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One of the most challenging issues in HIV-associated neurocognitive disorders (HAND) caused by HIV-1 virotoxins and drug abuse is the lack of understanding the underlying mechanisms that are commonly associated with disorders of the blood-brain barrier (BBB), which mainly consists of brain microvascular endothelial cells (BMEC). Here, we hypothesized that Glycoprotein 120 (gp120), methamphetamine (METH) and nicotine (NT) can enhance amyloid-beta (Aβ) accumulation in BMEC through Alpha7 nicotinic acetylcholine receptor (α7 nAChR). Both in vitro (human BMEC) (HBMEC) and in vivo (mice) models of BBB were used to dissect the role of α7 nAChR in up-regulation of Aβ induced by gp120, METH and NT. Aβ release from and transport across HBMEC were significantly increased by these factors. Methyllycaconitine (MLA), an antagonist of α7 nAChR, could efficiently block these pathogenic effects. Furthermore, our animal data showed that these factors could significantly increase the levels of Aβ, Tau and Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in mouse cerebrospinal fluid (CSF) and Aβ in the mouse brains. These pathogenicities were significantly reduced by MLA, suggesting that α7 nAChR may play an important role in neuropathology caused by gp120, METH and NT, which are the major pathogenic factors contributing to the pathogenesis of HAND.

2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.

Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.

Aim: We investigate how the α7 nicotinic acetylcholine receptor (α7 nAChR), an essential regulator of inflammation, contributes to the α7 agonist nicotine-enhanced Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMECs) through lipid rafts/caveolae-mediated signaling., Materials & Methods: α7 nAChR-mediated signaling and bacterial invasion were defined by lipid raft fractionation, immunofluorescence microscopy and siRNA knockdown., Results: Nicotine-enhanced bacterial invasion was dose-dependently inhibited by two raft-disrupting agents, nystatin and filipin. Significant accumulation of the lipid raft marker GM3 was observed in HBMEC induced by E. coli K1 and nicotine. The recruitment of α7 nAChR and related signaling molecules, including vimentin, and Erk1/2, to caveolin-1 enriched lipid rafts was increased upon treatment with E44 or E44 plus nicotine. Erk1/2 activation (phosphorylation), which is required for α7 nAChR-mediated signaling and E44 invasion, was associated with lipid rafts and nicotine-enhanced bacterial infection. Furthermore, E44 invasion, E44/nicotine-induced activation of Erk1/2 and clustering of α7 nAChR and caveolin-1 was specifically blocked by both siRNAs., Conclusion: α7 nAChR-mediated signaling through lipid rafts/caveolae is required for nicotine-enhanced E. coli K1 invasion of HBMEC.

IbeA in meningitic Escherichia coli K1 strains has been described previously for its role in invasion of BMECs (brain microvascular endothelial cells). Vimentin was identified as an IbeA-binding protein on the surface of HBMECs (human BMECs). In the present study, we demonstrated that vimentin is a primary receptor required for IbeA+ E. coli K1-induced signalling and invasion of HBMECs, on the basis of the following observations. First, E44 (IbeA+ E. coli K1 strain) invasion was blocked by vimentin inhibitors (withaferin A and acrylamide), a recombinant protein containing the vimentin head domain and an antibody against the head domain respectively. Secondly, overexpression of GFP (green fluorescent protein)-vimentin and GFP-VDM (vimentin head domain deletion mutant) significantly increased and decreased bacterial invasion respectively. Thirdly, bacterial invasion was positively correlated with phosphorylation of vimentin at Ser82 by CaMKII (Ca2+/calmodulin-dependent protein kinase II) and IbeA+ E. coli-induced phosphorylation of ERK (extracellular-signal-regulated kinase). Blockage of CaMKII by KN93 and inhibition of ERK1/2 phosphorylation by PD098059 resulted in reduced IbeA+ E. coli invasion. Fourthly, IbeA+ E. coli and IbeA-coated beads induced the clustering of vimentin that was correlated with increased entry of bacteria and beads. Lastly, IbeA+ E. coli K1 invasion was inhibited by lipid-raft-disrupting agents (filipin and nystatin) and caveolin-1 siRNA (small interfering RNA), suggesting that caveolae/lipid rafts are signalling platforms for inducing IbeA-vimentin-mediated E. coli invasion of HBMECs. Taken together, the present studies suggest that a dynamic and function-related interaction between IbeA and its primary receptor vimentin at HBMEC membrane rafts leads to vimentin phosphorylation and ERK-mediated signalling, which modulate meningitic E. coli K1 invasion.

