Viral meningitis by non-polio enteroviruses (NPEV) is a major public health burden causing fatal outcomes especially in the younger population. Strong evidence exists that the blood-cerebrospinal-fluid (CSF) barrier (BCSFB) serves as an entry point for enterovirus and leucocytes into the central nervous system (CNS). Moreover, analysis of clinical CSF specimens of patients with a NPEV infection revealed a predominance of polymorphonuclear granulocytes (PMN) in the early phase and mononuclear cells in the later course of meningitis. By applying a functional in vitro model of the BCSFB consisting of human choroid plexus papilloma (HIBCPP) cells, we aimed to analyse the mechanisms of sequential migration of PMN and naive CD3+ T lymphocytes following infection with Echovirus 30 (EV30). EV30 infection led to increased transmigration of PMN and naive CD3+ T lymphocytes. Transmigration of PMN was significantly enhanced in the presence of naive CD3+ T lymphocytes, but not vice versa. The barrier function was not differentially altered under the respective conditions. Infection with EV30 led to an upregulation of CXCL3 and CXCL11 on the RNA-level. Additional analysis of cytokine secretion revealed relatively high concentrations of IL-8, CCL20, CXCL3, CXCL10 and M-CSF. Overall, there was a predominantly polar direction of cytokine secretion to the basolateral side. IL-7 was the only cytokine which was strongly secreted to the apical side and that was enhanced following EV30 infection in our model. In conclusion, this study highlights the role of the choroid plexus and cytokines in regulating leucocyte entry into the CNS in the context of EV30 infection.

Echovirus-30 (E-30) is a non-polio enterovirus responsible for meningitis outbreaks in children worldwide. To gain access to the central nervous system (CNS), E-30 first has to cross the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCSFB). E-30 may use lipid rafts of the host cells to interact with and to invade the BCSFB. To study enteroviral infection of the BCSFB, an established in vitro model based on human immortalized brain choroid plexus papilloma (HIBCPP) cells has been used. Here, we investigated the impact of E-30 infection on the protein content of the lipid rafts at the BCSFB in vitro. Mass spectrometry analysis following E-30 infection versus uninfected conditions revealed differential abundancy in proteins implicated in cellular adhesion, cytoskeleton remodeling, and endocytosis/vesicle budding. Further, we evaluated the blocking of endocytosis via clathrin/dynamin blocking and its consequences for E-30 induced barrier disruption. Interestingly, blocking of endocytosis had no impact on the capacity of E-30 to induce loss of barrier properties in HIBCPP cells. Altogether, these data highlight the impact of E-30 on HIBCPP cells microdomain as an important factor for host cell alteration.

Although CD8(+) T cells have been implied in the pathogenesis of multiple sclerosis (MS), the molecular mechanisms mediating CD8(+) T-cell migration across the blood-brain barrier (BBB) into the central nervous system (CNS) are ill defined. Using in vitro live cell imaging, we directly compared the multistep extravasation of activated CD4(+) and CD8(+) T cells across primary mouse brain microvascular endothelial cells (pMBMECs) as a model for the BBB under physiological flow. Significantly higher numbers of CD8(+) than CD4(+) T cells arrested on pMBMECs under noninflammatory and inflammatory conditions. While CD4(+) T cells polarized and crawled prior to their diapedesis, the majority of CD8(+) T cells stalled and readily crossed the pMBMEC monolayer preferentially via a transcellular route. T-cell arrest and crawling were independent of G-protein-coupled receptor signaling. Rather, absence of endothelial ICAM-1 and ICAM-2 abolished increased arrest of CD8(+) over CD4(+) T cells and abrogated T-cell crawling, leading to the efficient reduction of CD4(+) , but to a lesser degree of CD8(+) , T-cell diapedesis across ICAM-1(null) /ICAM-2(-/-) pMBMECs. Thus, cellular and molecular mechanisms mediating the multistep extravasation of activated CD8(+) T cells across the BBB are distinguishable from those involved for CD4(+) T cells.

Echovirus 30 (E-30) is one of the most frequently isolated pathogens in aseptic meningitis worldwide. To gain access to the central nervous system (CNS), E-30 and immune cells have to cross one of the two main barriers of the CNS, the epithelial blood-cerebrospinal fluid barrier (BCSFB) or the endothelial blood-brain barrier (BBB). In an in vitro model of the BCSFB, it has been shown that E-30 can infect human immortalized brain choroid plexus papilloma (HIBCPP) cells.

