Azione antiossidante del sulforafane: analisi in cellule cardiache in coltura ed in un modello animale di esercizio fisico

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

Molekulare Charakterisierung der Centrin-Isoformen in der Retina von Säugetieren

Centrine sind Mitglieder einer hoch konservierten Überfamilie von Ca2+-bindenden Proteinen mit EF-Hand Motiven. Bislang sind vier Centrin-Isoformen bei Säugern beschrieben worden, die in diversen Zellen in der Regel mit Centriolen von Centrosomen oder Centrosomen-verwandten Strukturen assoziiert sind. Im Rahmen der vorliegenden Dissertation wurden die vier Centrin-Isoformen bezüglich der Expression in verschiedenen Geweben untersucht. Dabei lag der Hauptfokus auf Untersuchungen der Centrine in den Photorezeptorzellen der Retina. Analysen auf subzellulärer Ebene brachten Klarheit über die differenzielle Lokalisation der verschiedenen Isoformen in der Retina. Mit Hilfe von verschiedenen Methoden konnten Wechselwirkungspartner in der Retina identifiziert werden, die eine Rolle in der visuellen Signaltransduktionskaskade spielen. Dabei könnten Centrine einem Regelmechanismus angehören, der wichtige Translokationsprozesse dieser Proteine regelt. In den Photorezeptorzellen der Säugetierretina werden die vier Isoformen exprimiert, die in den Strukturen des Cilienapparates differenziell lokalisiert sind. Dabei beschränkt sich ihre Lokalisation entweder auf den Basalkörper (Centrin 4), auf das Verbindungscilium (Centrin 1) oder sie sind in beiden Strukturen zu finden (Centrin 2 und 3). In den nicht- Photorezeptorzellen der Retina sind die Isoformen Centrin 2 und 3 zudem an den Centriolen der Centrosomen lokalisiert. In der vorliegenden Arbeit wurde zum ersten Mal gezeigt, dass alle Centrin-Isoformen in ein und derselben Zelle, der Photorezeptorzelle, koexprimiert werden und dabei subzellulär kolokalisiert sind. Im Weiteren konnte die ubiquitäre Expression von Centrin 2 und 3 in allen untersuchten Geweben an Centrosomen bestätigt werden. Centrin 1 und 4 hingegen werden nur in Geweben mit Cilien-tragenden Zellen exprimiert. Die Funktion der Centrine wird nicht nur durch Bindung von Ca2+, sondern auch durch Phosphorylierungen reguliert. Alle Sequenzen der Centrine weisen diverse mögliche Phosphorylierungsstellen für unterschiedliche Proteinkinasen auf. Die Ergebnisse aller durchgeführten in vitro und ex vivo Phosphorylierungs „Assays“ zeigen eine licht-abhängige Phosphorylierung der Centrin-Isoformen in der Retina. Dabei war in der dunkel-adaptierten Retina die Phosphorylierung vor allem von Centrin 1 und 2 erhöht. Weiterführende Experimente mit Kinase-Inhibitoren wiesen darauf hin, dass vor allem die Proteinkinase CKII eine bedeutende Rolle bei der Centrin-Phosphorylierung in der Retina einnimmt. Centrine sind die ersten Cytoskelettkomponenten, deren Phosphorylierungsgrad lichtabhängig moduliert wird. Diese Ergebnisse weisen auf einen Signalweg, der zwischen der visuellen Signaltransduktionskaskade und der Regulation der Centrin-Aktivität vermittelt, hin. Bei der Suche nach Centrin-Bindungspartnern gelang mit Hilfe von Centrin 1 Blot „Overlay Assays“ der Durchbruch. Der neuartige Ansatz zeigte, dass ausschließlich Ca2+-aktiviertes Centrin 1 mit Proteinen aus der Retina interagierte. Nach der Identifikation eines 37 kDa-Proteins als die β-Untereinheit des visuellen G-Proteins Transducin wurden die Untersuchungen auf diesen Interaktionspartner fokussiert. Die Ergebnisse der hier durchgeführten biochemischen und biophysikalischen Protein-Protein Interaktionsexperimente zeigen insgesamt folgendes: ⇒ Alle vier Centrine interagieren mit Transducin, wobei Centrin 3 die geringste Affinität zu Transducin hat. ⇒ Die Assemblierung der Centrin•G-Protein-Komplexe ist strikt Ca2+-abhängig. ⇒ Die Centrine binden sowohl an das isolierte Gtβγ-Heterodimer als auch an den heterotrimeren Gt-holo-Proteinkomplex, nicht aber an Gtα. Die quantitativen immunoelektronenmikroskopischen Analysen zeigen im Weiteren, dass sich die Komplexe aus Transducin und Centrin 1 bis 3 wahrscheinlich in einer Subdomäne des Verbindungsciliums der Photorezeptorzellen ausbilden. Dabei dürfte die Ausbildung der Komplexe an der Regulation der lichtinduzierten Translokation von Transducin zwischen Innen- und Außensegment der Photorezeptorzellen beteiligt sein. Dieser Translokationsmechanismus wird als ein wichtiger Bestandteil der Langzeitadaption der Signaltransduktionskaskade der Säugerretina diskutiert. Der neuartige Regelmechanismus der molekularen Translokationen, in dem Centrine involviert sind, ist außergewöhnlich und dürfte über die speziellen Photorezeptorzellen hinaus von weit reichender Bedeutung sein.

