Human herpes virus and Alzheimer’s disease

Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder and according to the WHO it is estimated that 36 millions of people worldwide currently suffer from AD. Genetic and environmental factors interact in a complex interplay that might affect pathogenic mechanisms leading to age-related neurodegeneration. The hypothesis is that the presence of allelic polymorphisms in selected genes affecting individual brain susceptibility to infection by the herpes virus family during aging, may contribute to neuronal loss, inflammation and amyloid deposition. Herpes virus family show features relevant to AD, since they infect a large proportion of human population, develop a latent form persisting for several years, are difficult to eliminate by immune responses especially when latency has been established and are able to infect neurons. The association between AD and herpes viruses infection has been investigated. In particular the investigation focused on CMV, EBV and HHV-6 in DNA samples from peripheral blood of a large cohort of patients with clinical diagnosis of AD and age matched CTR, from a longitudinal population study, and DNA samples from brain tissue of patients with neuropathological diagnosis of definitive AD. An association between the presence of EBV and HHV-6 DNA from PBL positivity with the cognitive deterioration and progression to AD has been focused. Moreover, IgG plasma levels in CTR and AD to these viruses were tested. CMV and EBV IgG plasma levels were higher in elderly subjects that developed clinical AD at the end of the five year follow up. Our findings support the notion that persistent cycles of latency and reactivation of herpes viruses may contribute to impair systemic immune response and induce altered inflammatory process that in turn affect cognitive decline during aging.

Aktivierung der alpha-Sekretase ADAM10 als neuer therapeutischer Ansatz zur Behandlung der Alzheimer-Erkrankung

Die Stimulation der APP-prozessierenden α-Sekretase ADAM10 eröffnet eine vielversprechende Möglichkeit zur medizinischen Behandlung der Alzheimer-Krankheit. In dieser Arbeit wurden drei unterschiedliche Strategien zur therapeutischen Aktivierung von ADAM10 verfolgt: Die Aktivierung des G-Protein-gekoppelten Rezeptors PAC1 durch PACAP, die Gentherapie mit ADAM10-cDNA und die ADAM10-Promotorstimulation durch Retinoid-Rezeptor-Aktivierung. PACAP-38 stimuliert die α-Sekretase-vermittelte APPsα-Sekretion in humanen Neuroblastomzellen. Durch Aktivierung des PAC-1-Rezeptors via intranasal verabreichtem PACAP-38, konnte eine erhöhte α-sekretorische APP-Prozessierung bzw. verminderte Ablagerung von amyloiden Plaques in Mäusen gezeigt werden. Weiterhin sollte durch Immunoliposomen-basierte Transfektion die humane ADAM10-cDNA in den Neuronen der Maus überexprimiert werden. Hiefür wurde die DNA in Liposomen eingeschlossen, welche an ihrer Oberfläche mit anti-Transferrin-Antikörpern zur Überwindung der Blut-Hirn-Schranke gekoppelt waren. Für die Herstellung des DNA-Transportsystems wurden die Einzelschritte wie DNA-Einschluss mit einem Reportergen-Vektor, Konjugation mit verschiedenen Antikörpern und Größe der Liposomen erprobt und optimiert. Es konnte allerdings weder in vitro noch in vivo eine Immunoliposomen-vermittelte Transfektion nachgewiesen werden. In dieser Arbeit wurde zudem die Retinoid-basierte Expressionssteigerung von ADAM10 untersucht. Dafür wurden die beiden potentiellen Retinoid-Rezeptor-Bindestellen auf dem ADAM10-Promotor durch Verwendung selektiver nukleärer Rezeptor-Agonisten charakterisiert. Hierbei konnte erstmals gezeigt werden, dass der ADAM10-Promotor durch ein Dimer der nukleären Rezeptoren RAR und RXR aktiviert wird, wodurch eine erhöhte α-sekretorischen APP-Prozessierung in Neuroblastoma-Zellen resultiert. Weiterhin konnte gezeigt werden, dass die RAR/RXR-Heterodimeraktivierung sowohl auf dem humanen wie auf dem murinen ADAM10-Promotor identisch ist, so dass am Mausmodell entwickelte Retinoid-basierte Therapien auf den Menschen übertragbar sind. Für das Modell einer solchen Therapie wurde Acitretin verwendet, welches für die medizinische Behandlung humaner Hautkrankheiten seit Jahrzehnten eingesetzt wird. In dieser Arbeit konnte erstmals gezeigt werden, dass Acitretin in humanen und murinen Neuroblastoma-Zellen die Menge an ADAM10 erhöht, wodurch die α-sekretorische APP-Prozessierung gesteigert wird. Zudem wurden Mäuse mit Acitretin oral, subcutan und intranasal behandelt, wobei jedoch weder eine Veränderung in der APP-Prozessierung noch der Blut-Hirn-Transport von Acitretin eindeutig belegt werden konnten. Dennoch erschließt die α-Sekretase-erhöhende Eigenschaft von Acitretin einen neuen Therapieansatz, zur Behandlung von Demenzformen vom Typ des Morbus Alzheimer.

Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo

The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.

Comparative analysis of midline guidance and axon pathfinding in the chelicerates Achaearanea tepidariorum and Cupiennius salei

Die Neurogenese und axonale Wegfindung sind in den vergangenen Jahrzehnten Thema einer Vielzahl wissenschaftlicher Untersuchungen in den verschiedensten Organismen gewesen. Die zusammengetragenen Daten in Insekten und Crustaceen geben eine gute Übersicht darüber, wie das Nervensystem in Arthropoden aufgebaut wird. Die entwicklungsbiologischen Prozesse, die daran beteiligt sind, sind in den beiden genannten Gruppen sehr gut verstanden. In den Gruppen der Cheliceraten und Myriapoden jedoch wurden ähnliche Analysen bisher kaum durchgeführt. Das Hauptanliegen dieser Arbeit war es daher, Mechanismen in den Spinnen Achaearanea tepidariorum und Cupiennius salei, zwei Vertretern der Cheliceraten, zu untersuchen, die eine Rolle im Leitsystem der ventralen Mittellinie und bei der axonalen Wegfindung spielen. Eine Vorraussetzung hierfür sind Kenntnisse über die Architektur des Zentralnervensystems. In einem ersten Schritt beschrieb ich daher grundlegend die Morphologie des Nervensystems im Verlauf der gesamten Embryoalentwicklung. Ich konnte zeigen, dass in Spinnen ein für Arthropoden typisches Strickleiternervensystem gebildet wird. Dieses wird von segmental angelegten Neuronen geformt, wobei sowohl Gruppen von Zellen als auch einzelne Neurone daran beteiligt sind, die primären axonalen Trakte zu etablieren. Im Besonderen konnte ich eine Zelle identifizieren, die in Position, Projektionsmuster und der Expression des Markergens even-skipped vergleichbar zum PR2 Neuron in Drosophila ist, welches die posteriore Wurzel des Segmentalnervs anlegt.rnrnIn einem zweiten Ansatz untersuchte ich die ventrale Mittellinie in Spinnen im Bezug auf ihre mögliche Funktion in der axonalen Wegfindung. Es konnte gezeigt werden, dass es sich beim Epithel der Mittellinie, das die Lücke zwischen beiden Keimstreifhälften während des gesamten Prozesses der Inversion überspannt, um eine transiente Struktur handelt, die keine neuralen Zellen hervorbringt. Es ist daher vergleichbar mit der so genannten Floor plate in Vertebraten, die ebenfalls nur vorübergehend existiert. Die Untersuchung von single minded (sim) zeigte, dass es, anders als in Drosophila, wo sim ein wichtiges regulatorisches Gen für die korrekte Spezifizierung von Mittellinienzellen ist, nicht in den Zellen der Mittellinie, sondern in diesen benachbarten Zellen, exprimiert wird. Das ist vergleichbar mit Vertebraten. Zusätzlich konnte ich Expression von sim an den Basen der Gliedmassen und im Kopf nachweisen. Wie in Vertebraten könnte sim an der Musterbildung dieser Gewebe beteiligt sein. Dennoch spielt die Mittellinie in Spinnen eine wichtige Rolle als Organisator für auswachsende, kommissurale Axone. Diese Funktion teilt sie mit anderen Invertebraten und Vertebraten.rnrnDie Signaltransduktionskaskade, die an der axonalen Wegfindung an der Mittellinie beteiligt ist, ist in den verschiedensten Organismen hoch konserviert. In der vorliegenden Arbeit konnte ich sowohl in Achaearanea als auch in Cupiennius ein netrin Homolog identifizieren und eine konservierte Funktion des Wegfindungsmoleküls während der Bildung der Kommissuren aufzeigen. RNAi Experimente belegen, dass, wird die Funktion von netrin herunterreguliert, das Strickleiternervensystem nicht korrekt gebildet wird, ins Besondere die kommissuralen Faszikel. Des Weiteren konnte ich eine neue Funktion von netrin, die bisher in anderen Organsimen noch nicht beschrieben wurde, identifizieren. Neben seiner Rolle in der axonalen Wegfindung, scheint netrin auch an der epithelialen Morphogenese im zentralen Nervensystem beteiligt zu sein. In dieser Funktion scheint netrin in Gliazellen, die die epithelialen Vesikel der Invaginationsgruppen umhüllen, wichtig zu sein, um neurale Vorläuferzellen in einem undifferenzierten Zustand zu halten. Der Abbau von netrin Transkript durch RNA Interferenz führt zu einer verfrühten Segregation neuraler Vorläuferzellen aus dem epithelialen Verband der Invaginationsgruppen und zu einer Zunahme an Zellen, die den frühen Differenzierungsmarker islet exprimieren.

