Liver Monocytes and Kupffer Cells Remain Transcriptionally Distinct during Chronic Viral Infection

Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virus-induced chronic hepatitis

Liver monocytes and kupffer cells remain transcriptionally distinct during chronic viral infection

Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt cli

Dynamic Control of Nanoprecipitation in a Nanopipette

Studying the earliest stages of precipitation at the nanoscale is technically challenging but quite valuable as such phenomena reflect important processes such as crystallization and biomineralization. Using a quartz nanopipette as a nanoreactor, we induced precipitation of an insoluble salt to generate oscillating current blockades. The reversible process can be used to measure both kinetics of precipitation and relative size of the resulting nanoparticles. Counter ions for the highly water-insoluble salt zinc phosphate were separated by the pore of a nanopipette and a potential applied to cause ion migration to the interface. By analyzing the kinetics of pore blockage, two distinct mechanisms were identified: a slower process due to precipitation from solution, and a faster process attributed to voltage-driven migration of a trapped precipitate. We discuss the potential of these techniques in studying precipitation dynamics, trapping particles within a nanoreactor, and electrical sensors based on nanoprecipitation

Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

International audienceBACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader

Innate Sensing of HIV-Infected Cells

Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection

Polymer controlled mineralization of zinc phosphate hydrates and applications in corrosion protection, catalysis and biomedicine

