Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Modulation of parasitemia and antibody responce to Trypanosoma cruzy by cyclophosphamide in Calomys callosus (Rodentia, Cricetidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MODULATION OF PARASITEMIA AND ANTIBODY-RESPONSE TO TRYPANOSOMA-CRUZI BY CYCLOPHOSPHAMIDE IN CALOMYS-CALLOSUS (RODENTIA, CRICETIDAE)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan.Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. callosus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10(3) T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callosus to T. cruzi.

Controlling collapse in Bose-Einstein condensates by temporal modulation of the scattering length

A study was conducted on the dynamics of 2D and 3D Bose-Einstein condensates in the case when the scattering length in the Gross-Pitaevskii (GP) equation which contains constant (dc) and time-variable (ac) parts. Using the variational approximation (VA), simulating the GP equation directly, and applying the averaging procedure to the GP equation without the use of the VA, it was demonstrated that the ac component of the nonlinearity makes it possible to maintain the condensate in a stable self-confined state without external traps.

Modulation of cell proliferation, survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Prefrontal cortex modulation using transcranial DC stimulation reduces alcohol craving: A double-blind, sham-controlled study

Background: Functional neuroimaging studies have shown that specific brain areas are associated with alcohol craving including the dorsolateral prefrontal cortex (DLPFC). We tested whether modulation of DLPFC using transcranial direct current stimulation (tDCS) could alter alcohol craving in patients with alcohol dependence while being exposed to alcohol cues. Methods: We performed a randomized sham-controlled study in which 13 subjects received sham and active bilateral tDCS delivered to DLPFC (anodal left/cathodal right and anodal right/cathodal left). For sham stimulation, the electrodes were placed at the same positions as in active stimulation; however, the stimulator was turned off after 30 s of stimulation. Subjects were presented videos depicting alcohol consumption to increase alcohol craving. Results: Our results showed that both anodal left/cathodal right and anodal right/cathodal left significantly decreased alcohol craving compared to sham stimulation (p < 0.0001). In addition, we found that following treatment, craving could not be further increased by alcohol cues. Conclusions: Our findings showed that tDCS treatment to DLPFC can reduce alcohol craving. These findings extend the results of previous studies using noninvasive brain stimulation to reduce craving in humans. Given the relatively rapid suppressive effect of tDCS and the highly fluctuating nature of alcohol craving, this technique may prove to be a valuable treatment strategy within the clinical setting. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Modulation of epidermial growth factor receptor signaling in colon adenocarcinoma

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, monoclonal antibodies (mAbs) and EGFR tyrosine kinase inhibitors (TKIs) seemed to be the most promising. However they have demonstrated low utility in therapy, the former being effective at toxic doses, the latter resulting inefficient in colon cancer. This thesis work presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphtoquinone core as shikonin, an agent with great anti-tumor potential. In HT-29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 μM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer. In addition, surface plasmon resonance (SPR) investigation of the direct EGF/EGFR complex interaction using different experimental approaches is presented. A commercially available purified EGFR was immobilised by amine coupling chemistry on SPR sensor chip and its interaction to EGF resulted to have a KD = 368 ± 0.65 nM. SPR technology allows the study of biomolecular interactions in real-time and label-free with a high degree of sensitivity and specificity and thus represents an important tool for drug discovery studies. On the other hand EGF/EGFR complex interaction represents a challenging but important system that can lead to significant general knowledge about receptor-ligand interactions, and the design of new drugs intended to interfere with EGFR binding activity.

Identifizierung chemischer Transportinhibitoren zur Modulation (patho)biologischer Genprodukte

