Tesis sobre el tema "Brain microvascular endothelium"

MicroRNAs (miRs) are small non-coding regulatory RNAs that act through repression of protein translation and/or mRNA degradation at the post-transcriptional level. MiRs are critical players in the pathogenesis of many diseases, including 'neuroinflammatory disorders such as multiple sclerosis (MS), MS is characterized by leukocyte adhesion and infiltration subsequently leading to demyelination of nerve fibres. Leukocyte adhesion on brain endothelial cells (BEC) - the main cellular constituent of the blood-brain barrier (BBB) - is a complex multi-step process where activated BEC overexpress chemokines such as CCl 2 and endothelial adhesion molecules (CAM) such as selectins, VCAMl and tCAMl. Several therapies for MS target the common known mechanisms of leukocyte adhesion. Here, we studied whether specific endothelial miRs act as regulators of leukocyte adhesion to cultured human BEC in vitro, and hence whether they could be a potential therapeutic tool to prevent adhesion to endothelium, First, we characterised leukocyte adhesion using the monocytic (THP1) and T cell (Jurkat) lines under static conditions, interacting with the immortalized hCMEC/ 03 endothelial cell line as an in vitro model of the human BBB. Increased adhesion of both leukocytic cell lines to BEC was observed following treatment with TNFu and IFNy compared to unstimulated cells. Increased expression of both ICAMl and VCAMl by hCMEC/D3 cells was also observed following cytokine treatment. Cytokine-induced maximal VCAM1 and ICAM1 expression coincided with the observed maximal leukocyte adhesion to BEC at 24 h, Next, we established a novel flow-based leukocyte adhesion assay coupled with time lapse image acquisition, to mimic more closely the in vivo conditions. We successfully cultured and transfected hCMEC/D3 cells in six-channel chambers, connected to a flow system, to study leukocyte-endothelium interactions and firm adhesion, Second, we performed an initial screening of five cytokine-regulated BEC miRs, Of these five, miR- 126 and miR-155 appeared to have the most significant effects on leukocyte adhesion to hCMEC/D3cells, We further investigated the roles of miR-126, rn iR-126* (the complement of miR-126), and miR-155 in leukocyte adhesion to BEC. MiR-126 and - 126* were down-regulated in cytokine stimulated BEC low levels of miR-126 increased adhesion of both cell lines, while low levels of miR-l26* increased THP-l, but reduced Jurkat adhesion. Elevated miR-l26 and miR-126* levels significantly prevented Jurkat and THP-1 eel! adhesion to BEC both in unstimulated and cytokine-treated conditions. Furthermore, elevated miR-126 partially prevented cytokineinduced VCAM1 and CCl2 expression on BEC and an increased level of miR-126* partially prevented cytokine-induced E-selectin expression. In cytokine stimulated-BEC miR-155 was up-regulated, and decreasing the level of miR-155 reduced both T cell and monocyte adhesion to endothelium and VCAMl expression both in basal and in cytokine-stimulated conditions, The opposite effect on leukocyte adhesion was observed when miR-155 expression was increased in unstimulated hCMEC/D3 cells, but not in cytokine-stimulated endothelium. These data suggest that miR-155, miR-126 and miR-126* modulate leukocyte adhesion on human brain microvascular endothelium. To our knowledge, this study is the first to report a role for miR-155 and miR-l26* in the interactions between human brain endothelium and immune cells and the first to confirm the regulation of VCAM1 and CCL2 by miR-126 in brain endothelium.

Background: The brain is the most commonly affected organ during Toxoplasma gondii infection but the mechanisms utilized by this protozoan parasite for disrupting the brain's endothelial cells lining the blood–brain barrier (BBB) and moving to invade the brain are not yet understood. In the present study, we investigated the cellular pathogenicity of T. gondii infection in human brain microvascular endothelial cells (HBMECs), a fundamental component of the BBB. Methods: Intracellular development of T. gondii tachyzoites within HBMECs was characterized by using Acridine Orange (AO) staining. The integrity of HBMECs moolayer and tight junction permeability during T. gondii infection were assessed using transendothelial electrical resistance (TEER). Morphological changes associated with infection was assessed by scanning electron microscopy (SEM) and transmission electron microscope (TEM). Cell viability and metabolic changes associated with infection were identified using alamar blue and nuclear magnetic resonance (1H NMR). The changes in lipid content and fatty acid composition of the total phospholipids were evaluated using LipidTox staining and gas chromatography (GC). The changes in the content of trace elements in response to T. gondii infection was assessed using inductively coupled plasma mass spectrometry (ICP-MS). Results and Discussion: The invasion, growth, and replication of T. gondii tachyzoites within HBMECs are possible, with disruption of the integrity and viability of the host cell through the course of infection. AO staining of T. gondii tachyzoites infecting HBMECs showed a marked increase in the surface area of tachyzoites during infection, indicating that tachyzoites invade their host cell and form their own compartments (PV) in which tachyzoites proliferate with a generation time of 24 h, eventually leading to cell rupture and exit of the parasites. A decrease was noted in the TEER of infected cells compared to uninfected controls, indicating that the invasion of the HBMECs by T. gondii had detectable effects on the integrity HBMECs monolayer by increased tight junction permeability. Morphological analysis revealed that the intracellular development of the tachyzoites disruption of tight junctions HBMECs monolayer and reorganization of some organelles of the host cell, such as the mitochondria, endoplasmic reticulum, and Golgi apparatus around the Parasitophorous vacuole membrane (PVM) and remained stable throughout the growth of the tachyzoites. The tight association between the PVM and host organelles may provide lipids and other macromolecules for parasite survival, proliferation, membrane biogenesis, and energy requirement. A marked decrease was noted in cell viability of infected cells at 48 h PI, compared to uninfected controls by alamar blue assay, indicating that growth of the parasites that cause a metabolic burden on the host cells. Metabolite analysis of HBMECs infected with T. gondii revealed a drastic increase in lactate and glutamine levels, as well as a reduction in choline and myo-inositol levels with infection. A drastic increase in lactate and glutamine levels may be attributed to the fact that T. gondii requires energy from the host, primarily via glycolysis and glutaminolysis. It is believed that increased lactate and glutamine levels result in increased paracellular permeability. A drastic reduction in choline and myo-inositol levels suggests the use of host lipid fractions for increased membrane maintenance or parasite lipid anabolism. It is believed that increased phosphatidylcholine levels result in altered monolayer permeability. Fatty acid analysis of HBMECs infected with T. gondii revealed a significant increase in C18:0 and C18:1n9c. It is believed that increased monounsaturated and polyunsaturated fatty acids result in increased tight junction permeability via modulated occludin and ZO-1 localization. Trace element analysis of HBMECs infected with T. gondii revealed a significant increase in Zn, Fe, Mg, and Cd levels, as well as a reduction in Co levels in growth medium obtained from the infected cells compared to non-infected controls. It is possible that altered trace elements levels, whether a parasite-induced or host-cell response, is important for protection against cellular oxidative stress and DNA damage during infection, and for suppressing cell apoptosis. In conclusion, the results obtained show that HBMECs permit the invasion, growth and proliferation of T. gondii tachyzoites and that infection can disrupt tight junction permeability and cause multiple morphological changes in the relocation of the host cell organelles around the PV and changes in the levels of host cell metabolites and trace elements. These findings provide a more in depth understanding of how T. gondii replicate within the HBMECs during infection, which may lead to novel ways to prevent or treat this disease.

Ischaemic stroke is a leading cause of death and disability worldwide Successful therapies reside in a precise knowledge of brain function and pathology. Towards this end, previous work in the MMU laboratory used cDNA microarrays to examine gene expression in an experimental rat model of ischaemic stroke - permanent MCAO - and for the first time in human brain tissue from ischaemic stroke patients. Novel deregulated genes were identified. This study was intended to confirm the reproducibility of the in vivo data in vitro. The results showed that human foetal (cerebral cortical) neurons (HFN), human brain microvascular endothelial cells (HBMEC) and human astrocytes exposed to oxygen-glucose deprivation (OGD) and reperflision in most cases reproduced the gene expression profiling obtained in vivo. This suggests that HFN, HBMEC and astrocytes are good models to analyse gene deregulation after ischaemia in vitro. Further studies will help to determine the significance of such changes. Among the genes deregulated were prp and cdk5. The expression and localisation of these genes/proteins were explored in detail. Increased expression of PrPc gene and protein was found in HFN after OGD, but the significance of this expression change remains to be elucidated. Increased expression of Cdk5 and its activator p35, as well as nuclear translocation of Cdk5, p35 and p-Cdk5 (SI59) were observed in HFN and HBMEC upon ischaemic stimulation in vitro. Nuclear translocation occurred particularly in propidium iodide-positive cells, thus linking nuclear Cdk5 and p35 to cell damage in both cell types. This suggests a new role for Cdk5 in modulating endothelial cell survival following ischaemia.