New platforms for optical biosensing

Physicochemical experimental techniques combined with the specificity of a biological recognition system have resulted in a variety of new analytical devices known as biosensors. Biosensors are under intensive development worldwide because they have many potential applications, e.g. in the fields of clinical diagnostics, food analysis, and environmental monitoring. Much effort is spent on the development of highly sensitive sensor platforms to study interactions on the molecular scale. In the first part, this thesis focuses on exploiting the biosensing application of nanoporous gold (NPG) membranes. NPG with randomly distributed nanopores (pore sizes less than 50 nm) will be discussed here. The NPG membrane shows unique plasmonic features, i.e. it supports both propagating and localized surface plasmon resonance modes (p SPR and l-SPR, respectively), both offering sensitive probing of the local refractive index variation on/in NPG. Surface refractive index sensors have an inherent advantage over fluorescence optical biosensors that require a chromophoric group or other luminescence label to transduce the binding event. In the second part, gold/silica composite inverse opals with macroporous structures were investigated with bio- or chemical sensing applications in mind. These samples combined the advantages of a larger available gold surface area with a regular and highly ordered grating structure. The signal of the plasmon was less noisy in these ordered substrate structures compared to the random pore structures of the NPG samples. In the third part of the thesis, surface plasmon resonance (SPR) spectroscopy was applied to probe the protein-protein interaction of the calcium binding protein centrin with the heterotrimeric G-protein transducin on a newly designed sensor platform. SPR spectroscopy was intended to elucidate how the binding of centrin to transducin is regulated towards understanding centrin functions in photoreceptor cells.