Die intrazelluläre Lokalisation von Proteinen und Makromolekülen unterliegt in Eukaryoten einer strengen Regulation. Insbesondere erlaubt die Kompartimentierung eukaryotischer Zellen in Zellkern und Zytoplasma den simultanen Ablauf räumlich getrennter biochemischer Reaktionen, und damit die unabhängige Regulation zellulärer Programme. Da trotz intensiver Forschungsbemühungen bis dato die molekularen Details sowie die (patho)biologische Bedeutung von Kern-Zytoplasma-Transportprozessen noch immer nicht vollkommen verstanden sind, wurde im Rahmen der vorliegenden Arbeit ein Fokus auf die Identifizierung von chemischen Transportinhibitoren gelegt. Das zu diesem Zweck entwickelte Translokations-Biosensor-System basiert auf der Kombination von autofluoreszierenden Proteinen, sowie spezifisch ausgewählten Kernexport- und Kernimportsignalen. Nach Etablierung geeigneter Zellmodelle, die effizient und stabil die Translokations-Biosensoren exprimieren, wurde die 17 000 Substanzen umfassende Bibliothek der ChemBioNet-Initiative nach Kernexportinhibitoren mittels einer Fluoreszenzmikroskopie-basierten Hochdurchsatzanalyse-Plattform durchmustert. Zunächst wurden Translokations-Algorithmen, welche eine zuverlässige automatisierte Erkennung von Zellkern und Zytoplasma erlauben, optimiert. Im Folgenden konnten acht neue niedermolekulare Kernexport-Inhibitoren identifiziert werden, die sich in der Stärke, der Geschwindigkeit, sowie in der Beständigkeit der vermittelten Inhibition unterscheiden. Die Aktivität der Inhibitoren konnte auf den isolierten nukleären Exportsignalen (NES) von HIV-1 Rev und Survivin als auch auf den entsprechenden Volllängeproteinen mittels Mikroinjektionsexperimenten sowie durch umfassende in vitro und biochemische Methoden bestätigt werden. Zur Untersuchung der funktionellen Einheiten der Inhibitoren wurden homologe Substanzen auf Ihre Aktivität hin getestet. Dabei konnten für die Aktivität wichtige chemische Gruppen definiert werden. Alle Substanzen stellen neue Inhibitoren des Crm1-abhängigen Exports dar und zeigen keine nachweisbare NES-Selektivität. Interessanterweise konnte jedoch eine zytotoxische und Apoptose-induzierende Wirkung auf verschiedene Krebszellarten festgestellt werden. Da diese Wirkung unabhängig vom p53-Status der Tumorzellen ist und die Inhibitoren C3 und C5 die Vitalität nicht-maligner humaner Zellen signifikant weniger beeinträchtigen, wurden diese Substanzen zum internationalen Patent angemeldet. Da der nukleäre Export besonders für Tumorzellen einen wichtigen Überlebenssignalweg darstellt, könnte dessen reversible Hemmung ausgenutzt werden, um besonders in Kombination mit gängigen Krebstherapien eine therapeutisch relevante Tumorinhibition zu erzeugen. Eine weitere Anwendungsmöglichkeit der neuen Exportinhibitoren ist auf dem Gebiet der Infektionskrankheiten zu sehen, da auch die Aktivität des essentiellen HIV-1 Rev-Proteins inhibiert wird. Zusätzlich konnte in der Arbeit gezeigt werden, dass der zelluläre Kofaktor des Crm1-abhängigen Exports des HIV-1 Rev-Proteins, die RNA-Helikase DDX3, ein eigenes NES enthält. Der Nachweis einer direkten Interaktion des HIV-1 Rev- mit dem DDX3-Protein impliziert, dass multiple Angriffstellen für chemische Modulatoren hinsichtlich einer antiviralen Therapie gegeben sind. Da die Vielfalt des chemischen Strukturraums es unmöglich macht diesen experimentell vollständig zu durchmustern, wurden im Rahmen dieser Arbeit auch Naturstoffe als vielversprechende Wirkstoffquelle untersucht. Um zukünftig umfassend bioaktive Substanzen aus diesen hochkomplexen Stoffgemischen experimentell identifizieren zu können, wurde eine Fluoreszenzmikroskopie-basierte Hochdurchsatzanalyse-Plattform am Mainz Screening Center (MSC) etabliert. Damit konnte bereits ein weiterer, bisher unbekannter Exportinhibitor aus Cyphellopsis anomala identifiziert werden. Neben einer Anwendung dieser Substanz als chemisches Werkzeug zur Aufklärung der Regulation von Transportvorgängen, stellt sich auch die evolutionsbiologisch relevante Frage, wie es dem Pilzproduzenten gelingt die Blockierung des eigenen Kernexports zu umgehen. Weiterführende Projekte müssen sich neben der Aufklärung der molekularen Wirkmechanismen der gefundenen Substanzen mit der Identifizierung spezifischer chemischer „Funktionseinheiten“ beschäftigen. Neben einem verbesserten mechanistischen Verständnis von Transportvorgängen stellen die erarbeiteten Transportinhibitoren Vorstufen zur Weiterentwicklung möglicher Wirkstoffe dar. Die im Rahmen dieser Arbeit etablierte Technologie-Plattform und molekularen Werkzeuge stellen darüber hinaus eine wichtige Voraussetzung dar, um eine systematische Suche nach möglichen Wirkstoffen im Forschungsfeld der „Chemischen Biomedizin“ voranzutreiben.

