Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB

The Brain Microvascular Endothelium Supports T Cell Proliferation and Has Potential for Alloantigen Presentation

Endothelial cells (EC) form the inner lining of blood vessels and are positioned between circulating lymphocytes and tissues. Hypotheses have formed that EC may act as antigen presenting cells based on the intimate interactions with T cells, which are seen in diseases like multiple sclerosis, cerebral malaria (CM) and viral neuropathologies. Here, we investigated how human brain microvascular EC (HBEC) interact with and support the proliferation of T cells. We found HBEC to express MHC II, CD40 and ICOSL, key molecules for antigen presentation and co-stimulation and to take up fluorescently labeled antigens via macropinocytosis. In co-cultures, we showed that HBEC support and promote the proliferation of CD4+ and CD8+ T cells, which both are key in CM pathogenesis, particularly following T cell receptor activation and co-stimulation. Our findings provide novel evidence that HBEC can trigger T cell activation, thereby providing a novel mechanism for neuroimmunological complications of infectious diseases. © 2013 Wheway et al

Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells

BACKGROUND: Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. METHODOLOGY: Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. PRINCIPAL FINDINGS: Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. CONCLUSION: These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases

The relationship between smoking and quality of life in advanced lung cancer patients: a prospective longitudinal study.

PURPOSE: Smoking is a major cause of lung cancer, and continued smoking may compromise treatment efficacy and quality of life (health-related quality of life (HRQoL)) in patients with advanced lung cancer. Our aims were to determine (i) preference for treatments which promote quality over length of life depending on smoking status, (ii) the relationship between HRQoL and smoking status at diagnosis (T1), after controlling for demographic and clinical variables, and (iii) changes in HRQoL 6 months after diagnosis (T2) depending on smoking status. METHODS: Two hundred ninety-six patients with advanced lung cancer were given questionnaires to assess HRQoL (EORTC QLQ-C30), time-trade-off for life quality versus quantity (QQQ) and smoking history (current, former or never smoker) at diagnosis (T1) and 6 months later (T2). Medical data were extracted from case records. RESULTS: Questionnaires were returned by 202 (68.2 %) patients at T1 and 114 (53.3 %) at T2. Patients favoured treatments that would enhance quality of life over increased longevity. Those who continued smoking after diagnosis reported worse HRQoL than former smokers or those who never smoked. Smoking status was a significant independent predictor of coughing in T1 (worse in smokers) and cognitive functioning in T2 (better in never smokers). CONCLUSIONS: Smoking by patients with advanced lung cancer is associated with worse symptoms on diagnosis and poorer HRQoL for those who continue smoking. The results have implications to help staff explain the consequences of smoking to patients

In vivo assembly of the axon initial segment in motor neurons

International audienceThe axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and beta 4-spectrin only appear along AnkG(+) segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither beta 4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG

The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review

Background: The presence of circulating cell-free DNA from tumours in blood (ctDNA) is of major importance to those interested in early cancer detection, as well as to those wishing to monitor tumour progression or diagnose the presence of activating mutations to guide treatment. In 2014, the UK Early Cancer Detection Consortium undertook a systematic mapping review of the literature to identify blood-based biomarkers with potential for the development of a non-invasive blood test for cancer screening, and which identified this as a major area of interest. This review builds on the mapping review to expand the ctDNA dataset to examine the best options for the detection of multiple cancer types. Methods: The original mapping review was based on comprehensive searches of the electronic databases Medline, Embase, CINAHL, the Cochrane library, and Biosis to obtain relevant literature on blood-based biomarkers for cancer detection in humans (PROSPERO no. CRD42014010827). The abstracts for each paper were reviewed to determine whether validation data were reported, and then examined in full. Publications concentrating on monitoring of disease burden or mutations were excluded. Results: The search identified 94 ctDNA studies meeting the criteria for review. All but 5 studies examined one cancer type, with breast, colorectal and lung cancers representing 60% of studies. The size and design of the studies varied widely. Controls were included in 77% of publications. The largest study included 640 patients, but the median study size was 65 cases and 35 controls, and the bulk of studies (71%) included less than 100 patients. Studies either estimated cfDNA levels non-specifically or tested for cancer-specific mutations or methylation changes (the majority using PCR-based methods). Conclusion: We have systematically reviewed ctDNA blood biomarkers for the early detection of cancer. Pre-analytical, analytical, and post-analytical considerations were identified which need to be addressed before such biomarkers enter clinical practice. The value of small studies with no comparison between methods, or even the inclusion of controls is highly questionable, and larger validation studies will be required before such methods can be considered for early cancer detection

Role of KCNMA1 gene in breast cancer invasion and metastasis to brain

International audienceBACKGROUND: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion. METHODS: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX). RESULTS: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. CONCLUSION: Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy

Prenatal exposures and exposomics of asthma

This review examines the causal investigation of preclinical development of childhood asthma using exposomic tools. We examine the current state of knowledge regarding early-life exposure to non-biogenic indoor air pollution and the developmental modulation of the immune system. We examine how metabolomics technologies could aid not only in the biomarker identification of a particular asthma phenotype, but also the mechanisms underlying the immunopathologic process. Within such a framework, we propose alternate components of exposomic investigation of asthma in which, the exposome represents a reiterative investigative process of targeted biomarker identification, validation through computational systems biology and physical sampling of environmental medi