Charakterisierung funktioneller Domänen von Centrin-Isoformen

Centrine sind kleine Ca2+-bindende Proteine aus der Familie der EF-Hand Proteine. Erstmals wurden Centrine als Hauptbestandteil der kontraktilen Flagellenwurzeln von Grünalgen beschrieben. Mittlerweile konnten Centrine in nahezu allen eukaryotischen Organismen nachgewiesen werden. In Säugetieren wurden bis zu vier Isoformen identifiziert, die an Centrosomen oder davon abgeleiteten Strukturen, wie Spindelpolkörpern und Basalkörper, aber auch in Übergangszonen von Cilien exprimiert werden. In der vorliegenden Arbeit konnte gezeigt werden, dass die Centrine im zellulären Kontext der Photorezeptorzellen nicht nur durch die Bindung von Ca2+ reguliert werden, sondern auch durch reversible Phosphorylierungen. Die Phosphorylierung der Centrin-Isoformen findet in der Retina von Vertebraten lichtabhängig während der Dunkeladaption statt. Die Protein Kinase CK2 (CK2) ist für die beschriebenen lichtabhängigen Phosphorylierungen hauptverantwortlich. Obwohl alle Centrin-Isoformen mehrere mögliche Zielsequenzen für die CK2 besitzen, kommt es nur zur Phosphorylierung einer einzigen Aminosäure in Cen1p, Cen2p und Cen4p. Im Gegensatz dazu stellt die Isoform Cen3p kein Substrat für die CK2 dar. Zudem wurden hier erstmals Phosphatasen identifiziert, die in der Lage sind Centrine zu dephosphorylieren. Die Dephosphorylierung durch die PP2Cund PP2C ist sehr spezifisch, da keine andere Phosphatase der Retina die CK2-vermittelte Phosphorylierung der Centrine rückgängig machen kann. Hoch auflösende licht- und elektronenmikroskopische Analysen zeigten erstmals, dass die Centrine sowohl mit der CK2 als auch mit der PP2C im Verbindungscilium der Photorezeptorzellen colokalisiert sind. Cen1p und CK2 sind in der Lage, direkt an Mikrotubuli zu binden, was die notwendige räumliche Nähe zwischen Enzymen und Substrat herstellt. Bisherige Arbeiten zeigten, dass alle Centrine Ca2+-abhängig mit dem visuellen G-Protein Transducin interagieren. Diese Wechselwirkung dürfte an der Regulation der lichtabhängigen Translokation des visuellen G-Proteins Transducin zwischen dem Außen- und dem Innensegment der Photorezeptorzelle beteiligt sein. In der vorliegenden Arbeit zeigten Interaktionsstudien, dass die Bindungsaffinitäten der Centrine für Transducin durch die CK2-vermittelte Phosphorylierung drastisch verringert wurden. Dieser beobachtete Effekt beruht auf deutlich verringerten Ca2+-Affinitäten der Centrin-Isoformen nach der CK2-vermittelten Phosphorylierung. In der vorliegenden Arbeit wurde ein neuartiger Regulationsmechanismus der Centrine in den Photorezeptorzellen der Vertebraten beschrieben. Centrine werden nicht nur durch Ca2+-Bindung zur Bildung von Protein Komplexen stimuliert, sondern durch die Phosphorylierung zum Auflösen dieser Komplexe angeregt. Damit reguliert die CK2-vermittelte, lichtabhängige Phosphorylierung der Centrine möglicherweise ebenfalls die adaptive Translokation des visuellen G-Proteins Transducin zwischen dem Außen- und Innensegment der Photorezeptorzellen.

Host immune response in immunodeficient mice against infection with Cryptosporidium parvum

Immunantwort von immundefizienten Mäusen gegenüber Infektionen mit Cryptosporidium parvum. Cryptosporidium parvum ist ein intrazellulärer, protozoischer Krankheitserreger, der im immunkompromittierten Wirt zu lebensbedrohender Enteritis führen kann. CD4+ T-Zellen und Interferon (IFN)-γ spielen wesentliche Rollen bei der Wirtsimmunantwort gegen die Infektion. Dennoch sind die Effektormechanismen, die zur Resistenz führen nur wenig verstanden. In dieser Studie wurde die Immunantwort von IFN-γ- und Interleukin (IL)-12-Defektmäusen parallel zu Wildtypmäusen analysiert. Die Ergebnisse identifizierten IFN-γ als Schlüsselzytokin bei der natürlichen und erworbenen Immunität während der Erst- und Folgeinfektion mit C. parvum. Tumornekrosefaktor (TNF)-α ist möglicherweise ein Induktor der frühen IFN-γ-Antwort in IL-12 Knockout-Mäusen. Weiterhin tragen offenbar sowohl Th1- als auch Th2-Zytokine zur Überwindung der Primärinfektion bei, die ersten mehr als die letztgenannten. Zytokingene waren am Ort der Infektion (Ileum) dramatisch verändert, nicht aber in den lokalen Lymphknoten und der Milz. Nach Folgeinfektion ergab sich in Abwesenheit von IFN-γ eine signifikante Erhöhung der Th2-Zytokine IL-5 and IL-13. Die Ergebnisse zeigten weiterhin, dass das Th1-Zytokin IL-18 zur Resistenz gegenüber C. parvum beiträgt, möglicherweise durch verschiedene Immunfunktionen, wie der Regulation von Serum-IFN-γ während der Infektion und/oder der Erhaltung der Homeostase der Th1/Th2-Zytokine durch Regulation der Th2-Zytokine. Weiterhin zeigten diese Untersuchungen den Transfer von Resistenz gegenüber C. parvum von infizierten auf naïve Mäuse mittels stimulierter intraepithelialer Lymphozyten und CD4+ T-Zellen. Diese Ergebnisse weisen auf die Gegenwart von C. parvum-spezifischen CD4+ T-Zellen in anderen lymphatischen Geweben neben der Darmmukosa hin. Eine Stimulation der Spendertiere durch Infektion war notwendig für eine übertragbare schützende Immunität. Dennoch konnte die übertragene Immunität nicht die Infektion der Empfängertiere vollständig verhindern; eine Verdopplung der Spenderzellen führte zu keinem besseren Ergebnis. Weiterhin ergab der Transfer von CD4+ und CD8+ T-Zellen (Pan-T-Zellen) keinen erhöhten Schutz der naiven Empfängertiere als der alleinige Transfer von CD4+ T-Zellen. Dies weist auf die fehlende Bedeutung der CD8+ T-Zellen beim Schutz vor C. parvum-Infektion hin.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