Novel materials for direct X-ray detectors based on semiconducting organic polymers

Conventional inorganic materials for x-ray radiation sensors suffer from several drawbacks, including their inability to cover large curved areas, me- chanical sti ffness, lack of tissue-equivalence and toxicity. Semiconducting organic polymers represent an alternative and have been employed as di- rect photoconversion material in organic diodes. In contrast to inorganic detector materials, polymers allow low-cost and large area fabrication by sol- vent based methods. In addition their processing is compliant with fexible low-temperature substrates. Flexible and large-area detectors are needed for dosimetry in medical radiotherapy and security applications. The objective of my thesis is to achieve optimized organic polymer diodes for fexible, di- rect x-ray detectors. To this end polymer diodes based on two different semi- conducting polymers, polyvinylcarbazole (PVK) and poly(9,9-dioctyluorene) (PFO) have been fabricated. The diodes show state-of-the-art rectifying be- haviour and hole transport mobilities comparable to reference materials. In order to improve the X-ray stopping power, high-Z nanoparticle Bi2O3 or WO3 where added to realize a polymer-nanoparticle composite with opti- mized properities. X-ray detector characterization resulted in sensitivties of up to 14 uC/Gy/cm2 for PVK when diodes were operated in reverse. Addition of nanoparticles could further improve the performance and a maximum sensitivy of 19 uC/Gy/cm2 was obtained for the PFO diodes. Compared to the pure PFO diode this corresponds to a five-fold increase and thus highlights the potentiality of nanoparticles for polymer detector design. In- terestingly the pure polymer diodes showed an order of magnitude increase in sensitivity when operated in forward regime. The increase was attributed to a different detection mechanism based on the modulation of the diodes conductivity.

Effects of transcranial direct current stimulation (tDCS) on behaviour and electrophysiology of language production

Excitatory anodal transcranial direct current stimulation (A-tDCS) over the left dorsal prefrontal cortex (DPFC) has been shown to improve language production. The present study examined neurophysiological underpinnings of this effect. In a single-blinded within-subject design, we traced effects of A-tDCS compared to sham stimulation over the left DPFC using electrophysiological and behavioural correlates during overt picture naming. Online effects were examined during A-tDCS by employing the semantic interference (SI-)Effect – a marker that denotes the functional integrity of the language system. The behavioural SI-Effect was found to be reduced, whereas the electrophysiological SI-Effect was enhanced over left compared to right temporal scalp-electrode sites. This modulation is suggested to reflect a superior tuning of neural responses within language-related generators. After -(offline) effects of A-tDCS were detected in the delta frequency band, a marker of neural inhibition. After A-tDCS there was a reduction in delta activity during picture naming and the resting state, interpreted to indicate neural disinhibition. Together, these findings demonstrate electrophysiological modulations induced by A-tDCS of the left DPFC. They suggest that A-tDCS is capable of enhancing neural processes during and after application. The present functional and oscillatory neural markers could detect positive effects of prefrontal A-tDCS, which could be of use in the neuro-rehabilitation of frontal language functions.

Electrotonic modulation of cardiac impulse conduction by myofibroblasts

Structural remodeling of the myocardium associated with mechanical overload or cardiac infarction is accompanied by the appearance of myofibroblasts. These fibroblast-like cells express alpha-smooth muscle actin (alphaSMA) and have been shown to express connexins in tissues other than heart. The present study examined whether myofibroblasts of cardiac origin establish heterocellular gap junctional coupling with cardiomyocytes and whether ensuing electrotonic interactions affect impulse propagation. For this purpose, impulse conduction characteristics (conduction velocity [theta] and maximal upstroke velocity [dV/dtmax]) were assessed optically in cultured strands of cardiomyocytes, which were coated with fibroblasts of cardiac origin. Immunocytochemistry showed that cultured fibroblasts underwent a phenotype switch to alphaSMA-positive myofibroblasts that expressed connexin 43 and 45 both among themselves and at contact sites with cardiomyocytes. Myofibroblasts affected theta and dV/dtmax in a cell density-dependent manner; a gradual increase of myofibroblast-to-cardiomyocyte ratios up to 7:100 caused an increase of both theta and dV/dtmax, which was followed by a progressive decline at higher ratios. On full coverage of the strands with myofibroblasts (ratio >20:100), theta fell <200 mm/s. This biphasic dependence of theta and dV/dtmax on myofibroblast density is reminiscent of "supernormal conduction" and is explained by a myofibroblast density-dependent gradual depolarization of the cardiomyocyte strands from -78 mV to -50 mV as measured using microelectrode recordings. These findings suggest that myofibroblasts, apart from their role in structural remodeling, might contribute to arrhythmogenesis by direct electrotonic modulation of conduction and that prevention of their appearance might represent an antiarrhythmic therapeutic target.

Direct intrathecal implantation of mesenchymal stromal cells leads to enhanced neuroprotection via an NFkappaB-mediated increase in interleukin-6 production.