Neisseria meningitidis is the major cause of meningitis and sepsis, two kind of diseases that can affect children and young adults within a few hours, unless a rapid antibiotic therapy is provided. The meningococcal disease dates back to the 16th century. The first description of the disease caused by this pathogen was stated by Viesseux in 1805 as 33 deaths occurred in Geneva, Switzerland [1]. It took about seventy years before two Italians (Marchiafava and Celli) in 1884 identified micrococcal infiltrates within the cerebrospinal fluid [2]. The worldwide presence of meningococcal serogroups may vary within regions and countries. With the coming of antimicrobial agents, like sulphonamides, and with the development of an appropriate health care and prevention programme, the fatality rate cases has dropped from 14% to 9%, although 11% to 19% of patients continued to have post-infection issues such as neurological disorders, hearing or limb loss [3]. The bacteria can be divided into 13 different serogroups and, among these, up to 99% of infection is ascribed to the serogroups named A, B, C, 29E, W-135 and Y (Fig. 2). All the serogroups have been listed in 20 serotypes on the presence of PorB antigen, 10 serotypes on the presence of PorA antigen, and in other immunotypes on the presence of other bacterial proteins and on the presence of a characteristic lipopolysaccharide called LOS (lipooligosaccharide) [4]. The transmission from a carrier to an other person occurs by liquid droplet and the natural reservoir of Neisseria meningitidis is the human throat, in particular it usually invades the human nasopharynx where it can survive asymptomatically. The reported annual incidence goes from 1 to 5 cases per 100000 inhabitants in industrialized countries, while in non developed-countries the incidence goes up to 50 cases per 100000 inhabitants. More then 50% of cases occur within children below 5 years of age, and the peak regards those under the first year of age. This fact is due to the loss of maternal antibodies by the newborn. In non-epidemic period, the percentage of healthy carriers range from 10 to 20%, and notably the condition of chronic carrier is not so uncommon [5, 6]. Only in a small percentage of cases the colonization progresses until the insurgence of the pathogenesis. This happens because in the majority of cases specific antibodies or the human complement system are able to destroy the pathogens in the blood flow allowing a powerful impairment of the dissemination. In a small group of population the colonization of the upper respiratory tract is followed by a rapid invasion of the epithelial cells, and from there bacteria can reach the blood flow and invade the central nervous system (CNS), inducing the establishment of an acute inflammatory response. How the balance between being an healthy carrier or a infected patient can change so rapidly it is still unknown. Some factors that can play a role in this switch could be the virulence of the bacterial strain, the responsiveness of the host immune system, the mucosal integrity, and some environmental factors [7]. Neisserial heparin binding antigen (NHBA) is a surface- exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology [8]. NHBA in Nm has a predicted molecular weight of 51 kDa. The protein contains an Arg-rich region (-RFRRSARSRRS-) located at position 296–305 that is highly conserved among different Nm strains. The protein is specific for Neisseria species, as no homologous proteins were found in non redundant prokaryotic databases. Full length NHBA can be cleaved by two different proteases in two different manners: NalP, a neisserial protein with serine protease activity cleaves the entire protein at its C-terminal producing a 22 kDa protein fragment (commonly named C2) which starts with Ser293 and hence comprises the highly conserved Arg-rich region. The human proteases lactoferrin (hLf) cleaves NHBA immediately downstream of the Arg-rich region releasing a shorter fragment of approximately 21 kDa (commonly named C1) [9] . Although it is known that a crucial step in the pathogenesis of bacterial meningitidis is the disturbance of cerebral microvascular endothelial function, resulting in blood-brain barrier breakdown, the bacterial factor(s) produced by Nm responsible for this alteration remains to be established. The integrity of the endothelia is controlled by the protein VE-cadherin, mainly localized at cell-to-cell adherens junctions where it promotes cell adhesion and controls endothelial permeability [10]. It has been reported that alteration in the endothelial permeability can be ascribed to phosphorylation events induced by soluble factors such as VEGF or TGF-beta[11] [12]. Our work demonstrates that the NHBA- derived fragment C2 (but not C1) increases the endothelial permeability of HBMEC (human brain microvasculature endothelial cells) grown as monolayer onto the membrane of a transwell system. Indeed, the exposure of the apical domain of the endothelium to C2 allows the passage of the fluorescent tracer BSA-FITC, from the apical side to the basal one, early after the treatment. Interestingly, the effect of C2 on the endothelium integrity is such to allow the passage of bacteria, E. coli but, notably, also N. meningitidis MC58, from the apical to the basolateral side of the transwell and it depends on the production of mitochondrial ROS. Remarkably, we have found that the administration of C2 to endothelia results in a ROS-dependent reduction of the total VE-cadherin content. This event requires after VE-cadherin phosphorylation, the endocytosis and the subsequent degradation of the protein. Collectively our data suggest the possibility that C2 might be involved in the mechanisms of invasion owned by the bacterium to cross host tissues.
Neisseria meningitidis è uno dei patogeni in grado di causare meningite oltre che sepsi in soggetti infettati, due patologie che colpiscono maggiormente bambini e adolescenti entro poche ore dal contagio a meno di una tempestiva terapia antibiotica. La malattia meningococcica risale al sedicesimo secolo. La prima descrizione della malattia causata da questo agente patogeno avvenne ad opera di Viesseux nel 1805 come conseguenza di 33 decessi occorsi a Ginevra, Svizzera [1]. Circa 70 anni dopo, due italiani (Marchiafava e Celli) nel 1884 identificarono per la prima volta degli infiltrati meningococcichi nel fluido cerebrospinale [2]. La presenza di Neisseria meningitidis nel mondo varia in base a paesi e regioni e risulta essere ciclica. Grazie alla scoperta di agenti antimicrobicidi come i sulfonamidici e grazie alla diffusione di un adeguato protocollo di prevenzione sanitaria i casi di mortalita` dovuti a questo agente patogeno sono rapidamente diminuiti dal 14 al 9%. Ciò nonostante una percentuale compresa tra l’11 e il 19% dei soggetti ha continuato ad avere problemi post-infezione come disordini neurologici, o perdità dell’udito [3]. Esistono attualmente 13 sierogruppi e, di questi, il 99% delle infezioni è causato dai tipi A, B, C, 29E, W-135 e Y. I sierogruppi sono stati a loro volta classificati in 20 sierotipi sulla base della presenza dell’antigene proteico PorB, in 10 sierotipi sulla base dell’antigene PorA e in altri immunotipi a seconda della loro capacita` di indurre una risposta immunitaria nell’ospite grazie alla presenza di altre proteine batteriche del patogeno, e per la presenza di un particolare lipopolisaccaride chiamato LOS (lipooligosaccaride) [4]. Neisseria meningitidis è in grado di colonizzare l’epitelio della mucosa orofaringea, dove vi può sopravvivere in maniera asintomatica per l’ospite. La trasmissione inter-individuale avviene attraverso secrezioni dell’apparato respiratorio. L’ incidenza annuale risulta essere di 1- 5 casi ogni 100000 abitanti nei paesi industrializzati, mentre nei paesi ancora in via di sviluppo questa sale a 50 casi per 100000 abitanti. Più del 50% dei casi riguarda bambini sotto i 5 anni d’età, con un’elevata incidenza per coloro che hanno meno di un anno di vita. Questo fatto dipende dall’emivita degli anticorpi materni solitamente in grado di proteggere il neonato per circa 3-4 mesi dopo la nascita. In periodi definiti non-epidemici la percentuale dei portatori sani varia tra il 10 e il 20% della popolazione, e per l’appunto la condizione di portatore asintomatico non è poi così infrequente [5, 6]. Soltanto in un numero ristretto di casi la colonizzazione del batterio progredisce manifestando la patogenesi meningococcica: ciò è per la maggior parte dovuto alla presenza di specifici anticorpi, o per l’attività del sistema del complemento dell’ospite che è in grado di controllare ed eliminare il patogeno impedendone così la sua disseminazione attraverso il flusso sanguigno. Tuttavia, in un piccolo gruppo della popolazione, la colonizzazione del tratto respiratorio superiore è seguita da una rapida invasione delle cellule epiteliali della mucosa, da dove il batterio è in grado di entrare nel torrente ematico, e raggiungere il sistema nervoso centrale inducendo una forte risposta infiammatoria. Quale sia l’evento che perturbi l’equilibrio tra essere portatore asintomatico e paziente infetto ancora non è noto. Alcuni fattori sembrano giocare un ruolo chiave in questo cambiamento come la virulenza del ceppo batterico, la capacità della risposta immunitaria dell’ospite, l’integrità della mucosa e alcuni fattori ambientali [7]. La proteina NHBA, Neisserial Heparin Binding Antigen, è una lipoproteina esposta sulla superficie del batterio, originariamente identificata attraverso la tecnica della “reverse vaccinology” [8]. NHBA in Nm ha un peso molecolare predetto di 51 kDa. La proteina altresì contiene una regione ricca in Arginine (-RFRRSARSRRS-) localizzata in posizione 296 -305 ed altamente conservata in vari ceppi di Neisseria [9]. Tale proteina è altamente conservata in Neisseria e non ha omologie di sequenza con nessun’altra proteina registrata nei database procariotici. Due diverse proteasi possono tagliare la proteina intera NHBA producendo due frammenti differenti: nel primo caso la proteasi batterica NalP taglia la proteina intera in posizione C-terminale producendo un frammento di 22 kDa (comunemente chiamato C2) che inzia con la Ser293 e quindi comprendendo lo stretch di Arginine. Invece, nel secondo caso, la lattoferrina umana (hLf) taglia NHBA immediatamente a monte della sequenza di Arginine, producendo un frammento più corto di circa 21 kDa (comunemente chiamato C1). Sebbene sia risaputo che un passaggio cruciale nella patogenesi mediata da Neisseria meningitidis sia l’alterazione della funzione di barriera della microvascolatura encefalica, che può dunque risultare in una rottura della barriera emato- encefalica stessa, non è ancora chiaro quali siano i fattori rilasciati o prodotti dal batterio in grado di indurre un simile effetto. L’integrità dell’endotelio è controllata dalla proteina VE-caderina, localizzata sulle giunzioni aderenti che regolano il contatto cellula- cellula. Tale proteina promuove e regola dunque la permeabilità endoteliale [10]. E’ stato ben documentato che l’alterazione della permeabilità endoteliale può essere dovuta a processi di fosforilazione indotti da fattori solubili come VEGF o TGF-beta[11] [12]. Il nostro lavoro documenta come, a differenza del frammento C1, il frammento C2 prodotto dal taglio della proteina intera NHBA, sia in grado di aumentare la permeabilità delle cellule endoteliali HBMEC (human brain microvasculature endothelial cells) fatte crescere a monostrato sulla membrana di un sistema di transwell. L’esposizione della porzione apicale dell’endotelio polarizzato al frammento C2 consente il passaggio di un tracciante fluorescente, BSA-FITC, dal lato superiore a quello inferiore del transwell, in tempi rapidi a seguito del trattamento. E’ interessante notare che l’effetto di C2 sull’endotelio è tale da permettere il passaggio dal lato superiore a quello inferiore del transwell non solo di E. coli, usato come modello batterico preliminare, ma anche dello stesso Neisseria meningitidis MC58, in maniera ROS dipendente. Degno di nota è il fatto che abbiamo osservato che la somministrazione di C2 alle cellule endoteliali provoca una riduzione ROS dipendente del contenuto totale di VE-caderina. A seguito della sua fosforilazione, infatti, VE-caderina viene endocitata all’interno della cellula per poi essere degradata probabilmente attraverso il trasporto di essa verso il proteasoma. I nostri dati suggeriscono pertanto che C2 sia uno dei meccanismi di invasione possieduti da Neisseria per invadere i tessuti dell'ospite.

