The Pen Duick Escarpment off Morocco: A promising biogeochemically active carbonate mound laboratory (MiCROSYSTEMS)

Carbonate mud mounds, found in marine environments from shallow- to deep-water settings, span from Proterozoic to recent times. During the past decades, numerous active venting fields were discovered in deep marine environments and became a subject of extensive study for marine scientists. Mound building seems to be a fundamental but still enigmatic strategy for life. Various arguments suggest that microorganisms are playing a major role in the reef development, mound formation and biodiversity. Therefore, it is important to evaluate the microbial mediated processes of carbonate precipitation.Cold-water coral reefs thriving on carbonate mounds were discovered in the late 1990’s off western Ireland. An exploratory cruise of RV Belgica in 2002 off Morocco has led to the discovery of apparently juvenile mounds in water depths of 500-600 m, topping a cliff - the Pen Duick escarpment - flanked by giant mud volcanoes. Subsequent cruises have confirmed the colonization by deep-water corals and have unveiled extensive fields of seep-related carbonate crusts in the off-reef regions. Long cores taken in 2004 indicate that the ‘Pen Duick’ mounds, in which microbial action was demonstrated by a strong emission of hydrogen sulphide, may be considered as giant biogeochemical reactors. The mound sediments were dated 2 kyrs B.P. at the surface and 20 kyrs B.P. in a depth of 6 mbsf.A 450 cm long gravity core, coming from one of these juvenile mounds, was sampled and analyzed for mineralogy, stable isotopes composition, geochemistry, and microbial communities. Most of the sediment consists of calcite (coccoliths), quartz and dolomite. At a depth of 4 mbsf, we found hardened nodule-like structures, embedded in grey mud containing cold-water coral pieces. The presence of 20-30% of dolomite, in the carbonate phase, suggests a microbial influence during mineral formation. Preliminary results of the pore water geochemistry indicate a reactive sulphate – methane interface at 3.8 mbsf. In this layer we focused our studies on the microbial communities, such as methanogens, methanotrophs and sulphate reducers. The trend of the d13C values in digenetic carbonate supports the assumption of microbial activity in this section of the core. In order to define the primary microbial community involved in carbonate precipitation, we did direct culturing, DNA isolation and PCR analysis of three functional genes, the a subunit (mcrA) of the methyl-coenzyme M reductase (MCR), the a subunit (pmoA) of the particulate methane monooxygenase (MMO) and the a and ß subunits (dsrA and dsrB, respectively) of the dissimilatory sulfite reductase (DSR). These enzymes are involved in methanogenesis, methanotrophy and sulphate reduction biochemical pathways, respectively. In summary, our initial results demonstrate that the Pen Duick carbonate mound can be considered as a natural laboratory in which to study cold-water coral ecosystems associated with microbial activity

Authigenic carbonates from cold-water carbonate mounds in the Gulf of Cadiz: Microbial diversity and imprint on carbonate minerals

The Pen Duick Escarpment off Morocco consists of recent carbonate mounds in water depths of 500-600 m, flanked by giant mud volcanoes. These mounds are covered by mainly lifeless cold-water corals and have been associated with extensive fields of seep-related carbonates in off-reef regions. Three piston cores (from 350 to 640 cm long), coming from different sites on these juvenile mounds, were sampled and analyzed for mineralogy, stable isotopic composition of carbonates, geochemistry, and microbial communities. Most of the sediment comprises pelagic calcite (coccoliths), detrital quartz and authigenic dolomite, often observed encasing coccoliths. The decalcification of the sediment resulted in a dolomite dominated matrix that showed stable carbon istotope values of as low as -30 permil in contrast to the bulk sample values of -7 to -15 permil, which implies the involvement of microbes in the production of bicarbonate ions. Initial results from 16S rRNA gene clone libraries support the theory, that anaerobic oxidation of methane is one of the most important biogeochemical process leading to carbonate precipitation. Preliminary results of stable carbon isotopes of bulk samples from different carbonate mounds from the same area, indicates that the sulphate-methane transition zone moves in depth through time. We will show and discuss multidisciplinary data obtained after several cruises aimed to elucidate the impact of microorganisms on the construction of these carbonate mounds. The special emphasis in this research will be on the correlation between microbial ecosystems and their metabolic influence on mineral formation and diagenesis

Uterine artery pseudoaneurysm requiring embolization in pregnancy: a case report and review of the literature.

Background: Uterine Artery Pseudoaneurysm is a rare cause of pelvic pain and haemorrhage in pregnancy. It should be considered in the differential diagnosis of pregnant women presenting with abdominal pain and is readily diagnosed by colour Doppler ultrasound. If left untreated, they may bleed into the peritoneum causing severe pain and haemorrhagic shock and may progress to maternal and fetal death. Case presentation: We describe a case of a woman presenting with severe right iliac fossa pain at 26 weeks gestation attributed to a right uterine artery pseudoaneurysm diagnosed on duplex ultrasound which was successfully treated by uterine artery embolization at 28 weeks gestation without complication to the fetus. Conclusion: Uterine artery embolization appears to be a safe and effective method to treat pseudoaneurysm during pregnancy without compromising uteroplacental perfusion

Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion

Follicular Dendritic Cell-Specific Prion Protein (PrPc) Expression Alone Is Sufficient to Sustain Prion Infection in the Spleen

Prion diseases are characterised by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. Following peripheral exposure high levels of prion-specific PrPSc accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrPC is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrPC expression was specifically “switched on” or “off” only on FDC. We show that PrPC-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrPC-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrPC expression on FDC

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles

Increased Abundance of M cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases