[Show abstract][Hide abstract] ABSTRACT: Neonatal meningitis-causing Escherichia coli (NMEC) is the predominant Gram-negative bacterial pathogen associated with meningitis in newborn infants. High levels of heterogeneity and diversity have been observed in the repertoire of virulence traits and other characteristics among strains of NMEC making it difficult to define the NMEC pathotype. The objective of the present study was to identify genotypic and phenotypic characteristics of NMEC that can be used to distinguish them from commensal E. coli.
A total of 53 isolates of NMEC obtained from neonates with meningitis and 48 isolates of fecal E. coli obtained from healthy individuals (HFEC) were comparatively evaluated using five phenotypic (serotyping, serum bactericidal assay, biofilm assay, antimicorbial susceptibility testing, and in vitro cell invasion assay) and three genotypic (phylogrouping, virulence genotyping, and pulsed-field gel electrophoresis) methods.
A majority (67.92 %) of NMEC belonged to B2 phylogenetic group whereas 59 % of HFEC belonged to groups A and D. Serotyping revealed that the most common O and H types present in NMEC tested were O1 (15 %), O8 (11.3 %), O18 (13.2 %), and H7 (25.3 %). In contrast, none of the HFEC tested belonged to O1 or O18 serogroups. The most common serogroup identified in HFEC was O8 (6.25 %). The virulence genotyping reflected that more than 70 % of NMEC carried kpsII, K1, neuC, iucC, sitA, and vat genes with only less than 27 % of HFEC possessing these genes. All NMEC and 79 % of HFEC tested were able to invade human cerebral microvascular endothelial cells. No statistically significant difference was observed in the serum resistance phenotype between NMEC and HFEC. The NMEC strains demonstrated a greater ability to form biofilms in Luria Bertani broth medium than did HFEC (79.2 % vs 39.9 %).
The results of our study demonstrated that virulence genotyping and phylogrouping may assist in defining the potential NMEC pathotype.
[Show abstract][Hide abstract] ABSTRACT: Objective:
Blood brain barrier (BBB) breakdown and increased endothelial permeability is a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins.
Method and result:
To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells in-vitro, with a potent inflammatory mediator thrombin. By using confocal microscopy and transwell permeability assay, we show that pretreatment of brain endothelial cells with Ang-1 diminish thrombin mediated disruption of TJs and increase in endothelial permeability. We also found that Ang-1 inhibits thrombin induced tyrosine phosphorylation of Occludin and promote Occludin interaction with Zona Occludens-1 (ZO-1) to stabilize TJs. Interestingly, our study revealed that depletion of PTPN-2 by siRNAs abolishes Ang-1 ability to promote tyrosine dephosphorylation of Occludin, resulting Occludin disassociation from ZO-1 and endothelial hyperpermeability.
Collectively, our findings suggest that in brain endothelial cells blocking PTPN-2 mediated tyrosine phosphorylation of Occludin is a novel mechanism to maintain BBB function, and may offer a key therapeutic strategy for neuro-inflammatory disorders associated with BBB disruption.
PLoS ONE 06/2015; 10(6):e0130857. DOI:10.1371/journal.pone.0130857 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The choroid plexus (CP) is the source of cerebrospinal fluid (CSF) production and location of the blood-CSF barrier (BCSFB), which is constituted by the epithelial cells of the CP. Several infectious pathogens including viruses, bacteria, fungi and parasites cross the BCSFB to enter the central nervous system (CNS), ultimately leading to inflammatory infectious diseases like meningitis and meningoencephalitis. The CP responds to this challenge by the production of chemokines and cytokines as well as alterations of the barrier function of the BCSFB. During the course of CNS infectious disease host immune cells enter the CNS, eventually contributing to the cellular damage caused by the disease. Additional complications, which are in certain cases caused by choroid plexitis, can arise due to the response of the CP to the pathogens. In this review we will give an overview on the multiple functions of the CP during brain infections highlighting the CP as a multi-role player during infectious diseases of the CNS. In this context the importance of tools for investigation of these CP functions and a possible suitability of the CP as therapeutic target will be discussed.