Thesis (M.S.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed April 7, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 64-70).

Escherichia coli (E. coli) Kl is one of the commonest Gram negative bacteria causing neonatal bacterial meningitis in both developed and developing countries. Haematogenous spread is a key step in E. coli Kl meningitis; however, it is not clear how bacteria cross the brain endothelium to gain entry into the central nervous system. Previous studies have focussed mainly on the identification of bacterial virulence factors, as well as the signalling pathways that are activated for the recruitment of actin cytoskeleton to the bacterial adhesion site on the apical surface of human brain microvascular endothelial cells (HBMEC) and finally lead to bacterial uptake. However, the cellular requirements and mechanisms of post-invasion events are poorly understood. This study aims to further characterize E. coli KI entry, intracellular trafficking and the associated molecular mechanisms. To achieve this, a virulent fluorescent proteinexpressing E. coli K I strain was constructed. In a previous study, caveolin-l, a lipid raft marker associated with clathrin-independent endocytosis, was found associated with invading and intracellular bacteria in HBMEC. To further study the effect of caveolin-l on the bacterial entry, different caveolin-l mutants were applied here. Overexpression of caveolin-l Y 14A mutant and caveolin-l~, which is non-phosphorylatable, did not block E. coli Kl invasion of HBMEC. Furthermore, E. coli Kl invasion of caveolin-l knockout mouse lung endothelial cells (MLEC) was not blocked, which suggested that caveolin-l was not required for E. coli K 1 invasion of endothelial cells. The role of dynamin, a large GTPase that has been implicated in the membrane fission of caveolae buds, was also investigated. Based on quantitative microscopy scoring, no evidence of any inhibitory effect on the bacterial invasion was observed in cells overexpressing green fluorescent protein- (GFP) tagged dominant negative dynamin 2 [Dyn2(aa)K44A] and dominant negative dynamin 1 (DynlK44A). The experimental evidence from this study therefore suggests that E. coli Kl might invade HBMEC via a caveolae- and dynamin-independent endocytic pathway. To further explore the endocytosis pathway that the bacteria use to invade HBMEC, immunofluorescence staining of E. coli Kl infected HBMEC revealed colocalization of the bacteria with flotillin 1, another lipid raft marker associated with clathrin-independent endocytosis. However, E. coli K1 infection of flotillin 1 knockout MLEC demonstrated a significantly increased bacterial uptake. This observation suggests that E. coli K 1 uptake does not require flotillin 1. In parallel, the number of intracellular non-pathogenic E. coli K-12 recovered from the lysates of flotillin 1 knockout MLEC was also significantly higher than that recovered from the lysates of wild type MLEC. Further, overexpression of GFP-tagged flotillin 1 and flotillin 2 in HBMEC inhibited E. coli Kl invasion, which suggest flotillin might have a role as a regulatory cell barrier in host defence.

