Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis

Bacterial pathogens may breach the blood-brain barrier (BBB) and invade the central nervous system through paracellular and/or transcellular mechanisms. Transcellular penetration, e.g., transcytosis across the BBB has been demonstrated for Escherichia coli K1, group B streptococcus, Listeria monocytogenes, Citrobacter freundii and Streptococcus pneumonia strains. Genes contributing to invasion of brain microvascular endothelial cells include E. coli K1 genes ompA, ibeA, ibeB, and yijP. Understanding the mechanisms of bacterial penetration across the BBB may help develop novel approaches to preventing bacterial meningitis.     

Glycosaminoglycan binding facilitates entry of a bacterial pathogen into central nervous systems

Certain microbes invade brain microvascular endothelial cells (BMECs) to breach the blood-brain barrier (BBB) and establish central nervous system (CNS) infection. Here we use the leading meningitis pathogen group B Streptococcus (GBS) together with insect and mammalian infection models to probe a potential role of glycosaminoglycan (GAG) interactions in the pathogenesis of CNS entry. Site-directed mutagenesis of a GAG-binding domain of the surface GBS alpha C protein impeded GBS penetration of the Drosophila BBB in vivo and diminished GBS adherence to and invasion of human BMECs in vitro. Conversely, genetic impairment of GAG expression in flies or mice reduced GBS dissemination into the brain. These complementary approaches identify a role for bacterial-GAG interactions in the pathogenesis of CNS infection. Our results also highlight how the simpler yet genetically conserved Drosophila GAG pathways can provide a model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications.     

病毒穿越血脑屏障的两种方式与可能机制

血脑屏障是维持中枢神经系统内环境稳定的重要结构,限制血液中大多数病原体的入侵;但有些病毒可穿越血脑屏障入侵中枢神经系统,导致神经功能障碍及炎症性疾病。目前认为,病毒可通过细胞和细胞间隙两种方式穿越血脑屏障,前者为直接感染脑微血管内皮细胞和跨细胞途径,后者为破坏内皮细胞间紧密连接及"特洛伊木马"途径。本文就近年来病毒穿越血脑屏障的途径和机制进行综述。     

Entry of pathogens into the central nervous system

Abstract: The blood-brain barrier (BBB) is formed by the tight junctions of the cerebral capillary endothelium and the choroid plexus epithelium. The molecular anatomy of the tight junction resembles that of a polarized, transporting epithelium, suggesting some model cell culture systems can provide insight into traffic into the central nervous system. Pathogens target both the endothelium, causing encephalitis, and the choroid plexus, leading to meningitis. Routes of entry are diverse including paracellular and transcellular penetration. In addition, circulating microbial products can induce loss of BBB function. Understanding the heterogeneous molecular interactions between pathogens and the BBB may provide avenues to interrupt the devastating neurological sequelae that accompany central nervous system infections.     

Pathogen translocation across the blood–brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood–brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia ) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial–host interactions (receptor–ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo . This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.     

Viral disruption of the blood-brain barrier

The blood-brain barrier (BBB) provides significant protection against microbial invasion of the brain. However, the BBB is not impenetrable, and mechanisms by which viruses breach it are becoming clearer. In vivo and in vitro model systems are enabling identification of host and viral factors contributing to breakdown of the unique BBB tight junctions. Key mechanisms of tight junction damage from inside and outside cells are disruption of the actin cytoskeleton and matrix metalloproteinase activity, respectively. Viral proteins acting in BBB disruption are described for HIV-1, currently the most studied encephalitic virus; other viruses are also discussed.     

Mechanisms of Blood Brain Barrier Disruption by Different Types of Bacteria,and Bacterial–Host Interactions Facilitate the Bacterial Pathogen Invading the Brain

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria–host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.     

Development of the blood-brain barrier

The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over the substances that leave or enter the brain. An elaborate network of complex tight junctions (TJ) between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. Additonally, the lack of fenestrae and the extremely low pinocytotic activity of endothelial cells of the BBB inhibit the transcellular passage of molecules across the barrier. On the other hand, in order to meet the high metabolic needs of the tissue of the central nervous system (CNS), specific transport systems selectively expressed in the membranes of brain endothelial cells in capillaries mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS. Whereas the characteristics of the mature BBB endothelium are well described, the cellular and molecular mechanisms that control the development, differentiation and maintenance of the highly specialized endothelial cells of the BBB remain unknown to date, despite the recent explosion in our knowledge of the growth factors and their receptors specifically acting on vascular endothelium during development. This review summarizes our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.     

