemporal pattern of nearby inhibitory input which can drastically change the output firing mode of the neuron. This means that particular populations of interneurons embedded in the neocortical microcircuitry can more precisely control pyramidal cell output than has previously been thought. Furthermore, the output of any given neuron tends to feed back onto inhibitory circuits making the resultant network activity further dependent on inhibition. Network activity is therefore ultimately governed by the subcellular microcircuitry of the cortex and it is impossible to ignore the subcompartmentalization of inhibitory influence at the neuronal level in order to understand its effects at the network level. In this article, we summarize the inhibitory circuits that have been shown so far to act on specific dendritic compartments in vivo.

In vitro effects of thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture with Caco2 cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

Arginine vasopressin in vasodilatory shock: effects on metabolism and beyond

PURPOSE OF REVIEW: To describe the effects of arginine vasopressin other than its vasoconstrictive and antidiuretic potential in vasodilatory shock. RECENT FINDINGS: Arginine vasopressin influences substrate metabolism by stimulation of hepatic glucose release, gluconeogenesis, ureogenesis and fatty acid esterification. Although arginine vasopressin is a secretagogue of different hormones, only prolactin increases during arginine vasopressin therapy. Plasmatic and cellular coagulation are affected by arginine vasopressin, resulting in thrombocyte aggregation. Therefore, platelet count typically decreases following arginine vasopressin infusion in critically ill patients. In addition, arginine vasopressin reduces bile flow and may increase bilirubin concentrations. Despite its potential to decrease serum sodium, no change in electrolytes was observed in critically ill patients receiving arginine vasopressin. Although arginine vasopressin is an endogenous antipyretic, body temperature is not decreased by central venous arginine vasopressin infusion. In addition, arginine vasopressin modulates immune function through V1 receptors. Compared with norepinephrine, arginine vasopressin may have protective effects on endothelial function. Net arginine vasopressin effects on gastrointestinal motility seem to be inhibitory and are dose dependent. SUMMARY: Except for its antidiuretic and vasoconstrictive actions, the effects of arginine vasopressin in patients with vasodilatory shock have so far only been partially examined. Potential influences of arginine vasopressin on metabolism and immune, liver and mitochondrial function remain to be assessed in future studies.

In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites.

Upon the screening of 16 antiproliferative compounds against Toxoplasma gondii and Neospora caninum, two hydrolytically stable ruthenium complexes (compounds 16 and 18) exhibited 50% inhibitory concentrations of 18.7 and 41.1 nM (T. gondii) and 6.7 and 11.3 nM (N. caninum). To achieve parasiticidal activity with compound 16, long-term treatment (22 to 27 days at 80 to 160 nM) was required. Transmission electron microscopy demonstrated the rapid impact on and ultrastructural alterations in both parasites. These preliminary findings suggest that the potential of ruthenium-based compounds should thus be further exploited.

Being optimally aroused matters: Effects of a weak stress manipulation on children’s executive functions are moderated by temperament and age

We tested a core assumption of the bidirectional model of executive function (EF) (Blair & Ursache, 2011) indicating that EF is dependent on arousal. From a bottom-up perspective the performance on EF tasks is assumed to be curvilinearly related to arousal, with very high or low levels of arousal impairing EF. N = 107 4-and 6-year-olds’ performance on EF tasks was explored as a function of a weak stress manipulation aiming to raise children’s emotional arousal. EF (Stroop, Flanker, Go/no-go, and Backwards Color Recall) was assessed and stress was induced in half of the children by imposing a mild social evaluative threat. Furthermore, children’s temperament was assessed as a potential moderator. We found that stress effects on children’s EF performance were moderated by age and temperament: 4-year-olds with high Inhibitory Control and high Attentional Focusing were negatively affected by the stressor. However, it is unclear whether these effects were mediated by self-reported arousal. Our findings disconfirmed the hypotheses that adverse effects of the stressor are particularly high in children high on emotional reactivity aspects of temperament and low on self-regulatory aspects of temperament. Further, 6-year-olds did not show any stress effects. Results will be discussed within the framework of the Yerkes-Dodson law and with regard to stress manipulations in children.

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.