Acetylcholine (Ach) and glutamate (Glu) are two of the major excitatory neurotransmitters in the brain which increase cerebral blood flow by releasing nitric oxide (NO) from postsynaptic neurons and astrocytes and causing vasorelaxationin adjacent microvessels. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/Calmodulin endothelial NO synthase (eNOS). Surprisingly, the Ca2+-dependent mechanisms whereby Ach induces NO synthesis in brain endothelial cells (ECs) is still unclear. On the other hand, Glu stimulates NMDA receptors to activate eNOS, but it is able to cause a metabotropic increase in intracellular Ca2+ concentration in brain microvascular ECs. The present investigation sought to fill these gaps by analysing murine (bEND5) and human (hCMEC/D3) brain microvascular ECs. Herein, we first demonstrated that Ach induces NO release by triggering two different modes of Ca2+ signals in bEND5 and hCMEC/D3 cells. Of note, endoplasmic reticulum Ca2+ release via inositol-1,4,5-trisphosphate receptors and store-operated Ca2+ entry shapes the Ca2+ response to Ach in both cell types but their different Ca2+ toolkits result in two quite different waveforms, i.e. Ca2+ oscillations vs. biphasic Ca2+ elevation. Whatever its waveform, however, Ach-induced intracellular Ca2+ signals lead to robust NO release in both murine and human brain microvascular ECs. Likewise, we demonstrated for the first time that Glu activated metabotropic intracellular Ca2+ oscillation in bEND5 cells and a biphasic increase in intracellular Ca2+ concentration in hCMEC/D3 cells. We further showed that glutamate-dependent Ca2+ signals drive NO release in both types of cells. This NO signal is delayed as compared to the Ach-induced one and is likely to play a crucial role in the slower vasodilation that often follows brief neuronal activity or that sustains functional hyperemia during persistent synaptic transmission. This information has a potential clinical relevance as the decrease in neuronal activity-induced cortical CBF is involved in a growing number of neurodegenerative disorders, such as Alzheimer’s Disease. Understanding the underlying mechanisms could, therefore, be used in the future as target to rescue local blood perfusion in patients affected by neurodegenerative disorders.

Lipid oxidation is one of the leading causes of food quality degradation. Manufacturers typically add antioxidants or purge a productâ s package of oxygen to inhibit oxidation and the resulting off-flavors. Synthetic antioxidants (e.g. BHT, BHA) and some natural antioxidants (e.g. α-tocopherol) have found widespread use in this application. Unfortunately, the public views synthetic additives in a negative light and the current natural antioxidants have been unable to match the protection afforded by the synthetic antioxidants. The search for underutilized and natural antioxidants has led scientists to investigate many different plant-based extracts for use in food and in the treatment and prevention of disease. The objectives of this research were (1) to use ORAChromatography to identify peanut root extract fractions with high antioxidant capacity, (2) identification of compounds in peanut root extracts using HPLC and mass spectrometry, (3) test for the presence of aflatoxins in the extracts, (4) test peanut root extract in food model system for oxidation reduction capabilities, and (5) Testing peanut root extractâ s ability to decrease protein oxidation in cell culture. Crude peanut root extracts have high antioxidant activities that do not vary by cultivar. The ORAC activities of the peanut root fractions separated by HPLC with a C18 column varied (600.3 â 6564.4 μM TE/g dry extract), as did the total phenolic contents (23.1 â 79.6 mg GAE/g dry extract). Peanut root fractions had aflatoxins contamination well above the 20 ppb limit. Peanut root extracts and the known antioxidants tested were found to have no significant effect in inhibiting oxidation of peanut paste or HBMEC. Peanut root extracts were not shown to have any positive effects, but further research is necessary to eliminate peanut root extracts as a possible food ingredient and health supplement.
Ph. D.

Peanut skins are a low-value byproduct of peanut blanching operations. They have been shown to contain significant levels of phenolic compounds with demonstrated antioxidant properties. The effects of two types of extraction methods: solid-liquid extraction (SLE) and microwave-assisted extraction (MAE) on the recovery of phenolic compounds from peanut skins were investigated. Response surface methodology was used to optimize extraction conditions based on total phenolic content (TPC), ORAC (oxygen radical absorbance capacity) activity and trans-resveratrol content. The protective effect of peanut skin extracts (PSE) against hydrogen peroxide (H₂O₂)-induced oxidative stress in human brain microvascular endothelial cells (HBMEC) and the effect of PSE on lipid oxidation in commercial peanut butter were also evaluated. In the SLE method, EtOH was found to be the most efficient extraction solvent followed by MeOH, water and EA. Despite EtOH extracts having a higher TPC, samples extracted with MeOH demonstrated slightly higher ORAC activity. Resveratrol was identified in MeOH extracts but was not found in EtOH, water or EA extracts. In the MAE procedure, the maximum predicted TPC under the optimized conditions was 144 mg phenols/g skins compared to 118 mg/g with SLE. The maximum predicted ORAC activity was 2789 μmol TE/g as opposed to 2149 μmol TE/g with the SLE method. MAE was able to extract more phenolic compounds (with higher antioxidant activity) in a faster time than the SLE procedure. In addition, resveratrol was identified in PSE derived from MAE although at relatively low levels. PSE were found to have some protective effects against oxidative stress in HBMEC. Higher doses of PSE appeared to have a slightly cytotoxic effect. However, the data were highly variable which made it difficult to arrive at any definitive conclusions regarding the potential benefits of PSE in preventing oxidative damage to cells. In the PB experiment, hexanal levels over the storage period were not high enough for the samples to be considered oxidized. However, hexanal values of PB samples treated with PSE were lower than the control throughout storage, which suggests that PSE may provide some protection against oxidation of PB.
Ph. D.

Die bakterielle Meningitis ist trotz der Anwendung modernster Antibiotika mit einer hohen Letalität und neurologischen Spätkomplikationen verbunden. Ein entscheidendes Ereignis ist dabei der Zusammenbruch der Blut-Hirn-Schranke (BHS). Die genauen Mechanismen, die zu ihrer Schädigung führen, sind bis heute unklar. In dieser Arbeit wurde untersucht, ob lebende Pneumokokken in einem Zellkulturmodell der BHS zu einer apoptotischen Zellschädigung von zerebralen Endothelzellen, als wichtigstem zellulären Bestandteil der BHS, führen und damit zu ihrer strukturellen Schädigung beitragen. Mittels verschiedener Detektionsmethoden (TUNEL, Fluoreszenzmikroskopie, Elektronenmikroskopie) konnte nachgewiesen werden, daß Streptococcus pneumoniae zu einem apoptotischen endothelialen Zelltod führt. Eine Beteiligung von Caspasen konnte weder mit direkter Aktivitätsmessung noch mittels Inhibitionsexperimenten oder dem Nachweis von Caspase-spezifischen Substraten gezeigt werden. Insgesamt sind die Morphologie der Zellkerne und die spezifische Degradation der endothelialen DNS hinweisend für einen Apoptosis-Inducing-Factor-vermittelten Zelltod ohne Caspasenbeteiligung. Diese Form des Zelltodes ist bereits in anderen Zellmodellen, bisher jedoch nicht bei zerebralen Endothelzellen beschrieben worden. Auf Seiten des Bakteriums konnten Wasserstoffperoxid und Pneumolysin als Auslöser der Apoptose identifiziert werden. Die zytotoxische Potenz des Pneumolysins ist dabei an dessen Poren-formende Aktivität gebunden. Die Ergebnisse sind von potentieller klinischer Relevanz, da es bei einer Bakteriämie und während der Invasion der Pneumokokken in das ZNS zu einem direkten Kontakt zwischen Bakterien und zerebralen Endothelzellen kommt und sich daraus eine Möglichkeit zur Entwicklung adjuvanter Therapien ergeben könnte.
Despite sufficient antibiotic treatment, pneumococcal meningitis has remained a disease associated with high mortality and neurological sequelae. The disruption of the blood brain barrier (BBB) is regarded a key event in the initial phase of pneumococcal meningitis. However, the exact molecular mechanisms involved in this process are still unknown. The aim of this study was to determine if living pneumococci are able to induce apoptosis in cerebral endothelial cells - the main cellular component of BBB - and therefore might contribute to its damage. Using several different detection methods (TUNEL, fluorescence and electron microscopy), induction of apoptotic cell death of endothelial cells by pneumococci could be verified. An accompanying activation of caspases was not detectable, despite the use of specific detection techniques such as inhibition experiments, direct enzyme measurements and detection of caspase-specific protein cleavage. These results as well as the specific nuclear morphology and degradation of endothelial DNA suggest an involvement of the mitochondrial protein Apoptosis inducing factor (AIF). This is the first time this specific form of apoptotic, AIF-driven cell death has been described to be engaged in endothelial cells. On the part of the bacterium, pneumolysin and hydrogen peroxide were identified as the two main inducers of apoptosis. The cytotoxic potency of pneumolysin is related to its pore-forming activity. These results are of clinical relevance since pneumococci are known to reside in close proximity to cerebral endothelial cells during bacteriemia and their entry into the CNS. These findings could contribute to the development of adjuvant treatment of bacterial meningitis.

