Inflammatory Immune Responses and Gut Microbiota Changes Following Campylobacter coli Infection of IL-10 -/- Mice with Chronic Colitis

Human infections with the food-borne enteropathogens Campylobacter are progressively rising. Recent evidence revealed that pre-existing intestinal inflammation facilitates enteropathogenic infection subsequently exacerbating the underlying disease. Given that only little is known about C. coli-host interactions and particularly during intestinal inflammation, the aim of the present study was to survey gastrointestinal colonization properties, gut microbiota changes and pro-inflammatory sequelae upon peroral C. coli-infection of IL-10-/- mice with chronic colitis. C. coli colonized the gastrointestinal tract of mice with varying efficiencies until day 28 post-infection and induced macroscopic and microscopic inflammatory changes as indicated by shorter colonic lengths, more distinct histopathological changes in the colonic mucosa and higher numbers of apoptotic colonic epithelial cells when compared to mock-infected controls. Furthermore, not only colonic innate and adaptive immune cell responses, but also enhanced systemic TNF-α secretion could be observed following C. coli as opposed to mock challenge. Notably, C. coli induced intestinal inflammatory sequelae were accompanied with gut microbiota shifts towards higher commensal enterobacterial loads in the infected gut lumen. Moreover, the pathogen translocated from the intestinal tract to extra-intestinal tissue sites in some cases, but never to systemic compartments. Hence, C. coli accelerates inflammatory immune responses in IL-10-/- mice with chronic colitis

Correlative High-Resolution Imaging of Iron Uptake in Lung Macrophages

Detection of iron at the subcellular level in order to gain insights into its transport, storage, and therapeutic prospects to prevent cytotoxic effects of excessive iron accumulation is still a challenge. Nanoscale magnetic sector secondary ion mass spectrometry (SIMS) is an excellent candidate for subcellular mapping of elements in cells since it provides high secondary ion collection efficiency and transmission, coupled with high-lateral-resolution capabilities enabled by nanoscale primary ion beams. In this study, we developed correlative methodologies that implement SIMS high-resolution imaging technologies to study accumulation and determine subcellular localization of iron in alveolar macrophages. We employed transmission electron microscopy (TEM) and backscattered electron (BSE) microscopy to obtain structural information and high-resolution analytical tools, NanoSIMS and helium ion microscopy-SIMS (HIM-SIMS) to trace the chemical signature of iron. Chemical information from NanoSIMS was correlated with TEM data, while high-spatial-resolution ion maps from HIM-SIMS analysis were correlated with BSE structural information of the cell. NanoSIMS revealed that iron is accumulating within mitochondria, and both NanoSIMS and HIM-SIMS showed accumulation of iron in electrolucent compartments such as vacuoles, lysosomes, and lipid droplets. This study provides insights into iron metabolism at the subcellular level and has future potential in finding therapeutics to reduce the cytotoxic effects of excessive iron loading

Localisation and origin of the bacteriochlorophyll-derived photosensitizer in the retina of the deep-sea dragon fish Malacosteus niger

Most deep-sea fish have a single visual pigment maximally sensitive at short wavelengths, approximately matching the spectrum of both downwelling sunlight and bioluminescence. However, Malcosteus niger produces far-red bioluminescence and its longwave retinal sensitivity is enhanced by red-shifted visual pigments, a longwave reflecting tapetum and, uniquely, a bacteriochlorophyllderived photosensitizer. The origin of the photosensitizer, however, remains unclear. We investigated whether the bacteriochlorophyll was produced by endosymbiotic bacteria within unusual structures adjacent to the photoreceptors that had previously been described in this species. However, microscopy, elemental analysis and SYTOX green staining provided no evidence for such localised retinal bacteria, instead the photosensitizer was shown to be distributed throughout the retina. Furthermore, comparison of mRNA from the retina of Malacosteus to that of the closely related Pachystomias microdon (which does not contain a bacterichlorophyll-derived photosensitzer) revealed no genes of bacterial origin that were specifically up-regulated in Malacosteus. Instead up-regulated Malacosteus genes were associated with photosensitivity and may relate to its unique visual ecology and the chlorophyll-based visual system. We also suggest that the unusual longwave-reflecting, astaxanthin-based, tapetum of Malacosteus may protect the retina from the potential cytotoxicity of such a system

Reactivity of cyano- and isothiocyanatoborylenes: metal coordination, one-electron oxidation and boron-centred Brønsted basicity