Ruolo delle alterazioni mitocondriali indotte dall'ipossia nella patogenesi della malattia di alzheimer

Le alterazioni della funzionalità mitocondriale detengono un ruolo cruciale nella patogenesi della malattia di Alzheimer (AD), sostenendo il processo neurodegenerativo attraverso meccanismi quali la riduzione della disponibilità energetica e la iperproduzione di ROS. Alle numerose ipotesi di patogenesi dell’AD, si è recentemente affiancata la cosiddetta ipotesi vascolare. Nei soggetti AD è stata riscontrata una significativa riduzione della disponibilità di ossigeno a livello neuronale (ipossia neuronale). Da numerosi studi è poi emerso che l’ipossia gioca un ruolo fondamentale nello sviluppo dell’AD contribuendo a più vie patogenetiche contemporaneamente. Tuttavia, non sono stati ancora chiariti tutti i meccanismi attraverso cui l’ipossia esplica la sua azione di danno. Lo scopo di questo studio è stato quello di contribuire a chiarire il ruolo patologico dell’ipossia nell’AD, analizzando principalmente le alterazioni della funzionalità mitocondriale indotte dalla riduzione della disponibilità di ossigeno. Nella prima fase dello studio cellule PC12 sono state coltivate in presenza di β-amiloide e ipossia. In questo modello abbiamo osservato un potenziamento dei fenomeni di deplezione dell’ATP e di generazione delle ROS indotti dalla Aβ quando anche l’ipossia era presente come fonte di danno cellulare, ipotizzando per i due fattori un effetto congiunto di tipo additivo. Nella seconda fase abbiamo esposto all’ipossia fibroblasti prelevati da pazienti AD portatori di mutazioni a carico dei geni APP e PSEN. La presenza di mutazioni predisponenti ad un fenotipo AD era in grado di determinare un danno bioenergetico e ossidativo. Le alterazioni bioenergetiche riscontrate in normossia risultavano ulteriormente potenziate quando i fibroblasti erano coltivati in ipossia, mentre lo stato di stress ossidativo veniva evidenziato solo in condizioni ipossiche. Sulla base dei risultati finora conseguiti si può ipotizzare che uno dei meccanismi attraverso cui l’ipossia esplica la sua azione di danno nella AD, possa essere dovuto alla capacità di potenziare ulteriormente le alterazioni della funzionalità mitocondriale.