Die bakterielle Meningitis (BM) ist trotz antibiotischer Therapie eine Erkrankung mit einer hohen Mortalität und Morbidität. Kopfschmerzen und Meningismus sind Hauptsymtome und ein klinischer Hinweis für die Aktivierung trigeminaler Fasern. Ziel dieser Arbeit war es zu prüfen ob die freigesetzten Neuropeptide einen proinflammatorischen Effekt auf zerebrale Endothelzellen, einen wesentlichem Bestandteil der Blut-Hirn-Schranke haben. Wir verwendeten primär kultivierte zerebrale Kapillarendothelzellen (BMEC) der Ratte und als Stimulus Neuropeptide und/oder Pneumokokkenzellwände (PCW). Beide Neuropeptide, CGRP mehr als SP, verstärken den Effekt von PCW auf die mRNA Expression und Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und MIP-2 aus den BMEC. CGRP und SP haben nur eine geringe Wirkung. PCW regulieren die Dichte der CRLR (CGRP1-R) bzw. NK-1 Rezeptoren und erklären damit die kostimulatorische Wirkung. Zudem untersuchten wir den Effekt von PCW und/oder CGRP auf die Adrenomedullin (AM)- Synthese. AM ist ein vasodilatorisch wirkendes Peptid, dass vorwiegend in Endothelzellen konstitutiv gebildet wird und am CRLR Rezeptor wirkt. PCW und CGRP verstärken die Synthese von AM. Mit dieser Arbeit konnte gezeigt werden, dass PCW zur Hochregulation von Neuropeptidrezeptoren führt und CGRP und SP über diese Rezeptoren einen modulatorischen Effekt auf die Zytokinproduktion in BMEC haben. Ein genaues Verständnis dieser Interaktionen könnte die Entwicklung immunmodulatorischer Interventionen und damit eine Verbesserung der Prognose der bakteriellen Meningitis bewirken.
Despite antibiotic treatment bacterial meningitis is still associated with a high mortality and morbidity. Headache and meningismus as key symptoms, provide clear evidence for the activation of trigeminal nerve fibers. Aim of the study was to test whether the released neuropeptides have a proinflammatory effect in cerebral endothelial cells the major compartment of the blood brain barrier. We used primary brain microvascular endothelial cells of the rat (BMEC) which were stimulated with CGRP, SP and/or pneumococcal cell walls (PCW). Both neuropeptides CGRP more than SP enhanced PCW-induced mRNA expression and the release of TNF-alpha, IL-1-beta, IL-6, IL-10 and MIP-2. Neuropeptides alone were not able to induce these cytokines. PCW upregulate the density of CRLR receptor and regulate the NK-1 receptor and therefore may explain the costimulatory effect. Furthermore the effect of PCW and/or CGRP on adrenomedullin synthesis in BMEC was investigated. Adrenomedullin is a vasodilatatory peptide, which is constitutivly produced by endothelial cells and act on the CRLR receptor. PCW as well as CGRP enhance the synthesis of AM. Our data suggest that PCW upregulate neuropeptide receptors and modulate via these specific receptors the cytokine production. A detailed understanding of these interactions may open new immunmodulatory interventions and therefore may contribute to a better prognosis of bacterial meningitis.

The working hypothesis is that any stimulus acting on brain microvascular endothelium, might activate the neurovascular unit by intercellular crosstalk, and consequently could increase neuronal hyperexcitability. The first chapter presents the concept of neurovascular unit. Interactions between neurovascular unit components are also discussed, and their involvement in ictogenesis. The second chapter describes the aim of the thesis, the main objectives and the principal technical approaches used in the study. The third chapter is dedicated to the elusive interaction between Qtracker®800 vascular labels and brain endothelial microvascular endothelial cells. Results: (1) non-targeted PEGylated near-infrared emitting Qtracker®800 accumulate in brain vascular endothelium, (2) Qtracker®800 alters ‘normal’ calcium signalling in brain endothelial cells, (3) there are substantial inter-individual differences in human endothelial cells activation upon exposure to Qtracker®800, (4) Qtracker®800 may not be suitable for translational studies. In conclusion, although brain vasculature imaging techniques can greatly benefit from the use of nanoparticle labels, such labels may be internalized by and functionally interact with blood vessel endothelia, which raises obvious safety concerns. The fourth chapter makes a functional description of the muscarinic acetylcholine receptors in brain microvascular endothelial cells. Results: (1) all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells, (2) acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors, (3) The relative mRNA expression of M2-M5 correlates with their relative protein abundance, but a mismatch exists for M1 mRNA versus protein levels, (4) although M1 and M3 are the most abundant receptors, only a small fraction of the M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling, (5) bioinformatic analysis performed on eucaryotic species demonstrate the high degree of conservation of the orthosteric binding site and the great variability of the allosteric site, (6) muscarinic acetylcholine receptors represent potential pharmacological targets in future translational studies. In conclusion, our findings indicate that investigators should particularly focus on the allosteric binding sites of the M1 and M3 receptors. The fifth chapter describes molecular mechanisms of pilocarpine action at the level of brain microvascular endothelium. Results: (1) pilocarpine induces the in vivo and in vitro increase of the cytokines levels, (2) pilocarpine upregulates the expression of adhesion molecules in brain microvascular endothelial cells, (3) pilocarpine elicits calcium transients in brain microvascular endothelial cells but is not inducing epileptic-like discharges in hippocampal pyramidal neurons, (4) pilocarpine downregulates the expression of tight junction proteins and permeabilizes the monolayers of brain microvascular endothelial cells, (5) pilocarpine competes with acetylcholine on the same binding site of the muscarinic receptors and regulates their expression. In conclusion, our study indicates that brain endothelium is an important site of action for pilocarpine, and that neurons’ exposure to pilocarpine is not triggering seizure-like activity, therefore epileptogenesis mechanisms should be revisited.

博士
國立中興大學
生命科學系所
101
Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis-associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV-infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant-1, and regulated-upon-activation normal T-cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal-regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up-regulating cytosolic phospholipase A2, NF-κB, and cAMP response element binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.

Disruption of the blood-brain barrier (BBB) is hypothesized to play an important role in the disease biology of schizophrenia (SZ). Brain microvascular endothelial cells (BMECs) have paracellular and transcellular proteins, transporters, as well as important extracellular matrix proteins, which collectively contribute to maintaining proper BBB function. While previous studies have provided some insights into the role of the BBB in SZ pathophysiology, there is a significant gap in our understanding of the cellular-molecular underpinnings of its major component, BMECs. Human induced pluripotent stem cells (hiPSCs) provide an exciting new avenue for exploring the role of BMECs in SZ. We hypothesize that BMECs have intrinsic deficits that lead to BBB dysfunction in SZ. In this study, we first aimed to test whether the existing hiPSC-derived BMEC protocols work with our patient-specific hiPSC samples. Secondly, we sought to investigate any potential deficits between BMECs derived from healthy control (HC) and SZ subjects. We successfully adapted the established protocol and confirmed the identity of these hiPSC-derived BMECs with relevant cell markers such as CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. We also evaluate barrier function by measuring trans-endothelial electrical resistance (TEER) and efflux transporters activity of ABCB1 and ABCC1. We observed evidence of poor cellular adhesion and disrupted tight junctions in a subset of SZ hiPSC-derived BMECs, where approximately 70% of them demonstrated extensive BBB disruption (reduced TEER). These findings suggest that there may be cell-autonomous disease-specific deficits in BMECs in SZ that result in BBB dysfunction.
2022-06-07T00:00:00Z