[Osteoporosis diet]

Bone requires a wide variety of nutrients to develop normally and to maintain itself after growth. Most important--in the sense that bony abnormalities are associated with their deficiencies--are protein, calcium, phosphorus, vitamin D, C and K, zinc, manganese and copper. The nutrients most likely to be deficient in citizens of industrialized countries are calcium and vitamin D. In this review of the current literature about nutritional aspects of osteoporosis, we have focused on factors influencing calcium requirement: the principal interacting nutrients are sodium, protein, caffeine, fiber, oxalate, phytate, and the acid/alkaline ash character of the overall diet. Fiber and caffeine decrease calcium absorption from the gut and typically exert relatively minor effects, while sodium, protein and the acid/alkaline balance of the diet increase urinary excretion of calcium and are of much greater significance for the calcium homeostasis. Alkali buffers, whether vegetables or fruits reverse this urinary calcium loss. As long as accompanied by adequate calcium intake, protein-rich diet is not deleterious to bone: a calcium-to-protein ratio of 20:1 (mg calcium/g protein) is recommended. Whether a nutrition-based therapeutic approach to osteoporosis is feasible in the near future is yet unclear: at least there are some recent promising data from in-vitro as well as from rat studies showing that extracts taken from various vegetables, mainly from the onion family inhibit bone resorption in a dose-dependent manner.

Molecular Determinants Defining the Triggering Range of Prefusion F Complexes of Canine Distemper Virus

The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this "pocket'" appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE: With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering "stimulus" (attachment protein and receptor types) define a "triggering range," which governs the initiation of the membrane fusion process. A central "pocket" microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency.

A Normative Theory of Forgetting: Lessons from the Fruit Fly

Recent experiments revealed that the fruit fly Drosophila melanogaster has a dedicated mechanism for forgetting: blocking the G-protein Rac leads to slower and activating Rac to faster forgetting. This active form of forgetting lacks a satisfactory functional explanation. We investigated optimal decision making for an agent adapting to a stochastic environment where a stimulus may switch between being indicative of reward or punishment. Like Drosophila, an optimal agent shows forgetting with a rate that is linked to the time scale of changes in the environment. Moreover, to reduce the odds of missing future reward, an optimal agent may trade the risk of immediate pain for information gain and thus forget faster after aversive conditioning. A simple neuronal network reproduces these features. Our theory shows that forgetting in Drosophila appears as an optimal adaptive behavior in a changing environment. This is in line with the view that forgetting is adaptive rather than a consequence of limitations of the memory system.

Vaginal cytokines do not differ between postmenopausal women with and without symptoms of vulvovaginal irritation.