[Show abstract][Hide abstract] ABSTRACT: Carlo Agostoni, MD, and Kwang Sik Kim, MD, are the Guest Editors for this annual review issue on Nutrition and the Microbiome. Dr Agostoni is a Professor of Pediatrics at the Department of Clinical Sciences and Community Health, University of Milan, within the Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan. Dr Kim is Professor of Pediatrics and Molecular Microbiology and Immunology, Johns Hopkins University School of Medicine and Bloomberg School of Public Health; and Director, Division of Pediatric Infectious Diseases, Johns Hopkins Children's Center, Baltimore, MD.
Pediatric Research 01/2015; 77(1-2):113-4. DOI:10.1038/pr.2014.195 · 2.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NFκB and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NFκB dependent since it was blocked by inhibitors of NFκB activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NFκB inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-α was also inhibited by ShRNA mediated knockdown of NFκB. TNF-α treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NFκB-mediated induction of HLA-F following JEV infection and TNF-α exposure is being suggested for the first time.
[Show abstract][Hide abstract] ABSTRACT: The survivability of the naval ship is the capability of a warship to avoid or withstand a hostile environment. The survivability of the naval ship assessed by three categories (susceptibility, vulnerability and recoverability). The magnitude of susceptibility of a warship encountering with threat is dependent upon the attributes of detection equipment and weapon system. In this paper, as a part of a naval ship’s survivability analysis, an assessment process model for the ship’s susceptibility analysis technique is developed. Naval ship’s survivability emphasizing the susceptibility is assessed by the probability of detection, and the probability of hit. Considering the radar cross section (RCS), the assessment procedure for the susceptibility is described. It’s emphasizing the simplified calculation model based on the probability density function for probability of hit. Assuming the probability of hit given a both single-hit and multiple-hit, the susceptibility is accessed for a RCS and the hit probability for a rectangular target is applied for a given threat.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Central nervous system infection continues to be an important cause of mortality and morbidity worldwide. Our incomplete knowledge on the pathogenesis of how meningitis-causing pathogens cause CNS infection and emergence of antimicrobial resistance has contributed to the mortality and morbidity. An early empiric antibiotic treatment is critical for the management of patients with bacterial meningitis, but early recognition of bacterial meningitis continues to be a challenge.
This review gives an overview on current therapeutic strategies for CNS infection with a focus on recent literature since 2010 on bacterial meningitis. Bacterial meningitis is a medical emergency, requiring early recognition and treatment. The selection of appropriate empiric antimicrobial regimen, after incorporating the epidemiology of bacterial meningitis, impact of vaccination, emergence of antimicrobial-resistant bacteria, role of adjunctive therapy and the current knowledge on the pathogenesis of meningitis and associated neuronal injury are covered.
Prompt treatment of bacterial meningitis with an appropriate antibiotic is essential. Optimal antimicrobial treatment of bacterial meningitis requires bactericidal agents able to penetrate the blood-brain barrier, with efficacy in cerebrospinal fluid. Emergence of CNS-infecting pathogens with resistance to conventional antibiotics has been increasingly recognized, but development of new antibiotics has been limited. More complete understanding of the microbial and host factors that are involved in the pathogenesis of bacterial meningitis and associated neurologic sequelae is likely to help in developing new strategies for the prevention and therapy of bacterial meningitis.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II.
In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.
Journal of Virology 06/2014; 88(17). DOI:10.1128/JVI.01304-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen.
International Journal of Molecular Sciences 05/2014; 15(5):8639-55. DOI:10.3390/ijms15058639 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Though many essential oils from citrus peels are claimed to have several medicinal functions, the chemical composition and biological activities of the essential oils of Citrus flowers have not been well described. Therefore, this study intended to investigate the chemical composition and anti-inflammatory potential of essential oils from C. unshiu flower (CEO) to support its purported beneficial health effects. The chemical constituents of the CEO, analyzed by gas chromatography-mass spectrometry (GC-MS), included y-terpinene (24.7%), 2-beta-pinene (16.6%), 1-methyl-2-isopropylbenzene (11.5%), L-limonene (5.7%), beta3-ocimene (5.6%), and alpha-pinene (4.7%). The effects of the CEO on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were also examined. The results indicate that the CEO is an effective inhibitor of LPS-induced NO and PGE2 production in RAW 264.7 cells. Additionally, CEO was shown to suppress the production of inflammatory cytokines including interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6. Based on these results, CEO may be considered a potential anti-inflammatory candidate with human health benefits.
[Show abstract][Hide abstract] ABSTRACT: Copper (Cu) is a cofactor of various metalloenzymes and has a role in neurodegenerative diseases with disturbed Cu homeostasis, e.g., in Alzheimer's disease (AD) and Menkes disease. To address Cu imbalances, we synthesized two different dendritic nanoparticles (NP) for the transport of Cu ions across the blood-brain barrier (BBB). The synthesized NPs show low toxicity and high water solubility and can stabilize high amounts of Cu. The Cu-laden NPs crossed cellular membranes and increased the cellular Cu level. A human brain microvascular endothelial cell (HBMEC) model was established to investigate the permeability of the NPs through the BBB. By comparing the permeability×surface area product (PSe) of reference substances with those of NPs, we observed that NPs crossed the BBB model two times more effectively than 14C-sucrose and sodium fluorescein (NaFl) and up to 60 times better than Evans Blue labeled albumin (EBA). Our results clearly indicate that NPs cross the BBB model effectively. Furthermore, Cu was shielded by the NPs, which decreased the Cu toxicity. The novel design of the core-shell NP enabled the complexation of Cu in the outer shell and therefore facilitated the pH-dependent release of Cu in contrast to core-multishell NPs, where the Cu ions are encapsulated in the core. This allows a release of Cu into the cytoplasm. In addition, by using a cellular detection system based on a metal response element with green fluorescent protein (MRE-GFP), we demonstrated that Cu could also be released intracellularly from NPs and is accessible for biological processes. Our results indicate that NPs are potential candidates to rebalance metal-ion homeostasis in disease conditions affecting brain and neuronal systems.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to analyze the chemical composition of essential oil in 'Shiranuhi' immature fruit and to test their biological activities. 'Shiranuhi' immature essential oils (SIEO) were obtained by steam distillation from fruits collected from Jeju Island and were analyzed using gas chromatograph (GC)-flame ionization detectors (FID) and GC-MS. Fourteen components were identified in the essential oil. Limonene (75.21%) and terpineol (8.68%) were the major components in SIEO. Since acne vulgaris is the combined result of a bacterial infection and the inflammatory response to that infection, we examined whether SIEO possessed antibacterial against skin pathogens. As a result, SIEO showed excellent antibacterial activities against drug-susceptible and -resistant Propionibacterium acnes and Staphylococcus epidermidis, which are acne-causing bacteria. In this study, SIEO was examined on DPPH radical scavenging activities, which showed moderate antioxidant activity (, ). In order to determined whether SIEO can be safely applied to human skin, the cytotoxicity effects of SIEO were determined by colorimetric MTT assays in normal human fibroblasts and keratinocyte HaCaT cells. They exhibited low cytotoxicity at in both celllines. Based on these results, we suggest the possibility that essential oil of 'Shiranuhi' maybe considered as an antibacterial and antioxidant agent.
[Show abstract][Hide abstract] ABSTRACT: E. coli is the most common Gram-negative organism causing neonatal meningitis and neonatal meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis and pathophysiology contributes to such mortality and morbidity. Recent reports of neonatal meningitis caused by E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. E. coli invasion of the blood–brain barrier is a prerequisite for penetration into the brain, and requires specific microbial–host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial–host factors and host cell signaling molecules is shown to be efficient in preventing E. coli penetration into the brain, the essential step in the development of E. coli meningitis. Continued investigation of the microbial and host factors contributing to E. coli invasion of the blood–brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis, thereby limiting the exposure to emerging antimicrobial-resistant E. coli.
[Show abstract][Hide abstract] ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.