碩士
長庚科技大學
健康產業科技研究所
107
As the population of the world ages, the elderly population and aging-related disorders are also increasing. Therefore, in recent years, most studies have focused on the aging-related disorders, the central nervous system (CNS) degenerative diseases particularly. These CNS disorders include traumatic brain injury, stroke, neuroinflammation, and neurodegeneration. It is known that endothelial cells of Blood-Brain-Barrier (BBB) in patients with neurodegenerative diseases usually have an inflammatory state, but the mechanisms are still unclear. Here, we use the brain microvascular endothelial cells (bEnd.3) as a cell model to investigate the inflammatory responses induction by proinflammatory factors IL-1 and TNF- and then the effects of some natural products on the responses. First, we found that both IL-1 and TNF- can induce several inflammatory proteins’ expression, including MMP-9 by regulating their gene and protein. Moreover, the pharmacological inhibitors were used to investigate the mechanisms. By pretreatment of cells with reactive oxygen species (ROS) inhibitors, we demonstrated that IL-1 and TNF- induced inflammatory proteins’ expression via a ROS-dependent signaling pathway, including NADPH oxidase (NOX) or mitochondria. We further found that IL-1 and TNF- stimulated activation of many signaling molecules, such as c-Src, EGFR, AKT, and MAPKs (ERK1/2, p38, and JNK1/2), which may be involved in the regulatory pathway by the specific pharmacological inhibitors in bEnd.3 cells. In transcriptional factors, IL-1 and TNF- stimulated activation of NF-B and AP-1. Finally, we found that IL-1 and TNF- induced the configurational change of ZO-1, as a cell tight junction protein, in bEnd.3 cells by immunofluorescence stain. The induction of MMP-9 by IL-1 and TNF- may be involved in the event. Furthermore, many previous studies have indicated that some natural products have anti-inflammatory, anti-oxidative, or immune-enhancing effects, which may have fewer side effects. Therefore, these natural products could be used for the prevention or treatment of chronic inflammation-related diseases. However, the effects of most natural products still lack scientific evidence. Here, we found some natural products can attenuate IL-1- and TNF--induced MMP-9 expression, suggesting that these natural products may contain anti-inflammatory components to protect the brain BBB from damage. We will further explore the active ingredients of these natural products and their mechanisms in the future.

archives@tulane.edu
Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain injury. nNOS and eNOS have been shown to uncouple in pathological conditions to produce superoxide. Oxidative stress is believed to be the underlying cause of several cardiovascular diseases including ischemic stroke. However, the role of eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective of the study was to determine the effect of eNOS and nNOS inhibition on reactive oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain microvascular endothelial cells (BMECs) and rat cortical neurons following oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific inhibition of NOS in endothelial cells reduced ROS levels in BMECs but increased ROS levels in neurons under normoxia. This suggests that a pool of uncoupled NOS exists in the BMECs whereas the dominant functional NOS in neurons produces NO. We observed increased levels of ROS following OGD/R that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced mitochondrial respiration while it improved cell survival rate in both BMECs and neurons following OGD/R. Thus, it is possible that decreased mitochondrial respiration in the immediate aftermath (4 hours) of OGD/R could be protective against reoxygenation injury. Moreover, we identified the expression of nNOS in BMECs from rat, human, and mouse. We observed that the nNOS in the BMECs constitutively produces superoxide under physiological conditions instead of NO. In contrast, nNOS in the neurons produces NO and doesn’t contribute to ROS. We also confirmed the nNOS expression and its function in freshly isolated rat brain microvessels. In addition, we developed a novel method to measure mitochondrial respiration in freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We validated the method by demonstrating impaired mitochondrial respiration in cerebral microvessels isolated from old mice compared to young mice. In summary, the present doctoral research investigated the distinct role of NOS isoforms in BMECs and Neurons leading to the identification of novel functional variant of nNOS in BMECs and brain microvessels.
1
RAMARAO SVNL

碩士
國立中正大學
化學工程研究所
100
We constructed in vitro human blood-brain barrier (BBB) model using pericyte-condition medium (PCM) and astrocyte-condition medium and different ratio between human brain vascular pericytes (HBVPs) and human astrocytes (HAs) (1: 1, 1: 2, 1: 6) co-culture with human brain microvascular endothelial cells (HBMECs). After 7 days of co-culture of HBMECs, HBVPs, HAs (HBVPs:HAs=1: 2), PCM2, ACM2 (PCM: ACM= 1: 1), the transendothelial electrical resistance (TEER) increased to 319±16.67 Ω×cm2, reduced permeability of propidium iodide (PI) about 39 %. The activity of P-glycoprotein (P-gp) of the model with HBVPs: HAs= 1: 2 is higher than the model with HBVPs: HAs= 1: 1 (1.84-fold) and the model with HBVPs: HAs= 1: 6 (2.04-fold). We showed the co-culture models barrier integrity among three different ratio between HBVPs and HAs (1: 1, 1: 2, 1: 6) by TEER and propidium iodide permeability measurements and P-gp activity. The analysis of the three major barrier integrity modulators transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs) displayed higher TGF-β1 activity and lower levels VEGF, MMP-9 in the coculture with HBVPs: HAs= 1: 2. It suggest constructed a in vitro model match the in vivo coverage ratio of HBVPs on HBMECs is more representative. Finally, we used the TGF-β1 inhibitor SB431542, VEGF inhibitor asterric acid, and MMP-9 inhibitor CTT to discuss the relationship between barrier integrity modulators and the P-gp activity. In our study we find that TGF-β1 can up-regular P-gp activity and VEGF can down-regular P-gp activity. Keyword: blood-brain barrier, human brain microvascular endothelial cells, human astrocytes, P-glycoprotein, transforming growth factor-β1, ascular endothelial growth factor, matrix metalloproteinases.

Neisseria meningitidis ist einer der wichtigsten Erreger bakterieller Meningitiden und gefürchtet für das Potential Epidemien auszulösen. Die Meningokokken-Meningitis bleibt bis heute auch in Industrieländern mit hoher Mortalität verbunden. Um eine Meningitis verursachen zu können, müssen Meningokokken die Blut-Hirn/Liquor-Schranke überqueren. Dies erfolgt vermutlich über den transzellulären Weg durch die Endothelzellbarriere der Gehirnkapillare. Ereignisse unmittelbar vor der bakteriellen Internalisierung sind vielfach untersucht, noch wenig erforscht sind jedoch die in der Endothelzelle durch den initialen Kontakt des Erregers ausgelösten Signalkaskaden. Die Rolle des für eine ADP-Ribosyltransferase kodierenden narE Gens in der Pathogenese der Meningokokken-Infektion und die mögliche Bedeutung in der Aktivierung von Signaltransduktionsmechanismen wird diskutiert. Eine narE Insertionsmutante wurde hergestellt und charakterisiert. Anschließend wurde die Aktivierung der extracellular signal regulated kinase (ERK) im Verlauf von Infektionsassays in HBMEC (human brain microvascular endothelial cell) mittels Western Blot untersucht. Eine Zu- oder Abnahme in der Phosphorylierung von ERK und folglich eine Aktivierung oder Deaktivierung der ERK-vermittelten Signalkaskaden in HBMEC konnte jedoch im Laufe der Infektion bei der narE Mutante im Vergleich zum Wildtypstamm nicht festgestellt werden. Elektronenmikroskopische Aufnahmen zeigen Meningokokken intrazellulär einzeln aber auch zu mehreren in phagosomenähnlichen membranumgebenen Strukturen. Die Fähigkeit von N. meningitidis sich intrazellulär zu replizieren wurde mittels Infektions-assay untersucht. Bekapselte Meningokokken waren in der Lage, sich sowohl in Epithel- als auch in Endothelzellen zu replizieren, während unbekapselte Erreger intrazellulär abgetötet wurden. Bei Meningokokken wie auch beim Erreger neonataler Meningitiden E. coli K1 wird eine O-Acetylierung des Kapselpolysaccharids beobachtet. Die biologische Bedeutung der O-Acetylierung der Sialinsäure wurde in Infektionsassays mit einem nicht acetylierten E. coli K1 Stamm und einer isogenen konstitutiv acetylierten Mutante untersucht. In der Adhärenz an und Invasion in HBMEC konnten keine signifikanten Unterschiede festgestellt werden. Eine stärker ausgeprägte intrazelluläre Replikation wurde jedoch nach einer Verzögerung von mehreren Stunden bei dem nicht acetylierten Isolat beobachtet. Um die Neisseria containing Vacuole (NCV) näher zu charakterisieren und mögliche Interaktionen mit dem Endozytoseweg in HBMEC zu untersuchen, wurde eine dreifache Immunfluoreszenzfärbung zur simultanen Darstellung intrazellulärer Meningokken und spezifischer Marker des frühen bzw. späten Endosoms und Lysosoms etabliert. Eine Akquirierung des Transferrinrezeptors als Marker für das frühe Endosom und des Lamp-1 (lysosomal associated membrane protein 1) als Marker für das späte Endosom konnte durch Kolokalisationsstudien mittels Immunfluoreszenzmikroskopie gezeigt werden
In order to cause meningitis the extracellular pathogen Neisseria meningitidis has to traverse the blood-brain-barrier (BBB). It remains unclear, if the passage occurs through a transcellular or paracellular pathway. Postulating a transcellular passage, meningococci (MC) have been shown to adhere to and enter into BBB forming human brain microvascular endothelial cells (HBMECs). Furthermore, electron microscopy studies demonstrate that intracellular MC reside within membrane-bound compartments both solitary and in groups. Whether this is a result of simultaneous uptake or vacuole fusion or possible intracellular replication needs to be assessed. In order to investigate the ability of MC to survive and replicate intracellularly, prolonged gentamicin protection assays were performed using human epithelial (HEp-2) and endothelial (HBMEC) cells. Cells were infected with encapsulated and unencapsulated N. meningitidis serogroup B mutants in order to identify the potential role of the polysaccharide capsule for the intracellular survival. Encapsulated bacteria were found to be able to survive and, after an initial delay, to replicate within both endothelial and epithelial cells, whereas the number of intracellular capsule-deficient mutants decreased continuously. This strongly suggests that the capsule plays a pivotal role for the intracellular survival of MC both in epithelial and endothelial cells. Further investigations were initiated to characterise the membran-bound compartment, the Neisseria containing vacuole (NCV). Immunfluorescence microscopy studies showed that NCV acquire the early endosomal marker protein transferrin receptor and the lysosomal marker protein Lamp-1 respectively. The acquisition of further marker proteins as well as the kinetics of the association of these with NCV remain to be studied