Development of hematogenous pneumococcal meningitis in adult mice: the role of TNF-alpha

Bacterial penetration across the blood-brain barrier (BBB) into the central nervous system is the first step in development of meningitis. The role of tumor necrosis factor-alpha (TNF-alpha) in the penetration process was examined with peripheral infection of Streptococcus pneumoniae type 6. After intraperitoneal infection of S. pneumoniae type 6, the BBB opening was increased continuously from 6 h and the mice died of septic shock within 36 h due to bacterial overgrowth. The bacteria crossed the BBB and began to deposit in brain at 6 h post infection. There was strong staining of TNF-alpha on blood vessels of brain from 6 h to 24 h post infection. Anti-TNF-alpha antibody blocked both the BBB opening and the entrance of circulatory S. pneumoniae type 6 into brain, indicating that TNF-alpha played an important role in controlling the opening of BBB. Furthermore, an adult murine model of hematogenous pneumococcal meningitis was developed that is based on opening of the BBB by TNF-alpha and controlling the degree of bacteremia by cefazolin antibiotic. In conclusion, hematogenous meningitis developed as TNF-alpha initiated BBB opening, peripheral bacteria entered into the brain and formed bacterial emboli, and then progressed to meningitis.     

Microbial translocation of the blood-brain barrier

A major contributing factor to high mortality and morbidity associated with CNS infection is the incomplete understanding of the pathogenesis of this disease. Relatively small numbers of pathogens account for most cases of CNS infections in humans, but it is unclear how such pathogens cross the blood-brain barrier (BBB) and cause infections. The development of the in vitro BBB model using human brain microvascular endothelial cells has facilitated our understanding of the microbial translocation of the BBB, a key step for the acquisition of CNS infections. Recent studies have revealed that microbial translocation of the BBB involves host cell actin cytoskeletal rearrangements, most likely as the result of specific microbial-host interactions. A better understanding of microbial-host interactions that are involved in microbial translocation of the BBB should help in developing new strategies to prevent CNS infections. This review summarises our current understanding of the pathogenic mechanisms involved in translocation of the BBB by meningitis-causing bacteria, fungi and parasites.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

An insight into the ligand-receptor interactions involved in the translocation of pathogens across blood-brain barrier

Traversal of pathogen across the blood-brain barrier (BBB) is an essential step for central nervous system (CNS) invasion. Pathogen traversal can occur paracellularly, transcellularly, and/or in infected phagocytes (Trojan horse mechanism). To trigger the translocation processes, mainly through paracellular and transcellular ways, interactions between protein molecules of pathogen and BBB are inevitable. Simply, it takes two to tango: both host receptors and pathogen ligands. Underlying molecular basis of BBB translocation of various pathogens has been revealed in the last decade, and a plethora of experimental data on protein-protein interactions has been created. This review compiles these data and should give insights into the ligand-receptor interactions that occur during BBB translocation. Further, it sheds light on cell signaling events triggered in response to ligand-receptor interaction. Understanding of the molecular principles of pathogen-host interactions that are involved in traversal of the BBB should contribute to develop new vaccine and drug strategies to prevent CNS infections.     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood–brain barrier

Leukocyte transmigration across the blood–brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

链球菌突破血脑屏障的作用机制研究进展

血脑屏障(blood-brain barrier,BBB)是中枢神经系统(central nervous system,CNS)的天然结构和功能屏障之一,可有效阻止病原菌的入侵。然而病原菌能通过其自身毒力因子与脑内皮细胞相互作用,诱导宿主免疫应答反应,分泌大量细胞因子、趋化因子等,破坏紧密连接蛋白,最终突破血脑屏障,引起细菌性脑膜炎,产生不可逆的神经系统损伤。链球菌(Streptococcus)作为引起细菌性脑膜炎的重要病原菌,关于其突破血脑屏障分子机制研究已有显著进展。本文针对主要的链球菌,包括肺炎链球菌(Streptococcus pneumoniae)、猪链球菌(Streptococcus suis)、B型链球菌(group B Streptococcus,GBS)、马链球菌等突破血脑屏障的作用机制研究进展进行综述。     

Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIV_SF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.     

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.     

African trypanosome interactions with an in vitro model of the human blood-brain barrier

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.