Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier. Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis. In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs. / Eine bakterielle Meningitis tritt auf, wenn durch Blut übertragene Bakterien hochspezialisierte Hirnendothelzellen (BEC) durchdringen und Zugang zu den Meningen erhalten. Neisseria meningitidis (Nm) ist ein human-exklusiver Erreger, für dessen Untersuchung es an geeigneten In-vitro-Modellen mangelt. Bis vor kurzem war die Modellierung von BEC-Nm-Wechselwirkungen fast ausschließlich auf immortalisierte humane Zellen beschränkt, denen wichtige BEC-Phänotypen fehlen. Besonders hervorzuheben sind das Fehlen physiologischer Barriereeigenschaften durch unkontinuierliche dichte Zell-Zell-Verbindungen. Als alternative Modellorganismen können humanisierte Mäuse verwendet werden, die sich jedoch auf bekannte Wirt-Erreger-Wechselwirkungen stützen und durch Speziesunterschiede eine eingeschränkte Übersetzbarkeit aufweisen. Dies begründet die Notwendigkeit, neuartige humane In-vitro-BEC-Modelle zu etablieren, die physiologische Barrierephänotypen aufweisen, um Nm-BEC-Wechselwirkungen zu untersuchen. Kürzlich wurde ein humanes Modell entwickelt, welches auf aus induziert pluripotenten Stammzellen (iPSCs) abgeleiteten humanen BECs basiert und sich durch einen physiologischen Blut-Hirn-Schranken-Phänotyp auszeichnet. Die iPSC-BECs wurden in dieser Arbeit als neuartiges zelluläres Modell getestet, um Nm-Wirt-Pathogen-Wechselwirkungen zu untersuchen, wobei der Schwerpunkt auf Wirtsreaktionen auf Nm-Infektionen lag. Zwei Wildtypstämme und drei Mutantenstämme von Nm wurden verwendet, um zu bestätigen, dass diese ähnlichen Phänotypen wie in zuvor beschriebenen Modellen folgten. Hervorzuheben ist, dass die Rekrutierung des kürzlich veröffentlichten Pilus-Adhäsin-Rezeptors CD147 unter Meningokokken-Mikrokolonien in iPSC-BECs verifiziert werden konnte. Es wurde auch beobachtet, dass Nm die Expression der entzündungsfördernden und neutrophilen spezifischen Chemokine IL6, CXCL1, CXCL2, CXCL8 und CCL20 zu bestimmten Zeitpunkten der Infektion sowie die Sekretion von IFN-γ und RANTES durch iPSC-BECs signifikant erhöht. Es wurde zudem beobachtet, dass Nm die Tight Junction-Proteine ZO-1, Occludin und Claudin-5 zu späten Zeitpunkten der Infektion zerstört, verursacht durch die Infektion wurde ein ausgefranster und/oder diskontinuierlicher Tight Junction-Phänotyp beobachtet. Dieser Zerstörung geht die SNAI1-Aktivierung (ein Transkriptionsrepressor für Tight Junction-Proteine) voraus und könnte von ihr abhängig sein. In Übereinstimmung mit dem Verlust der Tight Junctions wurde zu späten Infektionszeitpunkten ein starker Verlust des transendothelialen elektrischen Widerstands und eine Zunahme der Natriumfluoreszein-Permeabilität beobachtet. Bemerkenswerterweise korrelierte die bakterielle Transmigration mit dem Verlust der Tight Junctions, was darauf hinweist, dass der parazelluläre Weg zur bakteriellen Überwindung von BECs eine entscheidende Rolle spielt. Schließlich wurde die RNA-Sequencing (RNA-Seq) von sortierten, infizierten iPSC-BECs durch die Verwendung von fluoreszenzaktivierten Zellsortiertechniken (FACS) nach der Infektion durchgeführt. Dies ermöglichte erstmalig den Nachweis von Expressionsdaten von Nm-responsiven Wirtsgenen, welche bei der Meningitidis eine Rolle zu spielen scheinen. Zusammenfassend konnte im Rahmen der vorliegenden Arbeit der Nutzen von iPSC-BECs In-Vitro-Modellen zur Untersuchung von Nm-Infektionen gezeigt werden. Dies ist das erste BEC-In-vitro-Modell, das alle wichtigen BEC-Tight Junctions exprimiert und ein hohes Barrierepotential aufweist. Insgesamt liefert das eingesetzte Modell neue Einblicke in die Nm-Pathogenese, einschließlich der Beeinflussung der Barriereeigenschaften und der Tight Junction-Komplexe durch Nm, und gibt erste Hinweise darauf, dass die parazelluläre Route zum Nm-Übertritt von BEC-Barrieren eine entscheidende Rolle spielt.

1. Clinical studies. Clinical diagnosis of dementia has been made largely on the basis of clinical findings supported by appropriate radiological and laboratory investigations. A minority of patients have treatable or reversible underlying causes for their dementing syndrome. It is important to distinguish between the two main forms of dementia Alzheimer's disease, senile dementia of Alzheimer type (AD/SDAT) and MID so that advantage can be taken of any future progress in treatments. In the clinical study significant differences between several diagnostic procedures were found between patients with AD/SDAT and MID. Blood pressure was significantly lower in the AD/SDAT group and focal neurological signs were seen in 70% of the MID patients but only in 6% of patients with AD/SDAT. Electrocardiogram was normal in all patients with AD/SDAT but pathological in 75% of the MID patients. Electroencephalogram showed generalized slow frequencies in 79% of the AD/SDAT patients and localized changes in 65% of the MID patients. Computerized tomography showed a significantly greater dilation of the ventricular system in MID patients compared to AD/SDAT patients and controls. Monoamine metabolites in the cerebrospinal fluid were lower in AD/SDAT patients and normal in MID patients. Psychopathological signs were found to be more variable and more pronounced in the AD/SDAT group compared with MID patients. 2. Etiological studies. Immunoglobulin and albumin were found changed in serum and CSF of both AD/SDAT and MID, indicating a more active immune response in MID and a less dense cerebrospinal fluid barrier in both MID and AD/SDAT. There appears to be a consumption of IgG in the central nervous system in patients with AD/SDAT. Abnormal chromosomes appearing as acentric fragments, i.e. without visible centromeres, were found in 90% of patients with AD/SDAT, 30% of patients with MID, and not at all in the control group. Increased aneuploidy was also seen both in patients with MID and AD/SDAT. Diabetes mellitus in old age and AD/SDAT do not seem to coexist. Furthermore, patients with AD/SDAT have changed carbohydrate metabolism with decreased fasting blood sugar concentrations, increased glucose tolerance and higher concentration of insulin during an oral glucose tolerance test. / <p>S. 1-47: sammanfattning, s. 49-144: 5 uppsatser</p> / digitalisering@umu.se

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suisinfected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.