Doubly base-stabilised cyano- and isothiocyanatoborylenes of the form LL′BY (L = CAAC = cyclic alkyl(amino)carbene; L′ = NHC = N-heterocyclic carbene; Y = CN, NCS) coordinate to group 6 carbonyl complexes via the terminal donor of the pseudohalide substituent and undergo facile and fully reversible one-electron oxidation to the corresponding boryl radical cations [LL′BY]˙+. Furthermore, calculations show that the borylenes have very similar proton affinities, both to each other and to NHC superbases. However, while the protonation of LL′B(CN) with PhSH yielding [LL′BH(CN)+][PhS−] is fully reversible, that of LL′B(NCS) is rendered irreversible by a subsequent B-to-CCAAC hydrogen shift and nucleophilic attack of PhS− at boron

Ultrastrukturelle Untersuchung der Melanogenese im adulten humanen retinalen Pigmentepithel nach adenoviraler Transduktion mit dem Tyrosinasegen

Until recently, it was widely accepted that melanogenesis does not occur in the adult retinal pigment epithelium (RPE), since the typical hallmarks of melanogenesis, the premelanosome and the expression of melanogenic proteins like tyrosinase and melanocyte-associated protein 17 (PMEL17), were absent post-natal. In the meantime, active tyrosinase has been observed in the adult RPE of different animal species, e.g. after phagocytosis of retinal photoreceptor outer segments (ROS). The aim of this thesis was to investigate whether melanogenesis can be induced in adult human RPE cells in response to ROS phagocytosis or after transduction with a tyrosinase vector. The role of the melanogenic proteins PMEL17 and TRP1 and the classical melanosomal stages, known from pre-natal melanin synthesis, were also to determine. As a model system tyrosinase transduced amelanotic RPE cells were used to study tyrosinase function and melanogenesis and the influence of phagocytosis in adult RPE. The presences of the melanogenic proteins tyrosinase, tyrosinase-related protein 1 (TRP1) and PMEL17 were investigated using immunocytochemistry. Tyrosinase activity and loclisation was further studied with electron microscopical DOPA histochemistry. The ultrastructural morphology of melanogenic stages was compared to that of pigmented melanoma cells (MNT-1), which were used as a positive control for typical melanogenesis. Melanin synthesis was detected with HPLC analysis. MTT tests confirmed that viability was not affected after tyrosinase transduction and melanin synthesis. Post-natal RPE melanogenesis was also studied in animal experiments after subretinal injection of the tyrosinase vector in rats and rabbits. Compared to controls, tyrosinase active cells had a redifferentiated cobblestone morphology, were pigmented and had an improved phagocytosis rate. Tyrosinase trafficking was different to the classical model found in MNT-1 cells, since a DOPA reaction was not observed in Golgi-derived vesicles, but as membrane-less, small DOPA granules free-floating in the cytoplasm. Melanogenesis occurred without the involvement of TRP1, PMEL17 and typical striated premelanosomes and melanogenic stages. In contrast, melanin was synthesised in lysosome-like organelles. Thus, a new pathway of melanogenesis is described for this model system. Transduced RPE cells of living rats and rabbits and cultured cells of human donors showed also a similar morphology of melanogenesis as the ARPE-19 cells. Interestingly, control MNT-1 cells contained similar melanosomes in addition to the classical stages of melanogenesis. Although a transport of ingested material to newly-formed ARPE-19 melanosomes was not observed, phagocytosis led to an improved tyrosinase activity and to an accelerated melanogenesis, compared to non-fed transduced cells. In conclusion, tyrosinase transduction in combination with phagocytosis led to a morphological reorganisation and functional improvement of cultured ARPE-19 cells. Additionally, melanogenesis has been induced, which is independent of premelanosome formation. It can be transferred to the in vivo situation by gene therapy.Bis vor kurzem war es allgemein akzeptiert, dass die Melansomen