The role of NPxY domains in LRP1 receptor maturation and function

Analyses of low density lipoprotein receptor-related protein 1 (LRP1) mutant mouse embryonic fibroblasts (MEFs) generated from LRP1 knock-in mice revealed that inefficient maturation and premature proteasomal degradation of immature LRP1 is causing early embryonic lethality in NPxY1 and NPxY1+2 mutant mice. In MEFs, NPxY2 mutant LRP1 showed efficient maturation but, as expected, decreased endocytosis. The single proximal NPxY1 and the double mutant NPxY1+2 were unable to reach the cell surface as an endocytic receptor due to premature degradation. In conclusion, the proximal NPxY1 motif is essential for early sorting steps in the biosynthesis of mature LRP1.rnThe viable NPxY2 mouse was used to provide genetic evidence for LRP1-mediated amyloid-β (Aβ) transport across the blood-brain barrier (BBB). Here, we show that primary mouse brain capillary endothelial cells (pMBCECs) express functionally active LRP1. Moreover, demonstrate that LRP1 mediates [125I]-Aβ1-40 transcytosis across pMBCECs in both directions, whereas no role for LRP1-mediated Aβ degradation was detected. Aβ transport across pMBCECs generated from NPxY2 knock-in mice revealed a reduced Aβ clearance in both directions compared to WT derived pMBCECs. Finally, we conclude that LRP1 is a bona-fide receptor involved in bidirectional transcytosis of Aβ across the BBB.rn

Microglial involvement in brain physiopathology: in vitro studies using rat primary cultures

Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Der Einfluss des Endocannabinoidsystems auf die Alzheimer Pathologie

Die Alzheimer Krankheit ist eine der häufigsten neurodegenerativen Erkrankungen, deren Ursache, abgesehen von einem geringen Prozentsatz vererbter Formen, bisher nicht bekannt ist. Ein wichtiges Ziel der Grundlagenforschung liegt derzeit in der Modulation der APP-spaltenden Enzyme. Durch die Modulation dieser Enzyme könnten weniger schädigende Amyloid β-Peptide entstehen. Die Aktivität des ECS ist in vielen neurodegenerativen Krankheiten verändert. Protektive Eigenschaften der Cannabinoidrezeptoren wurden bei der Alzheimer Krankheit beschrieben. Deshalb sollte in dieser Arbeit der Einfluss des ECS auf die Pathogenese der Alzheimer Erkrankung untersucht werden. In Zellkultursystemen wurde der Einfluss von Cannabinoiden auf die Prozessierung des Amyloid-Vorläuferproteins analysiert. Durch Inkubation der Zellen mit CB1-Rezeptor Agonisten konnte die APP-Prozessierung zugunsten von sAPPα moduliert werden. Gleichzeitig führte die Inkubation mit Cannabinoiden zur reduzierten Amyloid β Menge im Medium der Zellen. In dieser Arbeit konnte die APP-Prozessierung durch die Aktivierung des CB1-Rezeptors zugunsten des nicht-amyloiden Wegs moduliert werden.rnIn einem Tiermodell wurde der Einfluss des CB1-Rezeptors in APP23 transgenen Mäusen untersucht. Der Knockout des CB1-Rezeptors führte in APP23 transgenen Tieren zu weitreichenden biochemischen Veränderungen. APP23/CB1-/--Tiere zeigten eine erhöhte Mortalität und ein sehr geringes Durchschnittsgewicht. Im Vergleich zu APP23/CB1+/+-Tieren führte der CB1-Rezeptor Knockout zur Reduktion der APP-Expression und dessen Prozessierungsprodukten. In den histologischen Untersuchungen wurde eine reduzierte Anzahl an amyloiden Plaques, sowie eine reduzierte Neuroinflammation ermittelt. Biochemische Untersuchungen zeigten, dass der CB1-Rezeptor einen möglichen regulatorischen Einfluss auf die Expression und Prozessierung von APP ausübt. Die Tiere mit der geringsten Plaque-Menge (APP23/CB1-/-) und einer reduzierten Prozessierung von sAPPα- und den CTFs zeigten die schlechteste Lernleistung im Morris Water-Maze. Deshalb müssen andere Faktoren (z.B. die Degradation der Myelinschicht) für die schlechte Lernleistung verantwortlich sein. Mit einem zweiten Tiermodell könnte in CB1-Knockout Mäusen durch den viral-vermittelten Gentransfer eine mögliche Toxizität von Aβ Peptiden untersucht werden. Die in dieser Arbeit ermittelten Ergebnisse zeigen, dass der CB1-Rezeptor an der Regulation der APP-Prozessierung beteiligt ist und zu proteinbiochemischen Veränderungen im Zell- und Tiermodell führt.