Mesenchymal stromal cell (MSC) therapy has shown promise for the treatment of traumatic brain injury (TBI). Although the mechanism(s) by which MSCs offer protection is unclear, initial in vivo work has suggested that modulation of the locoregional inflammatory response could explain the observed benefit. We hypothesize that the direct implantation of MSCs into the injured brain activates resident neuronal stem cell (NSC) niches altering the intracerebral milieu. To test our hypothesis, we conducted initial in vivo studies, followed by a sequence of in vitro studies. In vivo: Sprague-Dawley rats received a controlled cortical impact (CCI) injury with implantation of 1 million MSCs 6 h after injury. Brain tissue supernatant was harvested for analysis of the proinflammatory cytokine profile. In vitro: NSCs were transfected with a firefly luciferase reporter for NFkappaB and placed in contact culture and transwell culture. Additionally, multiplex, quantitative PCR, caspase 3, and EDU assays were completed to evaluate NSC cytokine production, apoptosis, and proliferation, respectively. In vivo: Brain supernatant analysis showed an increase in the proinflammatory cytokines IL-1alpha, IL-1beta, and IL-6. In vitro: NSC NFkappaB activity increased only when in contact culture with MSCs. When in contact with MSCs, NSCs show an increase in IL-6 production as well as a decrease in apoptosis. Direct implantation of MSCs enhances neuroprotection via activation of resident NSC NFkappaB activity (independent of PI3 kinase/AKT pathway) leading to an increase in IL-6 production and decrease in apoptosis. In addition, the observed NFkappaB activity depends on direct cell contact.

The role of BCL-2 in regulation of apoptosis susceptibility by modulation of glutathione

The role of oxidative stress and apoptosis has recently been recognized as an important determinant in the development of a variety of diseases known to man. The oncogene BCL-2 is known to regulate sensitivity to induction of apoptosis and appears to function in an antioxidant pathway by regulating glutathione. We have investigated various steps in the oxidative stress cascade to determine possible sites of action for BCL-2. The fluorescent probes H2DCFDA, dihydroethidium and cis-parinaric acid were used to quantitate generation of peroxides, superoxide and lipid peroxidation, respectively. While each of these agents was able to detect substantial increases in oxidative stress following exposure of cells to ionizing radiation, there was no significant difference between cells expressing high or low levels of BCL-2. Investigation of mitochondrial dysfunction during apoptosis revealed a possible site of bcl-2 intervention, but, analysis of kinetic events occurring during apoptosis suggested that the observed effect is not in the direct apoptotic effector pathway. When glutathione was studied, localization to the nucleus was observed in cells overexpressing BCL-2 that did not occur in cells lacking BCL-2. Additionally, nuclear accumulation of glutathione was sufficient to block granzyme b-mediated nuclear DNA fragmentation, poly (ADP-ribose) polymerase cleavage and caspase activity suggesting that nuclear accumulation of glutathione via a bcl-2 dependent process is functionally relevant to suppression of apoptosis. Thus, a model system emerges where BCL-2 is able to regulate a cell's ability to prevent apoptosis by modifying the cell's antioxidant systems at the organelle level to compensate for oxidative stresses placed upon it. ^

Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model

Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial direct current stimulation could reduce the infarct size and the number of depolarizations, focal ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15min on and 15min off) starting 45min after middle cerebral artery occlusion and lasting 4h. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but starting soon after middle cerebral artery occlusion and lasting 6h. In both experiments controls were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p=0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048). Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.

Diminishing risk-taking behavior by modulating activity in the prefrontal cortex: a direct current stimulation study.

Studies have shown increased risk taking in healthy individuals after low-frequency repetitive transcranial magnetic stimulation, known to transiently suppress cortical excitability, over the right dorsolateral prefrontal cortex (DLPFC). It appears, therefore, plausible that differential modulation of DLPFC activity, increasing the right while decreasing the left, might lead to decreased risk taking, which could hold clinical relevance as excessively risky decision making is observed in clinical populations leading to deleterious consequences. The goal of the present study was to investigate whether risk-taking behaviors could be decreased using concurrent anodal transcranial direct current stimulation (tDCS) of the right DLPFC, which allows upregulation of brain activity, with cathodal tDCS of the left DLPCF, which downregulates activity. Thirty-six healthy volunteers performed the risk task while they received either anodal over the right with cathodal over the left DLPFC, anodal over the left with cathodal over the right DLPFC, or sham stimulation. We hypothesized that right anodal/left cathodal would decrease risk-taking behavior compared with left anodal/right cathodal or sham stimulation. As predicted, during right anodal/left cathodal stimulation over the DLPFC, participants chose more often the safe prospect compared with the other groups. Moreover, these participants appeared to be insensitive to the reward associated with the prospects. These findings support the notion that the interhemispheric balance of activity across the DLPFCs is critical in decision-making behaviors. Most importantly, the observed suppression of risky behaviors suggests that populations with boundless risk-taking behaviors leading to negative real-life consequences, such as individuals with addiction, might benefit from such neuromodulation-based approaches.