des retinalen Pigmentepithel (RPE) nur pränatal gebildet werden und danach nicht erneuert werden können. Typische Merkmale der Melanogenese, wie die Bildung von Prämelanosomen und melanogenetischen Proteinen (Tyrosinase, Melanozyten-assoziiertes Protein 17 (PMEL17)), waren adult nicht auffindbar. Zwischenzeitlich konnte allerdings das aktive Enzym Tyrosinase im adulten RPE verschiedener Spezies nachgewiesen werden, z.B. nach Phagozytose von retinalen Stäbchenaußensegmenten (ROS). Die Fragestellung dieser Doktorarbeit was es herauszufinden, ob eine Neusynthese des Melanins in adulten humanen RPE-Zellen induziert werden kann, indem sie mit ROS gefüttert werden oder mittels eines adenoviralen Vektors eine Tyrosinase-Überexpression induziert wird. Außerdem sollte die Rolle der melanogenetischen Proteine PMEL17 und Tyrosinase-assoziiertes Protein 1 (TRP1) und der typischen Melanogenesestadien I-IV, wie sie aus der pränatalen Melaninsynthese bekannt sind, evaluiert werden. Die Experimente wurden an tyrosinase-transduzierten amelanotischen RPE-Kulturen (ARPE-19-Zellline) durchgeführt. Diese Zellen dienten als Modellsystem zur Untersuchung der Funktion der Tyrosinase, der Melanogenese und des Einflusses der Fütterung auf das adulte RPE. Das Vorhandensein der melanogenetischen Proteine Tyrosinase, TRP1 und PMEL17 wurde immunhistologisch geprüft. Die Aktivität und die Lokalisierung der Tyrosinase in der Zelle wurden mittels DOPA-Histologie im Elektronenmikroskop untersucht. Die Ultrastruktur der auftretenden Stadien der Melanogenese¬ wurde mit der von pigmentierten Melanomzellen (MNT-1) verglichen. MNT-1 Zellen dienten ebenfalls als Positivkontrollen für die histo-logischen Färbungen. Die Neusynthese des Melanins wurde durch HPLC-Analysen bestätigt. MTT-Tests zeigten, dass die Viabilität der tyrosinase-transduzierten Zellen gegenüber Kontrollzellen auch nach der Melanisierung nicht beeinträchtigt war. Desweiteren wurde die postnatale Melanogenese des RPE nach subretinaler Injektion des Vektors in Albino-Ratten und Kaninchen untersucht. Verglichen mit den nicht-transduzierten ARPE-19 Zellen zeigten die tyrosinase-transduzierten ARPE-19 Zellen eine redifferenzierte epithelartige Morphologie, waren tyrosinase-positiv und pigmentiert und zeigten außerdem eine stärkere Phagozytose. Verglichen mit den MNT-1 Melanomzellen, welche als Positivkontrolle für eine typische Melanogenese dienten, zeigten die tyrosinase-transduzierten ARPE-19-Zellen einen stark veränderten Tyrosinasetransport und eine andersartige Morphologie der melanosomalen Vorstufen. Golgi-Apparate und deren Transportvesikel waren wider Erwarten tyrosinasefrei, statt dessen zeigte sich eine positive DOPA-Reaktion in nicht-membranumhüllten, kleinen Granula im Zytoplasma. TRP1 und PMEL17, Prämelanosomen und typische Melanogenese¬stadien wurden nicht gefunden. Statt dessen wurde das Melanin in lysosomenartigen Organellen synthetisiert und gespeichert. Interessanterweise zeigten selbst die MNT-1 Zellen neben den klassischen Melanogenesestadien auch eine Melaninsynthese in lysosomen¬artigen Organellen. Anhand dieser Daten konnte ich einen neuen Verlauf der Melanin¬synthese beschreiben. Transduzierte RPE Zellen von lebenden Ratten und Kaninchen und auch kultivierte und transduzierte Spender-RPE Zellen zeigten eine ähnliche Melanogenese wie die transduzierten ARPE-19 Zellen. Obwohl der Transport von phagozytiertem Material in neu gebildete Melanosomen für die ARPE-19-Zellen hier nicht einwandfrei nachgewiesen wurde, konnte doch gezeigt werden, dass eine Phagozytose zu einer verstärkten Tyrosinaseaktivität und zu einer schnelleren Melaninsynthese gegenüber den ungefütterten transduzierten Zellen führte. Fazit: Tyrosinase-Transduktion in Kombination mit Fütterung von retinalen Stäbchen-außensegmenten führt zu einer morphologischen Reorganisation und zu einer Verbesserung der Zellfunktion in ARPE-19-Zellen. Die Melanin-Neusynthese hier ist unabhängig von der Bildung von typischen Prämelanosomen. Die gefunden Ergebnisse lassen sich mittels Gentherapie auch in die in vivo-Situation übertragen