OBJECTIVE The aim of this exploratory pilot study was to determine if there are differences in vaginal cytokine levels between postmenopausal women with and without vulvovaginal irritative symptoms (itching, burning, or pain). METHODS Postmenopausal women (n = 34) not using hormone therapy and presenting with or without symptoms of vulvovaginal irritation were asked to volunteer for this study. Each participant underwent a vaginal examination and screening for vaginitis using Amsel criteria, pH, and light microscopy. A vaginal lavage with 5.0 mL of sterile saline was carried out, and a peripheral blood sample was obtained. The vaginal lavage and serum samples were assayed for interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α by specific enzyme-linked immunosorbent assays. Results were adjusted for total protein concentration and presented as the amount of cytokines per protein (pg/μg protein). Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). The means and SDs of all variables among women with and without vulvovaginal irritation were compared using independent-samples Student's t test. RESULTS A total of 26 postmenopausal women were enrolled into the study (symptomatic, n = 15; asymptomatic, n = 11). The mean (SD) vaginal pH for all participants was 5.9 (1.2). There were no significant differences (P > 0.05) in age, age at menopause, vaginal pH, and vaginal and serum cytokines and chemokines (IL-1β, IL-6, IL-8, and tumor necrosis factor-α) among symptomatic versus asymptomatic women. IL-8 was the most abundant vaginal cytokine, with mean (SD) vaginal IL-8 levels being 4.1 (3.4) and 3.1 (3.9) pg/μg protein in the symptomatic versus asymptomatic groups, respectively (P = 0.55). There were no significant linear correlations (P > 0.05) between serum and vaginal cytokine levels for all endpoints. CONCLUSIONS The presence or absence of postmenopausal vulvovaginal symptoms does not significantly differentiate vaginal inflammatory markers. Serum and vaginal cytokines are not significantly linearly correlated among postmenopausal women with and without symptoms commonly associated with vaginal atrophy, implying that this is a local reaction.

Development of a novel marker vaccine platform for protection against Bluetongue Virus (BTV)

Bluetongue virus (BTV) is an economically important member of the genus Orbivirus and closely related to African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV). Currently, 26 different serotypes of BTV are known. The virus is transmitted by blood-feeding Culicoides midges and causes disease (bluetongue [BT]) in ruminants. In 2006/2007, BTV serotype 8 (BTV-8) caused widespread outbreaks of BT amongst livestock in Europe, which were eventually controlled employing a conventionally inactivated BTV vaccine. However, this vaccine did not allow the discrimination of infected from vaccinated animals (DIVA) by the commonly used VP7 cELISA. RNA replicon vectors based on propagation-incompetent recombinant vesicular stomatitis virus (VSV) represent a novel vaccine platform that combines the efficacy of live attenuated vaccines with the safety of inactivated vaccines. Our goal was to generate an RNA replicon vaccine for BTV-8, which is safe, efficacious, adaptable to emerging orbivirus infections , and compliant with the DIVA principle. The VP2, VP5, VP3 and VP7 genes encoding the BTV-8 capsid proteins, as well as the non-structural proteins NS1 and NS3 were inserted into a VSV vector genome lacking the essential VSV glycoprotein (G) gene. Infectious virus replicon particles (VRP) were produced on a transgenic helper cell line providing the VSV G protein in trans. Expression of antigens in vitro was analysed by immunofluorescence using monoclonal and polyclonal antibodies. In a pilot study, sheep were immunized with two different VRP-based vaccine candidates, one comprising the BTV-8 antigens VP2, VP5, VP3, VP7, NS1, and NS3, the other one containing antigens VP3, VP7, NS1, and NS3. Control animals received VRPs containing an irrelevant antigen. Virus neutralizing antibodies and protection after BTV-8 challenge were evaluated and compared to animals immunized with the conventionally inactivated vaccine. Full protection was induced only when the two antigens VP2 and VP5 were included in the vaccine. To further evaluate if VP2 alone, a combination of VP2 and VP5 or VP5 alone were necessary for complete protection, we performed a second animal trial. Interestingly, VP2 as well as the combination of VP2 and VP5 but not VP5 alone conferred full protection in terms of neutralizing antibodies, and protection from clinical signs and viremia after BTV-8 challenge. These results show that the VSV replicon system represents a safe, efficacious and DIVA-compliant vaccine against BTV as well as a possible platform for protection against other Orbiviruses, such as AHSV and EHDV.