碩士
長庚大學
生物醫學研究所
104
Endothelial cells (ECs) exist in all the organ systems and regulate the flow of diverse nutrient substances and biologically active molecules. The brain is lined by continuous ECs connected by tight junctions that help to maintain the blood-brain barrier (BBB). The abnormal function(s) of bEnd.3Cs, causing by inflammation, oxidative stress or other kinds of diseases, will result in malfunction of BBB. HO-1 catalyzes the cleavage of heme molecule to produce carbon monoxide (CO), bilirubin and iron. The HO-1-catalyzed heme degradation also plays a critical role in antioxidant, and cytoprotective function during inflammatory disorders. HO-1 has been proposed to play an obligatory role in endogenous defense against oxidative stress. The direct activated HO-1 during condition of cerebral oxidative stress is critical for neuroprotection. The 15-Deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of PPARs, can modulate several cellular responses including growth arrest, apoptosis, differentiation, or suppression of macrophage activation and inflammation through an interaction with the PPARγ. It is important to note that pretreatment with PPARγ antagonist failed to suppress 15d-PGJ2-induced HO-1 expression. Based on these findings, the results suggested that HO-1 induction by 15d-PGJ2 is independent on PPARγ. In neuronal protection, 15d-PGJ2 can decrease oxidative/nitrosative mediators, and suppress inflammatory signaling, mediate neuronal autophagy after cerebral ischemia-reperfusion injury, to protect brain from ischemia-reperfusion injury. In this study, mouse brain microvascular endothelial cells (bEnd.3Cs) were used as a model to investigate the mechanisms underlying 15d-PGJ2-induced HO-1 expression. The preliminary results indicated that 15d-PGJ2 can induce the HO-1 protein expression through de-novo protein synthesis in a time- and concentration-dependent manner. Furthermore, the 15d-PGJ2-induced HO-1 expression is mediated by NADPH oxidase, which was attenuated by pretreatment with the inhibitor of NADPH oxidase (DPI, MitoTempo, and Rotenone) in bEnd.3Cs. 15d-PGJ2-induced HO-1 protein expression is also involved in receptor tyrosine kinase JNK1/2, PI3K/Akt and PKC, which was attenuated by pretreatment with the inhibitor of PI3K (LY294002), PYK2(PF431396), Src (PP1), Akt (AktVIII), PKC (Ro318220 and Rottlerin), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (Mithramycin A). Moreover, the downstream stream transcription factors and cofactors also involved in the HO-1 induction such as Sp1, FoxO1 and c-Jun. Based on these findings, we demonstrated that 15d-PGJ2 activates the cooperation of PI3K/NOX/ROS/ c-Src/PYK2/Akt/FoxO1 and Sp1、PI3K/NOX/ROS/PKCδ/Sp1、PI3K/NOX/ ROS/PKC/c-Jun pathway through nuclear translocation of FoxO1 and Sp1 and leading to HO-1 expression. The induction of HO-1 by 15d-PGJ2 exerts anti-inflammatory effects on brain neuroinflammation. It will facilitate the development of therapeutic strategies for the treatment of brain neuroinflammation

碩士
國立中正大學
化學工程所
95
The study aims to the electrophoretic mobility and the softness of solid lipid nanoparticles (SLN) composed of cocoa butter and tripalmitin as lipid phase, L-arginine loaded SLN (R-SLN), and human brain microvascular endothelial cells (HBMECs) were investigated under the influences of electromagnetic field with various power. The size of SLN and R-SLN were measured by zetasizer and confirmed by the images of FE-SEM and TEM. The softness, fixed charge density, and Donnan potential were estimated from the electrophoretic mobility of capillary electrophoresis and the thickness of polyelectrolyte layer of small angle X-ray scattering (SAXS) with the soft particle electrokinetic theory. The results revealed that, the size and the electrophoretic mobility of SLN increased with an increase in the content of CB, but the softness of SLN decreased. The electrophoretic mobility of R-SLN reduced with an increase in L-arginine adsorbed onto SLN surface, but the softness of R-SLN raised. For EMF effects, the electrophoretic mobility of SLN, R-SLN, and HBMECs decreased, but the softness increased.

