Добірка наукової літератури з теми "Brain microvascular endothelium"

Abstract Background. The blood-brain barrier represents the selective diffusion barrier at the level of the cerebral microvascular endothelium. Other functions of blood-brain barrier include transport, signaling and osmoregulation. Endothelial cells interact with surrounding astrocytes, pericytes and neurons. These interactions are crucial to the development, structural integrity and function of the cerebral microvascular endothelium. Dysfunctional blood-brain barrier has been associated with pathologies such as acute stroke, tumors, inflammatory and neurodegenerative diseases. Conclusions. Blood-brain barrier permeability can be evaluated in vivo by perfusion computed tomography - an efficient diagnostic method that involves the sequential acquisition of tomographic images during the intravenous administration of iodinated contrast material. The major clinical applications of perfusion computed tomography are in acute stroke and in brain tumor imaging.

The regulatory interplay between laminar shear stress and proinflammatory cytokines during homeostatic maintenance of the brain microvascular endothelium is largely undefined. We hypothesized that laminar shear could counteract the injurious actions of proinflammatory cytokines on human brain microvascular endothelial cell (HBMvEC) barrier properties, in-part through suppression of cellular redox signaling. For these investigations, HBMvECs were exposed to either shear stress (8 dynes/cm2, 24 hours) or cytokines (tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6), 0 to 100 ng/mL, 6 or 18 hours). Human brain microvascular endothelial cell ‘preshearing’ ± cytokine exposure was also performed. Either cytokine dose–dependently decreased expression and increased phosphorylation (pTyr/pThr) of interendothelial occludin, claudin-5, and vascular endothelial-cadherin; observations directly correlating to endothelial barrier reduction, and in precise contrast to effects seen with shear. We further observed that, relative to unsheared cells, HBMvECs presheared for 24 hours exhibited significantly reduced reactive oxygen species production and barrier permeabilization in response to either TNF-α or IL-6 treatment. Shear also downregulated NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activation in HBMvECs, as manifested in the reduced expression and coassociation of gp91phox and p47phox. These findings lead us to conclude that physiologic shear can protect the brain microvascular endothelium from injurious cytokine effects on interendothelial junctions and barrier function by regulating the cellular redox state in-part through NADPH oxidase inhibition.

ABSTRACTThe Gram-positive bacteriumStreptococcus pneumoniaeis the main causative agent of bacterial meningitis.S. pneumoniaeis thought to invade the central nervous system via the bloodstream by crossing the vascular endothelium of the blood-brain barrier. The exact mechanism by which pneumococci cross endothelial cell barriers before meningitis develops is unknown. Here, we investigated the role of PECAM-1/CD31, one of the major endothelial cell adhesion molecules, inS. pneumoniaeadhesion to vascular endothelium of the blood-brain barrier. Mice were intravenously infected with pneumococci and sacrificed at various time points to represent stages preceding meningitis. Immunofluorescent analysis of brain tissue of infected mice showed that pneumococci colocalized with PECAM-1. In human brain microvascular endothelial cells (HBMEC) incubated withS. pneumoniae, we observed a clear colocalization between PECAM-1 and pneumococci. Blocking of PECAM-1 reduced the adhesion ofS. pneumoniaeto endothelial cellsin vitro, implying that PECAM-1 is involved in pneumococcal adhesion to the cells. Furthermore, using endothelial cell protein lysates, we demonstrated thatS. pneumoniaephysically binds to PECAM-1. Moreover, bothin vitroandin vivoPECAM-1 colocalizes with theS. pneumoniaeadhesion receptor pIgR. Lastly, immunoprecipitation experiments revealed that PECAM-1 can physically interact with pIgR. In summary, we show for the first time that blood-borneS. pneumoniaecolocalizes with PECAM-1 expressed by brain microvascular endothelium and that, in addition, they colocalize with pIgR. We hypothesize that this interaction plays a role in pneumococcal binding to the blood-brain barrier vasculature prior to invasion into the brain.

SummaryEndothelial cells produce platelet-activating factor (PAF), which is the key process in the interactions between the vascular wall and blood cells. To examine the production of PAF in brain microvasculature we have cultured brain endothelial cells and performed a comparative study with aortic endothelial cells. Fresh porcine brain was homogenized, and microvascular endothelial cells were separated by enzyme digestion. The cells were cultured in medium containing epidermal growth factor and bovine brain extract. Endothelial cells from the aorta of the same animal were cultured in a similar manner. Production of PAF was assessed by ǀ3Hǀacetate incorporation into phospholipids or by radioimmunoassay. Prostacyclin was measured by radioimmunoassay of 6-ketoprostaglandin F1α. The cells produced 1760 ± 403 and 2892 ± 347 dpm/106 cells (n = 4) of PAF when stimulated with brady- kinin and calcium ionophore A23187, each at 1 μM, respectively. Aortic endothelial cells produced 3911 ± 2006 and 8052 ± 2270 dpm/106 cells (n = 4), respectively, and these values were significantly higher than those in brain endothelial cells (p<0.01, U-test). Prostacyclin production was also higher in aortic cells as compared to brain microvascular endothelial cells. In aortic endothelial cells both Ca ionophore A23187 and bradykinin significantly stimulated PMN adherence whereas in brain microvascular cells only Ca ionophore enhanced the adherence. Brain microvascular endothelial cells produce smaller amount of PAF and prostacyclin as compared to aortic endothelial cells, and this fact may imply that the functional integrity of the brain microvascular endothelium is maintained at a low level.