Molecular control of cardiac sodium homeostasis in health and disease.

INTRODUCTION Cardiac myocytes utilize three high-capacity Na transport processes whose precise function can determine myocyte fate and the triggering of arrhythmias in pathological settings. We present recent results on the regulation of all three transporters that may be important for an understanding of cardiac function during ischemia/reperfusion episodes. METHODS AND RESULTS Refined ion selective electrode (ISE) techniques and giant patch methods were used to analyze the function of cardiac Na/K pumps, Na/Ca exchange (NCX1), and Na/H exchange (NHE1) in excised cardiac patches and intact myocytes. To consider results cohesively, simulations were developed that account for electroneutrality of the cytoplasm, ion homeostasis, water homeostasis (i.e., cell volume), and cytoplasmic pH. The Na/K pump determines the average life-time of Na ions (3-10 minutes) as well as K ions (>30 minutes) in the cytoplasm. The long time course of K homeostasis can determine the time course of myocyte volume changes after ion homeostasis is perturbed. In excised patches, cardiac Na/K pumps turn on slowly (-30 seconds) with millimolar ATP dependence, when activated for the first time. In steady state, however, pumps are fully active with <0.2 mM ATP and are nearly unaffected by high ADP (2 mM) and Pi (10 mM) concentrations as may occur in ischemia. NCX1s appear to operate with slippage that contributes to background Na influx and inward current in heart. Thus, myocyte Na levels may be regulated by the inactivation reactions of the exchanger which are both Na- and proton-dependent. NHE1 also undergo strong Na-dependent inactivation, whereby a brief rise of cytoplasmic Na can cause inactivation that persists for many minutes after cytoplasmic Na is removed. This mechanism is blocked by pertussis toxin, suggesting involvement of a Na-dependent G-protein. Given that maximal NCX1- and NHE1-mediated ion fluxes are much greater than maximal Na/K pump-mediated Na extrusion in myocytes, the Na-dependent inactivation mechanisms of NCX1 and NHE1 may be important determinants of cardiac Na homeostasis. CONCLUSIONS Na/K pumps appear to be optimized to continue operation when energy reserves are compromised. Both NCX1 and NHE1 activities are regulated by accumulation of cytoplasmic Na. These principles may importantly control cardiac cytoplasmic Na and promote myocyte survival during ischemia/reperfusion episodes by preventing Ca overload.

IN VITRO INDUCTION OF XENOIMMUNE RESPONSES TO LIPOSOMES CONTAINING HUMAN COLON TUMOR ANTIGENS

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl(,2) to a dried lipid film. Cytoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposomes also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposomes were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens.^ The adjuvant effect of liposomes was shown to be mediated primarily by tumor antigens exposed on the outer surface of liposomes. Trypsinization of the liposomes which eliminated 96% of the surface protein reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes of increasing protein concentration showed that while 10 (mu)g protein was threshold, 100 (mu)g protein induced maximal responses. In addition, membrane fluidity studies revealed that rigid liposomes were significantly (P < 0.05) more efficacious than fluid liposomes in inducing cytotoxicity.^ The induction of the primary response required the presence of nonadherent splenocytes bearing the Thy-1, Lyt-1, and Lyt-2 surface markers. The role of a Lyt-123 subpopulation was suggested by the inability of both the Lyt-1 and Lyt-2 depleted populations to completely restore the cytolytic levels to normal. In addition, the interaction of I-A('+) spleen adherent cells with liposomes for at least 8 hours was required to generate maximal cytotoxic activity. The phenotype of the cytotoxic effector was Thy-1('+), Lyt-2('+), and I-A('d-).^ Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select specific immune responses, as well as to identify colon tumor-specific determinants.^

Familial aggregation, candidate genes and genome scans: Analyzing the role of genetics in stroke