Les cellules endothéliales (EC) constituent une première barrière physique à la dissémination de virus pléiotropiques circulant par voie hématogène mais leur contribution à la défense innée anti-virale est peu connue. Des dysfonctions des EC de la barrière hémato-encéphalique (BMEC) et des sinusoïdes hépatiques (LSEC) ont été rapportées dans des neuropathologies et des hépatites aiguës ou chroniques d’origine virale, suggérant que des atteintes à leur intégrité contribuent à la pathogenèse. Les sérotypes de coronavirus de l’hépatite murine (MHV), se différenciant par leur capacité à induire des hépatites et des maladies neurologiques de sévérité variable et/ou leur tropisme pour les EC, représentent des modèles viraux privilégiés pour déterminer les conséquences de l’infection des EC sur la pathogenèse virale. Lors d’infection par voie hématogène, le sérotype MHV3, le plus virulent des MHV, induit une hépatite fulminante, caractérisée par une réponse inflammatoire sévère, et des lésions neurologiques secondaires alors que le sérotype moins virulent, MHV-A59, induit une hépatite modérée sans atteintes secondaires du système nerveux central (SNC). Par ailleurs, le sérotype MHV3, à la différence du MHV-A59, démontre une capacité à stimuler la production de cytokines par la voie TLR2. Les variants atténués du MHV3, les virus 51.6-MHV3 et YAC-MHV3, sont caractérisés par un faible tropisme pour les LSEC et induisent respectivement une hépatite modérée et subclinique. Compte tenu de l’importance des LSEC dans le maintien de la tolérance hépatique et de l’élimination des pathogènes circulants, il a été postulé que la sévérité de l’hépatite et de la réponse inflammatoire lors d’infections par les MHV est associée à la réplication virale et à l’altération des propriétés tolérogéniques et vasculaires des LSEC. Les désordres inflammatoires hépatiques pourraient résulter d’une activation différentielle du TLR2, plutôt que des autres TLR et des hélicases, selon les sérotypes. D’autre part, compte tenu du rôle des BMEC dans la prévention des infections du SNC, il a été postulé que l’invasion cérébrale secondaire par les coronavirus est reliée à l’infection des BMEC et le bris subséquent de la barrière hémato-encéphalique (BHE). À l’aide d’infections in vivo et in vitro par les différents sérotypes MHV, chez des souris ou des cultures de BMEC et de LSEC, nous avons démontré, d’une part, que l’infection in vitro des LSEC par le sétotype MHV3, à la différence des variants 51.6- et YAC-MHV3, altérait la production du facteur vasodilatant NO et renversait leur phénotype tolérogénique en favorisant la production de cytokines et de chimiokines inflammatoires. Ces dysfonctions se traduisaient in vivo par une réponse inflammatoire incontrôlée et une dérégulation du recrutement intrahépatique de leucocytes, favorisant la réplication virale et les dommages hépatiques. Nous avons aussi démontré, à l’aide de souris TLR2 KO et de LSEC dont l’expression du TLR2 a été abrogée par des siRNA, que la sévérité de l’hépatite et de la réponse inflammatoire induite par le sérotype MHV3, dépendait en partie de l’induction et de l’activation préférentielle du TLR2 par le virus dans le foie. D’autre part, la sévérité de la réplication virale au foie et des désordres dans le recrutement leucocytaire intrahépatique induits par le MHV3, et non par le MHV-A59 et le 51.6-MHV3, corrélaient avec une invasion virale subséquente du SNC, au niveau de la BHE. Nous avons démontré que l’invasion cérébrale du MHV3 était associée à une infection productive des BMEC et l’altération subséquente des protéines de jonctions serrées occludine, VE-cadhérine et ZO-1 se traduisant par une augmentation de la perméabilité de la BHE et l’entrée consécutive du virus dans le cerveau. Dans l’ensemble, les résultats de cette étude mettent en lumière l’importance du maintien de l’intégrité structurale et fonctionnelle des LSEC et des BMEC lors d’infections virales aigües par des MHV afin de limiter les dommages hépatiques associés à l’induction d’une réponse inflammatoire exagérée et de prévenir le passage des virus au cerveau suite à une dissémination par voie hématogène. Ils révèlent en outre un nouveau rôle aggravant pour le TLR2 dans l’évolution de l’hépatite virale aigüe ouvrant la voie à de nouvelles avenues thérapeutiques visant à moduler l’activité inflammatoire du TLR2.
Endothelial cells (EC) act as a physical barrier against invasion by pleiotropic blood borne viruses but their contribution in innate antiviral defense is poorly known. Dysfunctions in blood-brain barrier EC (BMECs) and liver sinusoidal EC (LSECs) have been reported in viral neuropathologies and hepatitis, suggesting that loss of ECs integrity may contribute to the pathogenesis. Mouse hepatitis coronaviruses (MHV), differing in their ability to induce severe to subclinical hepatitis and neurological diseases and / or their tropism for ECs, are relevant viral models to study the consequences of EC infection in viral pathogenesis. Following hematogenous infection, the MHV3 serotype, the most virulent MHV, induces fulminant hepatitis, characterized by severe inflammatory response, followed by neurological damage whereas the less virulent MHV-A59 serotype induces milder hepatitis but does not invade the central nervous system (CNS). In addition, MHV3, in contrast to MHV-A59, shows ability to induce TLR2-dependent cytokine response. The attenuated MHV3 variants, 51.6-MHV3 and YAC-MHV3, are characterized by a weak tropism for LSECs and induce moderated and subclinical hepatitis respectively. Given the importance of LSECs in hepatic tolerance and the elimination of circulating pathogens, it has been postulated that the severity of hepatitis and inflammatory response induced by MHVs correlates with infection and alterations in vascular and tolerogenic properties of LSECs. Hepatic inflammatory disorders may result from differential activation of TLR2, rather than other TLRs and helicases, according to serotypes. Moreover, given the role of BMECs in preventing CNS infections, it has been postulated that secondary cerebral invasion by coronaviruses is related to infection of BMECs and subsequent breakdown of the blood-brain barrier (BBB). Through in vitro and in vivo infections of isolated BMECs, LSECs or mice with the different MHVs, we demonstrated, first, that in vitro productive infection of LSECs by the highly virulent MHV3 serotype, in contrast to 51.6- et YAC-MHV3 variants, altered their production of vasoactive factors and overthrew their intrinsic tolerogenic properties by promoting inflammatory cytokines and chemokines production. These disturbances were reflected in vivo by an uncontrolled inflammatory response and a deregulation of intrahepatic leukocyte recruitment, favoring viral replication and liver damages. We demonstrated, using TLR2 KO mice and LSECs treated with siRNA for TLR2 that the abnormal inflammatory response induced by MHV3 depended in part on preferential induction and activation of TLR2 by the virus on the surface of hepatic cells. Moreover, the severity of the primary viral replication in the liver and disorders in intrahepatic leucocyte recruitment induced by MHV3, but not by MHV-A59 and 51.6-MHV3, correlated with a subsequent brain invasion at the BBB level. Such invasion was related to productive infection of BMECs and subsequent IFN--dependent disruption of tight junction proteins occludin, VE-cadherin and ZO-1, resulting in an increase of BBB permeability and further viral entry into the CNS. Overall, the results of this study highlight the importance of structural and functional integrity of LSECs and BMECs during acute viral infections by MHVs to limit liver damages associated with viral-induced exacerbation of inflammatory response and prevent brain invasion by MHVs following viral spread through the bloodstream. They also reveal a new worsening role for TLR2 in the evolution of acute viral hepatitis paving the way for new therapies targeting TLR2-induced inflammatory activity.

La barrière hémato-encéphalique (BHE) est une structure retrouvée au niveau des capillaires cérébraux. Elle représente un véritable obstacle pour les actifs qui doivent se rendre au cerveau pour y exercer un effet pharmacologique. Durant les étapes du développement du médicament, des modèles cellulaires in vitro sont utilisés pour l’évaluation de la perméabilité au cerveau des nouveaux médicaments. Le modèle assemblé avec des cellules endothéliales (CEs) isolées des capillaires des cerveaux de souris présente un intérêt particulier pour la recherche en raison de sa facilité d’obtention et sa pertinence pour le criblage des médicaments. Le but de ce projet a été de construire et de caractériser un modèle monocouche de CEs primaires de souris. En parallèle, un modèle monocouche de la lignée murine b.End3 a été investigué. L’évaluation de ces modèles a été basée sur les valeurs de TEER et de perméabilité aux marqueurs fluorescents, ainsi que sur la présence des protéines spécifiques de la BHE. La validation du modèle a été établie par la corrélation des résultats de perméabilité obtenus avec le modèle développé (in vitro) avec ceux obtenus chez la souris (in vivo). L’intégrité et l’expression des protéines spécifiques de la BHE du modèle primaire se sont montrées supérieures au modèle bEnd.3. La corrélation in vitro/in vivo du modèle primaire a abouti à un r2 = 0,765 comparé au r2 = 0,019 pour le modèle bEnd.3. Ce travail de recherche montre que le modèle primaire monocouche issu de cellules endothéliales cérébrales de souris est un modèle simple et fiable pour la prédiction de la perméabilité des actifs à travers la BHE.
The blood-brain barrier (BBB), a central nervous system structure, is found in the cerebral capillaries. It represents a major obstacle for the drugs that have to reach the brain in order to exercise their pharmacological effect. In the early stages of the drug development, in vitro cell models are used to evaluate the brain permeability of new drugs. Models assembled using primary endothelial cells (ECs) isolated from mouse brain capillaries are of particular interest for research, as for their ease of obtaining and relevance for the drug screening. Thus, the goal of this project was to build and characterize a primary mouse monolayer model. At the same time, a murine b.End3 cell line monolayer model was investigated. The evaluation of these models was based on the TEER and fluorescent marker permeability values, as well as on the presence of the BBB hallmark proteins. The model validation was established by the correlation of the permeability data obtained with the in vitro model and the data obtained in mice (in vivo). As a result, the primary mouse model showed superior monolayer integrity and higher expression of the tight junction and membrane transporter proteins when compared with the bEnd.3 cell line model. The in vitro/in vivo correlation of the primary model resulted in r2 = 0.765 compared to the bEnd.3 model with r2 = 0.019. This research work shows that the primary monolayer mouse model is a simple and reliable model for predicting the drug permeability across the BBB.