The endothelium plays an important role in the modulation of vascular tone and blood cell activation. Extensive work has demonstrated that the release of endothelium-derived relaxing factor (EDRF) from the endothelium is evoked by a number of physical and chemical stimuli requiring Ca2+. Because endothelial cells do not express voltage-dependent Ca2+ channels, Ca2+ influxes following receptor activation may be facilitated by cell hyperpolarizations mediated by the activation of K+ conductances. There has been recent interest in the role of ATP-sensitive K+ channels (KATP) suggesting that KATP may play a role in the regulation of blood flow. We have investigated the electrophysiological properties of an ATP-sensitive K+ conductance in whole cell and membrane patches from rat aorta and brain microvascular endothelial cells. Whole cell as well as single-channel currents were increased by either intracellular dialysis of ATP or application of glucose-free/NaCN (2 mM) solutions. Both currents were reversibly blocked by glibenclamide (1-100 microM). The KATP channel opener pinacidil (30 microM) caused activation of an outward current in the presence of physiological intracellular ATP concentrations. In inside-out patches, 10 microM-1 mM ATP invariably caused a dramatic decrease in channel activity. We conclude that both rat aorta and brain microvascular endothelial cells express KATP channels. KATP may play a role in the regulation of endothelial cell resting potential during impaired energy supply and therefore modulate EDRF release and thus cerebral blood flow.

Microvascular endothelial cells at the blood–brain barrier exhibit a protective phenotype, which is highly induced by biochemical and biomechanical stimuli. Amongst them, shear stress enhances junctional tightness and limits transport at capillary-like levels. Abnormal flow patterns can reduce functional features of macrovascular endothelium. We now examine if this is true in brain microvascular endothelial cells. We suggest in this paper a complex response of endothelial cells to aberrant forces under different flow domains. Human brain microvascular endothelial cells were exposed to physiological or abnormal flow patterns. Physiologic shear (10–20 dyn/cm2) upregulates expression of tight junction markers Zona Occludens 1 (1.7-fold) and Claudin-5 (more than 2-fold). High shear stress (40 dyn/cm2) and/or pulsatility decreased their expression to basal levels and altered junctional morphology. We exposed cells to pathological shear stress patterns followed by capillary-like conditions. Results showed reversible recovery on the expression of tight junction markers. Flow protection of barrier phenotype commensurate with junctional signaling pathways decrease (Src, 0.25-fold, ERK, 0.77-fold) when compared to static conditions. This decrease was lost under high shear and pulsatile flow. In conclusion, abnormal shear stress inherent to systemic vascular disease leads to barrier impairment, which could be reverted by hemodynamic interventions.

The luminal surface of vascular endothelium contains glycocalyx residues that establish an overall negative charge. Recent evidence has suggested that local endothelial surface charge properties may account for the permeability properties of various macromolecules. It has also been suggested that altered membrane charge on the luminal side may play a role in thrombogenesis and atherogenesis. The relationship of macromolecule charge to endothelial cell permeability was examined in vitro using mouse brain microvessel endothelial cells grown to confluence on a nitrocellulose filter separating a double-chamber system. Endothelial permeability to 4K and 10K fluorescein-labeled neutral dextrans was compared with the permeability to 4K and 10K fluorescein-labeled anionic dextrans (sulfated). After 1 h, there was significantly greater permeability of neutral fluorescein-labeled dextran than of anionic fluorescein-labeled dextran in each particle size. In addition, there was significantly greater permeability of 4K than 10K fluorescein-labeled dextrans of either charge. The findings indicate that charge in addition to size plays an important role in the movement of macromolecules across cultured microvascular endothelial cells.

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1–stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1–mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO–ET-1 interactions may contribute to regulation of microvascular function.

Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.

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.

The leading cause of death in human patients with malignant cancer is the dissemination of the primary tumor to secondary sites throughout the body. It is well known that cancers metastasize to certain tissues (e.g. breast cancer typically spreads to the lungs. brain and bone), in a pattern that cannot be explained by blood flow from the primary tumor or simple mechanical arrest. Circulating tumor cells usually arrest in the microvasculature of target tissues. At these sites, they must adhere to the endothelium, survive, proliferate and extravasate in order to form a secondary tumor. In vitro tools that appropriately mimic the microvasculature in which cancer metastasis occurs have been largely unavailable. With the advent of microfluidic and nanotechnology, we can now more accurately model the complexity of the microvascular environment, in terms of representative endothelial cells, geometry, shear stress and exposure to organ-specific environmental cues. This talk will focus on the use of microfluidic devices to explore mechanisms involved in tumor-endothelial cell interactions that govern cancer metastasis to organ specific sites.