In Vitro Study of the Effects of Angiostrongylus cantonensis Larvae Extracts on Apoptosis and Dysfunction in the Blood-Brain Barrier (BBB)

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease

Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage

A hematopoietic contribution to microhemorrhage formation during antiviral CD8 T cell-initiated blood-brain barrier disruption

<p>Abstract</p> <p>Background</p> <p>The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS.</p> <p>Methods</p> <p>PIFS was induced by intravenous injection of VP2_121-130peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer.</p> <p>Results</p> <p>C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2_121-130peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system.</p> <p>Conclusion</p> <p>C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.</p

Quantification of myelin loss in frontal lobe white matter in vascular dementia, Alzheimer's disease, and dementia with Lewy bodies

The aim of this study was to characterize myelin loss as one of the features of white matter abnormalities across three common dementing disorders. We evaluated post-mortem brain tissue from frontal and temporal lobes from 20 vascular dementia (VaD), 19 Alzheimer’s disease (AD) and 31 dementia with Lewy bodies (DLB) cases and 12 comparable age controls. Images of sections stained with conventional luxol fast blue were analysed to estimate myelin attenuation by optical density. Serial adjacent sections were then immunostained for degraded myelin basic protein (dMBP) and the mean percentage area containing dMBP (%dMBP) was determined as an indicator of myelin degeneration. We further assessed the relationship between dMBP and glutathione S-transferase (a marker of mature oligodendrocytes) immunoreactivities. Pathological diagnosis significantly affected the frontal but not temporal lobe myelin attenuation: myelin density was most reduced in VaD compared to AD and DLB, which still significantly exhibited lower myelin density compared to ageing controls. Consistent with this, the degree of myelin loss was correlated with greater %dMBP, with the highest %dMBP in VaD compared to the other groups. The %dMBP was inversely correlated with the mean size of oligodendrocytes in VaD, whereas it was positively correlated with their density in AD. A two-tier regression model analysis confirmed that the type of disorder (VaD or AD) determines the relationship between %dMBP and the size or density of oligodendrocytes across the cases. Our findings, attested by the use of three markers, suggest that myelin loss may evolve in parallel with shrunken oligodendrocytes in VaD but their increased density in AD, highlighting partially different mechanisms are associated with myelin degeneration, which could originate from hypoxic–ischaemic damage to oligodendrocytes in VaD whereas secondary to axonal degeneration in AD

Endothelium-Based Biomarkers Are Associated with Cerebral Malaria in Malawian Children: A Retrospective Case-Control Study

Differentiating cerebral malaria (CM) from other causes of serious illness in African children is problematic, owing to the non-specific nature of the clinical presentation and the high prevalence of incidental parasitaemia. CM is associated with endothelial activation. In this study we tested the hypothesis that endothelium-derived biomarkers are associated with the pathophysiology of severe malaria and may help identify children with CM.Plasma samples were tested from children recruited with uncomplicated malaria (UM; n = 32), cerebral malaria with retinopathy (CM-R; n = 38), clinically defined CM without retinopathy (CM-N; n = 29), or non-malaria febrile illness with decreased consciousness (CNS; n = 24). Admission levels of angiopoietin-2 (Ang-2), Ang-1, soluble Tie-2 (sTie-2), von Willebrand factor (VWF), its propeptide (VWFpp), vascular endothelial growth factor (VEGF), soluble ICAM-1 (sICAM-1) and interferon-inducible protein 10 (IP-10) were measured by ELISA. Children with CM-R had significantly higher median levels of Ang-2, Ang-2:Ang-1, sTie-2, VWFpp and sICAM-1 compared to children with CM-N. Children with CM-R had significantly lower median levels of Ang-1 and higher median concentrations of Ang-2:Ang-1, sTie-2, VWF, VWFpp, VEGF and sICAM-1 compared to UM, and significantly lower median levels of Ang-1 and higher median levels of Ang-2, Ang-2:Ang-1, VWF and VWFpp compared to children with fever and altered consciousness due to other causes. Ang-1 was the best discriminator between UM and CM-R and between CNS and CM-R (areas under the ROC curve of 0.96 and 0.93, respectively). A comparison of biomarker levels in CM-R between admission and recovery showed uniform increases in Ang-1 levels, suggesting this biomarker may have utility in monitoring clinical response.These results suggest that endothelial proteins are informative biomarkers of malarial disease severity. These results require validation in prospective studies to confirm that this group of biomarkers improves the diagnostic accuracy of CM from similar conditions causing fever and altered consciousness

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability

Serum Angiopoietin-1 and -2 Levels Discriminate Cerebral Malaria from Uncomplicated Malaria and Predict Clinical Outcome in African Children

BACKGROUND: Limited tools exist to identify which individuals infected with Plasmodium falciparum are at risk of developing serious complications such as cerebral malaria (CM). The objective of this study was to assess serum biomarkers that differentiate between CM and non-CM, with the long-term goal of developing a clinically informative prognostic test for severe malaria. METHODOLOGY/PRINCIPAL FINDINGS: Based on the hypothesis that endothelial activation and blood-brain-barrier dysfunction contribute to CM pathogenesis, we examined the endothelial regulators, angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), in serum samples from P. falciparum-infected patients with uncomplicated malaria (UM) or CM, from two diverse populations--Thai adults and Ugandan children. Angiopoietin levels were compared to tumour necrosis factor (TNF). In both populations, ANG-1 levels were significantly decreased and ANG-2 levels were significantly increased in CM versus UM and healthy controls (p<0.001). TNF was significantly elevated in CM in the Thai adult population (p<0.001), but did not discriminate well between CM and UM in African children. Receiver operating characteristic curve analysis showed that ANG-1 and the ratio of ANG-2:ANG-1 accurately discriminated CM patients from UM in both populations. Applied as a diagnostic test, ANG-1 had a sensitivity and specificity of 100% for distinguishing CM from UM in Thai adults and 70% and 75%, respectively, for Ugandan children. Across both populations the likelihood ratio of CM given a positive test (ANG-1<15 ng/mL) was 4.1 (2.7-6.5) and the likelihood ratio of CM given a negative test was 0.29 (0.20-0.42). Moreover, low ANG-1 levels at presentation predicted subsequent mortality in children with CM (p = 0.027). CONCLUSIONS/SIGNIFICANCE: ANG-1 and the ANG-2/1 ratio are promising clinically informative biomarkers for CM. Additional studies should address their utility as prognostic biomarkers and potential therapeutic targets in severe malaria