During the development of Escherichia coli K1 meningitis, interaction between E. coli invasion protein IbeA and surface protein(s) on human brain microvascular endothelial cells (HBMEC) is required for invasion and IbeA-mediated signaling. Here, an IbeA-binding protein was identified as polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF). The specific binding was confirmed by ligand overlay assay. The cell surface-expressed PSF was verified by the confocal microscopy. Recombinant PSF blocked E. coli K1 invasion of HBMEC effectively. Overexpression of PSF in the lentivirus-transducted HBMEC significantly enhanced E. coli K1 invasion. These results suggest that IbeA interacts with PSF for the E. coli K1 invasion of HBMEC.

Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. C. neoformans has a predilection to the brain, resulting in devastating meningoencephalitis. We have previously shown the invasion of C. neoformans into the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Here, we demonstrated that C. neoformans invasion of HBMEC was enhanced by HIV-1 gp41 protein. Peptide mapping defined its functional domain around the disulfide-bond linkage of gp41 molecule (a.a. 579-611). Recombinant protein gp41-I90 (a.a. 550-639) can also enhance the binding activity. The enhancement of C. neoformans binding to HBMEC is a strain-independent manner, suggesting that gp41 ectodomain peptide exerts its function directly on HBMEC. Importantly, the enhancement could be observed in mouse animal model. Our results suggest that HIV-1 gp41 ectodomain and C. neoformans may follow a similar invasion mechanism, possibly actin reorganization and/or membrane activation, during pathogen infections on HBMEC.

Escherichia coli K1 is the most common gram-negative bacteria that cause meningitis during the neonatal period. The ibeA gene product in E. coli K1 has been characterized as a virulence factor that contributes to the binding to and invasion of brain microvascular endothelial cells (BMEC). Here, we identified a surface protein on human BMEC, vimentin, that interacts with the E. coli invasion protein IbeA. The binding sites of the IbeA-vimentin interaction are located in the 271-370 residue region of IbeA and the vimentin head domain. The regulatory protease factor Xa is able to cleave IbeA between R297 and K298 residues, and this cleavage abolishes the IbeA-vimentin interaction.

The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK-->LIMK-->cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients' sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind (125)I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as epsilon -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood-brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.

Although epidemiological studies have shown that exposure to tobacco smoking significantly increases the risk of bacterial meningitis, heretofore the pathogenic effects of smoking on this disease have been poorly understood. In order to dissect this issue, we have investigated the effects of nicotine, the major component of tobacco, on E. coli invasion of human brain microvascular endothelial cells (HBMEC). Our studies showed that E. coli invasion of HBMEC was significantly enhanced by nicotine in a dose-dependent manner. The nicotine-mediated enhancement was associated with actin cytoskeleton rearrangement and morphological changes in the eukaryotic host cell that are essential for bacterial entry. The recombinant IbeA protein and alpha-bungarotoxin (a nicotinic acetylcholine receptor antagonist) were able to efficiently block the nicotine-mediated cellular effects, suggesting the involvement of the IbeA and nicotinic receptors. Blocking of phosphatidylinositol 3-kinase (PI3K) by LY294002 abolished the entry of E. coli in HBMECs treated with nicotine in a dose-dependent manner. Inhibition of PI3K was associated with decreased phosphorylation of Akt and actin cytoskeletal rearrangement. In contrast to PI3K, blockage of Rho kinase (ROCK) by Y27632 upregulated both nicotine- and E. coli-mediated cellular responses. Thus, this study provides experimental evidence for the first time that the major component of tobacco, nicotine, enhances meningitic E. coli invasion of HBMEC through modulation of cytoskeleton.

Presents information on a study which assessed the role of the ibeA gene in brain microvascular endothelial cell invasion by characterizing the ibeA gene and its invasion protein by chromosomal gene replacement, complementation, in vitro translation, and in vivo protein expression. Materials and methods; Isolation of subcloning of large DNA fragment that carries full-length ibeA gene; Construction of complementation of the ibeA deletion mutant ZD1.