The physiological role of APP in the activation of neuroprotective signaling mechanisms

Accumulating evidence indicates that loss of physiological amyloid precursor protein (APP) function leads to enhanced susceptibility of neurons to cellular stress during brain aging. This study investigated the neuroprotective function of the soluble APP ectodomain sAPPα. Recombinant sAPPα protected primary hippocampal neurons and neuroblastoma cells from cell death induced by trophic factor deprivation. This protective effect was abrogated in APP-depleted neurons, but not in APLP1-, APLP2- or IGF1-R-deficient cells, indicating that expression of holo-APP is required for sAPPα-dependent neuroprotection. Strikingly, recombinant sAPPα, APP-E1 domain and the copper-binding growth factor-like domain (GFLD) of APP were able to stimulate PI3K/Akt survival signaling in different wildtype cell models, but failed in APP-deficient cells. An ADAM10 inhibitor blocking endogenous sAPPα secretion exacerbated neuron death in organotypic hippocampal slices subjected to metabolic stress, which could be rescued by exogenous sAPPα. Interestingly, sAPPα-dependent neuroprotection was unaffected in neurons of APP-ΔCT15 mice which lack the intracellular C-terminal YENPTY motif of APP. In contrast, sAPPα-dependent Akt signaling was completely abolished in APP mutant cells lacking the C-terminal G-protein interaction motif and by specifically blocking Gi/o-dependent signaling with pertussis toxin. Collectively, the present thesis provides new mechanistic insights into the physiological role of APP: the data suggest that cell surface APP mediates sAPPα-induced neuroprotection via Go-protein-coupled activation of the Akt pathway.

Blood-CNS barriers in neurodegenerative diseases

The blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) separate the brain and the spinal cord from the circulating blood and are important for the maintenance of the CNS homeostasis. They build a physical barrier thereby protecting the CNS from pathogens and toxic agents, and their disruption plays a crucial role in the pathogenesis of several CNS disorders. In this thesis, the blood-CNS-barriers were studied via in vitro models in two case studies for neurodegenerative disorders, in particular Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The first model evaluates treatment possibilities of AD using nanotechnology-based strategies. Since the toxic amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of AD, reduced generation or enhanced clearance of Aβ42 peptides are expected to modify the disease course in AD. Therefore, several Aβ42-lowering drugs like flurbiprofen had been tested in clinical trials, but most of them failed due to their low brain penetration. Here, flurbiprofen was embedded in polylactide (PLA) nanoparticles and its transport was examined in an in vitro BBB model. The embedding of flurbiprofen into the nanoparticles disguised its cytotoxic potential and enabled the administration of higher drug concentrations which resulted in a sufficient transport of the drug across an endothelial cell monolayer. These results demonstrate that non-permeable drugs can be transported efficiently via nanoparticles and that these nanotechnology-based strategies are a promising tool to generate novel therapeutic options for AD and other CNS diseases. rnThe focus of the second project was to investigate the impaired integrity of the BSCB in a mouse model for ALS. About 20% of all familial ALS cases are associated with missense mutations or small deletions in the gene that encodes Cu/Zn-superoxide dismutase 1 (SOD1). To date, the molecular mechanisms resulting in ALS are still unknown, but there is evidence that the disruption of the BSCB is one of the primary pathological events. In both familial and sporadic ALS patients, loss of endothelial integrity and endothelial cell damage was observed, and studies with SOD1 transgenic mice demonstrated that the BSCB disruption was found prior to motor neuron degeneration and neurovascular inflammation. Thus, an in vitro model for ALS endothelial cells was generated which exhibited comparable integrity characteristics and tight junction (TJ) protein expression profiles as isolated primary endothelial cells of the BSCB of SOD1 transgenic mice. In this, an alteration of the βcat/AKT/FoxO1 pathway, which regulates the expression of the TJ protein claudin-5, could be observed. These data furthermore indicate that ALS is a neurovascular disease, and understanding of the primary events in ALS pathogenesis will hopefully provide ideas for the development of new therapeutic strategies. rn