The genetic etiology of stroke likely reflects the influence of multiple loci with small effects, each modulating different pathophysiological processes. This research project utilized three analytical strategies to address the paucity of information related to the identification and characterization of genetic variation associated with stroke in the general population. ^ First, the general contribution of familial factors to stroke susceptibility was evaluated in a population-based sample of unrelated individuals. Increased risk of subclinical cerebral infarction was observed among individuals with a positive parental history of stroke. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. ^ The need to identify specific gene variation associated with stroke in the general population was addressed by evaluating seven candidate gene polymorphisms in a population-based sample of unrelated individuals. Three polymorphisms were significantly associated with increased subclinical cerebral infarction or incident clinical ischemic stroke risk. These relationships include the G-protein β3 subunit 825C/T polymorphism and clinical stroke in Whites, the lipoprotein lipase S/X447 polymorphism and subclinical and clinical stroke in men, and the angiotensin I-converting enzyme Ins/Del polymorphism and subclinical stroke in White men. These associations did not appear to be obfuscated by the stroke risk factors adjusted for in the analysis models specifically blood pressure levels or anti-hypertensive medication use. ^ The final research strategy considered, on a genome-wide scale, the idea that genetic variation may contribute to the occurrence of hypertension or stroke through a common etiologic pathway. Genomic regions were identified for which significant evidence of heterogeneity was observed among hypertensive sibpairs stratified by family history of stroke information. Regions identified on chromosome 15 in African Americans, and chromosome 13 in Whites and African Americans, suggest the presence of genes influencing hypertension and stroke susceptibility. ^ Insight into the role of genetics in stroke is useful for the potential early identification of individuals at increased risk for stroke and improved understanding of the etiology of the disease. The ultimate goal of these endeavors is to guide the development of therapeutic intervention and informed prevention to provide a lasting and positive impact on public health. ^

Relación entre la composición química corporal, la fertilidad y la prolificidad en conejas primíparas.

En este trabajo se ha estudiado el efecto de la composición química corporal, estimada mediante el método de impedancia bioeléctrica (BIA) en la primera y segunda inseminación artificial (IA), sobre la fertilidad y la prolificidad de conejas primíparas. Se utilizaron los datos de 137 conejas procedentes de dos granjas que utilizaban dos genéticas distintas (UPV e Hyplus) y tres ritmos reproductivos (R4: inseminación a los 4 días tras el parto y destete a los 28 días, R11: inseminación a los 11 días postparto y destete a 35 días y R14: inseminación a los 14 días tras el parto y destete a los 42 días). La fertilidad media observada en el segundo parto fue de un 56,2% y el porcentaje de nacidos vivos, sobre los nacidos totales, fue de un 87,5%, no encontrándose diferencias entre los ritmos utilizados. El aumento de la proporción de proteína corporal, grasa y energía en la primera IA tuvieron un efecto positivo (P=0,007; P=0,03 y P<0,001, respectivamente) sobre la fertilidad del segundo parto. Igualmente, un mayor contenido en grasa (P<0,001) y energía (P<0,001) en la segunda IA también incrementaron la fertilidad del segundo parto. El incremento del ratio proteína/energía corporal de las conejas, determinado en las dos IA, tuvo un efecto negativo sobre la fertilidad en el segundo parto (P<0,001), observándose mejores resultados en conejas con 12 g proteína/MJ respecto a otras con valores superiores. El porcentaje de nacidos vivos (P<0,001), sobre los nacidos totales, aumentó con la proporción de proteína de las conejas en el momento de la segunda IA, pero esta última no tuvo efecto sobre la fertilidad. This study evaluated the effect of chemical body composition at first and second artificial inseminations (AI), determined by bioelectric impedance technique (BIA), on fertility and prolificacy of primiparous rabbit does. Data of 137 does from two farms using two different genetics (UPV and Hyplus) and three breeding systems were analyzed. Fertility was 56.2 % and the percentage of kits born alive over total born was 87.5 %. Breeding system had no effect on fertility or the percentage of kits born alive. Higher body protein, fat and energy of the rabbit doe at first artificial insemination (AI) increased the fertility in the second parity (P=0.007, P=0.03 and P<0.001 respectively). Fat and energy content of the doe at the second AI affected positively fertility in the second parity (P<0.001 and P<0.001 respectively), while protein influenced positively only prolificacy (P<0.001). The increase of the ratio protein/energy in both AI moments had a negative influence on fertility in the second parity (P<0.001), rabbit does with 12 g protein/MJ showed a higher fertility than those with higher values