Inhaltszusammenfassung in der Originalsprache des Dokuments (deutsch) ZUSAMMENFASSUNG Die Tauglichkeit von Hybridmaterialien auf der Basis von Zinkphosphathydrat-Zementen zum Einsatz als korrosionshemmende anorganische Pigmente oder zur prothetischen und konservierenden Knochen- und Zahntherapie wird weltweit empirisch seit den neunziger Jahren intensiv erforscht. In der vorliegenden Arbeit wurden zuerst Referenzproben, d.h. alpha-und beta-Hopeite (Abk. a-,b-ZPT) dank eines hydrothermalen Kristallisationsverfahrens in wässerigem Milieu bei 20°C und 90°C hergestellt. Die Kristallstruktur beider Polymorphe des Zinkphosphattetrahydrats Zn3(PO4)2 4 H2O wurde komplett bestimmt. Einkristall-strukturanalyse zeigt, daß der Hauptunterschied zwischen der alpha-und beta-Form des Zinkphosphattetrahydrats in zwei verschiedenen Anordnungen der Wasserstoffbrücken liegt. Die entsprechenden drei- und zweidimensionalen Anordnungen der Wasserstoffbrücken der a-und b-ZPT induzieren jeweils unterschiedliches thermisches Verhalten beim Aufwärmen. Während die alpha-Form ihr Kristallwasser in zwei definierten Stufen verliert, erzeugt die beta-Form instabile Dehydratationsprodukt. Dieses entspricht zwei unabhängigen, aber nebeneinander ablaufenden Dehydratationsmechanismen: (i) bei niedrigen Heizraten einen zweidimensionalen Johnson-Mehl-Avrami (JMA) Mechanismus auf der (011) Ebene, der einerseits bevorzugt an Kristallkanten stattfindet und anderseits von existierenden Kristalldefekten auf Oberflächen gesteuert wird; (ii) bei hohen Heizraten einem zweidimensionalen Diffusionsmechanismus (D2), der zuerst auf der (101) Ebene und dann auf der (110) Ebene erfolgt. Durch die Betrachtung der ZPT Dehydratation als irreversibele heterogene Festkörperstufenreaktion wurde dank eines „ähnlichen Endprodukt“-Protokolls das Dehydratationsphasendiagramm aufgestellt. Es beschreibt die möglichen Zusammenhänge zwischen den verschiedenen Hydratationszuständen und weist auf die Existenz eines Übergangszustandes um 170°C (d.h. Reaktion b-ZPT a-ZPT) hin. Daneben wurde auch ein gezieltes chemisches Ätzverfahren mit verdünnten H3PO4- und NH3 Lösungen angewendet, um die ersten Stufe des Herauslösens von Zinkphosphat genau zu untersuchen. Allerdings zeigen alpha- und beta-Hopeite charakteristische hexagonale und kubische Ätzgruben, die sich unter kristallographischer Kontrolle verbreitern. Eine zuverlässige Beschreibung der Oberfächenchemie und Topologie konnte nur durch AFM und FFM Experimente erfolgen. Gleichzeitig konnte in dieser Weise die Oberflächendefektdichte und-verteilung und die Volumenauflösungsrate von a-ZPT und b-ZPT bestimmt werden. Auf einem zweiten Weg wurde eine innovative Strategie zur Herstellung von basischen Zinkphosphatpigmenten erster und zweiter Generation (d.h. NaZnPO4 1H2O und Na2ZnPO4(OH) 2H2O) mit dem Einsatz von einerseits oberflächenmodifizierten Polystyrolatices (z.B. produziert durch ein Miniemulsionspolymerisationsverfahren) und anderseits von Dendrimeren auf der Basis von Polyamidoamid (PAMAM) beschritten. Die erhaltene Zeolithstruktur (ZPO) hat in Abhängigkeit von steigendem Natrium und Wassergehalt unterschiedliche kontrollierte Morphologie: hexagonal, würfelförmig, herzförmig, sechsarmige Sterne, lanzettenförmige Dendrite, usw. Zur quantitativen Evaluierung des Polymereinbaus in der Kristallstruktur wurden carboxylierte fluoreszenzmarkierte Latices eingesetzt. Es zeigt sich, daß Polymeradditive nicht nur das Wachstum bis zu 8 µm.min-1 reduzierten. Trotzdem scheint es auch als starker Nukleationsbeschleuniger zu wirken. Dank der Koordinationschemie (d.h. Bildung eines sechszentrigen Komplexes L-COO-Zn-PO4*H2O mit Ligandenaustausch) konnten zwei einfache Mechanismen zur Wirkung von Latexpartikeln bei der ZPO Kristallisation aufgezeigt werden: (i) ein Intrakorona- und (ii) ein Extrakorona-Keimbildungsmechanismus. Weiterhin wurde die Effizienz eines Kurzzeit- und Langzeitkorrosionschutzes durch maßgeschneiderte ZPO/ZPT Pigmente und kontrollierte Freisetzung von Phosphationen in zwei Näherungen des Auslösungsgleichgewichts abgeschätzt: (i) durch eine Auswaschungs-methode (thermodynamischer Prozess) und (ii) durch eine pH-Impulsmethode (kinetischer Prozess. Besonders deutlich wird der Ausflösungs-Fällungsmechanismus (d.h. der Metamorphismus). Die wesentliche Rolle den Natriumionen bei der Korrosionshemmung wird durch ein passendes zusammensetzungsabhängiges Auflösungsmodell (ZAAM) beschrieben, das mit dem Befund des Salzsprühteste und der Feuchtigkeitskammertests konsistent ist. Schließlich zeigt diese Arbeit das herausragende Potential funktionalisierter Latices (Polymer) bei der kontrollierten Mineralisation zur Herstellung maßgeschneiderter Zinkphosphat Materialien. Solche Hybridmaterialien werden dringend in der Entwicklung umweltfreundlicher Korrosionsschutzpigmente sowie in der Dentalmedizin benötigt. -------------------------------------------------------------------------------- Inhaltszusammenfassung in einer weiteren Sprache (englisch) ABSTRACT Materials research devoted to the development of new or improved materials based on zinc phosphate hydrate cements with enhanced mechanical and chemical stability, suitable as “environmental friendly” corrosion inhibitor for partial or permanent restoration of bone and teeth attracts much interest at the interface between medicine, inorganic and physical chemistry