Hormonally defined pancreatic and duodenal neuroendocrine tumors differ in their transcription factor signatures: expression of ISL1, PDX1, NGN3, and CDX2

We recently identified the transcription factor (TF) islet 1 gene product (ISL1) as a marker for well-differentiated pancreatic neuroendocrine tumors (P-NETs). In order to better understand the expression of the four TFs, ISL1, pancreatico-duodenal homeobox 1 gene product (PDX1), neurogenin 3 gene product (NGN3), and CDX-2 homeobox gene product (CDX2), that mainly govern the development and differentiation of the pancreas and duodenum, we studied their expression in hormonally defined P-NETs and duodenal (D-) NETs. Thirty-six P-NETs and 14 D-NETs were immunostained with antibodies against the four pancreatic hormones, gastrin, serotonin, calcitonin, ISL1, PDX1, NGN3, and CDX2. The TF expression pattern of each case was correlated with the tumor's hormonal profile. Insulin-positive NETs expressed only ISL1 (10/10) and PDX1 (9/10). Glucagon-positive tumors expressed ISL1 (7/7) and were almost negative for the other TFs. Gastrin-positive NETs, whether of duodenal or pancreatic origin, frequently expressed PDX1 (17/18), ISL1 (14/18), and NGN3 (14/18). CDX2 was mainly found in the gastrin-positive P-NETs (5/8) and rarely in the D-NETs (1/10). Somatostatin-positive NETs, whether duodenal or pancreatic in origin, expressed ISL1 (9/9), PDX1 (3/9), and NGN3 (3/9). The remaining tumors showed labeling for ISL1 in addition to NGN3. There was no association between a particular TF pattern and NET features such as grade, size, location, presence of metastases, and functional activity. We conclude from our data that there is a correlation between TF expression patterns and certain hormonally defined P-NET and D-NET types, suggesting that most of the tumor types originate from embryologically determined precursor cells. The observed TF signatures do not allow us to distinguish P-NETs from D-NETs.

Pentraxins and atherosclerosis: the role of PTX3

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we will concisely review the general properties of PTX3 in the context of the pentraxin superfamily and discuss recent data suggesting that PTX3 plays a cardiovascular protective effect. PTX3 may represent a new marker in vascular pathology which correlates with the risk of developing vascular events.

Squirrel Monkeys (Saimiri sciureus) Infected with the Agent of Bovine Spongiform Encephalopathy Develop Tau Pathology

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

Ways of coping and biomarkers of an increased atherothrombotic cardiovascular disease risk in elderly individuals

Objective. To investigate the relationship between coping and atherothrombotic biomarkers of an increased cardiovascular disease (CVD) risk in the elderly. Methods. We studied 136 elderly caregiving and noncaregiving men and women who completed the Ways of Coping Checklist to assess problem-focused coping, seeking social support (SSS), blamed self, wishful thinking, and avoidance coping. They had circulating levels of 12 biomarkers measured. We also probed for potential mediator and moderator variables (chronic stress, affect, health behavior, autonomic activity) for the relation between coping and biomarkers. Results. After controlling for demographic and CVD risk factors, greater use of SSS was associated with elevated levels of serum amyloid A (P = 0.001), C-reactive protein (CRP) (P = 0.002), vascular cellular adhesion molecule (VCAM)-1 (P = 0.021), and D-dimer (P = 0.032). There were several moderator effects. For instance, greater use of SSS was associated with elevated VCAM-1 (P < 0.001) and CRP (P = 0.001) levels in subjects with low levels of perceived social support and positive affect, respectively. The other coping styles were not significantly associated with any biomarker. Conclusions. Greater use of SSS might compromise cardiovascular health through atherothrombotic mechanisms, including elevated inflammation (i.e., serum amyloid A, CRP, VCAM-1) and coagulation (i.e., D-dimer) activity. Moderating variables need to be considered in this relationship.

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.