and ceramic science. Reference probes, namely alpha-and beta-hopeite (i.e. a-, b-ZPT) have been synthesized by hydrothermal crystallization from aqueous solution at 20°C and 90°C respectively. The crystal structure of these polymorphic forms of zinc phosphate tetrahydrate (ZPT) Zn3(PO4)2· 4 H2O has been completely resolved. Single crystal analysis proves that the main difference between the alpha- and beta-form of zinc phosphate tetrahydrate stems from two different hydrogen bonding patterns. The respective three dimensional and two dimensional hydrogen bonding patterns of a- and b-ZPT induce unique thermal behaviour. Upon heating ZPT polymorphs release crystal water differently: while the alpha-form looses water two by two in two well defined stages, the beta-form exhibits a dehydration pathway involving intermediary products of hydration. This corresponds to concurring mechanisms of dehydration: on one hand at low heating rate a two dimensional JMA mechanism on the (011) surface, induced by surface crystal defects and starting preferentially from crystal edges is privileged, and on the other hand at high heating rate a two dimensional diffusion mechanism (D2) first on the (101) plan and then on the (110) surface takes over. Furthermore, considering dehydration of ZPT a multistep irreversible heterogeneous solid state (solid-gas) reaction and applying a “same final thermodynamic state” procedure, the thermodynamic stability phase diagram pointing out the possible interrelations between the various hydrate states and phases was built and an intermediary activation state (transformation from b- to a-ZPT at 170°C) was introduced. Besides, targeted chemical etching with diluted H3PO4 and NH3 solutions proved to be successful in providing key information about the first stage of surface dissolution. Indeed, a- and b-ZPT display characteristic hexagonal to cubic etch pits propagating under crystallographic control. Complementary AFM and surface FFM experiments/analysis point out various types of surface defects (point defects, screw dislocations) and allow the simultaneous evaluation of the defect density and of the bulk dissolution rates of both a- and b-ZPT, thus offering a representative description of their surface topology and chemistry. Moreover, a novel approach of polymer controlled mineralization employing functionalized latexes obtained by radical miniemulsion polymerization and PAMAM based dendrimers proved to be efficient at producing basic zinc phosphate pigments of 1st and 2nd generation (i.e. NaZnPO4· 1 H2O and Na2ZnPO4(OH) 2 H2O). These zeolitic (ZPO) structures of increasing sodium and water contents exhibit different morphologies: hexagonal, cubic, heart-like, six-arm star, multidimensional lancets, etc. Using fluorescence labelled particles, carboxylated PS-based latexes were shown not only to reduce monotonically the crystal growth rate to around 8 nm.min-1 but also revealed to be strong nucleation promoter. On this basis, two simple mechanisms referring to coordination chemistry (i.e. formation of a six-atom complexL-COO-Zn-PO4*H2Ocrl with ligand exchange) are tentatively proposed in order to highlight the primary role of negatively charged latexes on ZP nucleation: an intra-corona nucleation mechanism and an extra-corona nucleation mechanism. Finally, the short and long-term anticorrosion efficiency of the tailor-made ZPO/ZPT pigments and the controlled release of phosphate ions was evaluated using two approaches of the solubility equilibrium: a leaching method (thermodynamic approach), and a pH response method (kinetic approach). Furthermore the overwhelming mechanism of dissolution-reprecipitation (metamorphism) and the primordial role of sodium ions in the corrosion efficacy of acid and basic zinc phosphate pigments appeared conspicuously in employing first a composition dependent dissolution model (CDD model) developed on purpose and secondly further validated (i.e. emphasis of blistering effects governed by the sodium concentration in the coating) by combined salt spray tests and humidity chamber tests. Further, the studies described in this thesis demonstrate the capability of the functionalized latex (polymer) controlled mineralization process to produce tailor-made materials on zinc phosphate basis. Such organic/inorganic hybrid materials are urgently needed to develop both advanced formulations of “environmental friendly” anticorrosive pigments and reconstructive orthopaedic dental cements with improved service lifetime

Organizational forms and risk of listed private equity

This article investigates the stock performance of listed private equity and develops a classification of vehicles according to their organizational structure. The authors identify and examine 274 liquid listed private equity entities in the period from 1986 to 2008. The listed private equity shows a Dimson beta of 1.7 without any significant excess return, and vehicles differ strongly depending on their organizational form. Market risk is high in internally managed vehicles but low in externally managed ones. The authors conclude that different sources of cash flows, such as management fees and carried interest, can account for these risk characteristics. Precautions must be taken, therefore, when using specific listed private equity vehicles as a proxy for traditional private equity funds