The central nervous system (CNS) is protected by several barriers, including the blood-brain (BBB) and blood-cerebrospinal fluid (BCSFB) barriers. Understanding how cancer cells circumvent these protective barriers to invade the CNS is of crucial interest, since brain metastasis during cancer is often a fatal event in both children and adults. However, whereas much effort has been invested in elucidating the process of tumor cell transmigration across the BBB, the role of the BCSFB might still be underestimated considering the significant number of meningeal cancer involvement. Our work aimed to investigate the transmigration of neuroblastoma cells across the BCSFB in vitro.

Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology.

The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6+ Th17 cells during the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). To advance our understanding of the importance of the ChP in orchestrating CNS immune cell entry during neuroinflammation, we here directly compared the accumulation of CD45+ immune cell subsets in the ChP, the brain and spinal cord at different stages of EAE by flow cytometry. We found that the ChP harbors high numbers of CD45int resident innate but also of CD45hi adaptive immune cell subsets including CCR6+ Th17 cells. With the exception to tissue-resident myeloid cells and B cells, numbers of CD45+ immune cells and specifically of CD4+ T cells increased in the ChP prior to EAE onset and remained elevated while declining in brain and spinal cord during chronic disease. Increased numbers of ChP immune cells preceded their increase in the cerebrospinal fluid (CSF). Th17 but also other CD4+ effector T-cell subsets could migrate from the basolateral to the apical side of the blood-cerebrospinal fluid barrier (BCSFB) in vitro, however, diapedesis of effector Th cells including that of Th17 cells did not require interaction of CCR6 with BCSFB derived CCL20. Our data underscore the important role of the ChP as CNS immune cell entry site in the context of autoimmune neuroinflammation.

The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood-brain barrier has been intensely investigated, cellular diapedesis through the blood-CSF barrier (BCSFB) is incompletely understood. To investigate how B cells interact with the choroid plexus to transmigrate into the CSF we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. We found, that spontaneous transmigration of HC- and MS-derived B cells was scant, yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5, and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Our findings highlight the relevance of the BCSFB as important gate for the entry of potentially harmful activated B cells into the CSF.

The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP).

Echovirus-30 (E-30) is responsible for the extensive global outbreaks of meningitis in children. To gain access to the central nervous system, E-30 first has to cross the epithelial blood-cerebrospinal fluid barrier. Several meningitis causing bacteria preferentially infect human choroid plexus papilloma (HIBCPP) cells in a polar fashion from the basolateral cell side. Here, we investigated the polar infection of HIBCPP cells with E-30. Both apical and basolateral infections caused a significant decrease in the transepithelial electrical resistance of HIBCPP cells. However, to reach the same impact on the barrier properties, the multiplicity of infection of the apical side had to be higher than that of the basolateral infection. Furthermore, the number of infected cells at respective time-points after basolateral infection was significantly higher compared to apical infection. Cytotoxic effects of E-30 on HIBCPP cells during basolateral infection were observed following prolonged infection and appeared more drastically compared to the apical infection. Gene expression profiles determined by massive analysis of cDNA ends revealed distinct regulation of specific genes depending on the side of HIBCPP cells' infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood-cerebrospinal fluid barrier leading to central nervous system inflammation.

Febrile seizures (FS) affect up to 5% of children. The pathogen etiology in regard of viral loads has never been investigated. In a prospective cohort study we investigated the correlation between virus type and quantity in nasopharyngeal aspirates (NPAs) and the clinical characteristics in pediatric patients with a FS. From January 2014 to April 2016, 184 children with a FS were prospectively enrolled. The mean age of all included children was 26.7 ± 18.3 months with a male to female ratio of 1.4:1. Males with an acute disease and a short duration or absence of prior symptoms had a higher risk for complex FS. The majority of patients with FS presented with a generalized convulsion (180; 98%) and was admitted to hospital (178; 97%). Overall, 79 (43%) single and in 59 (32%) co-infections were detected. Human herpes virus 6 (HHV6), influenza, adenovirus (AV) and rhinovirus (RV) were the dominant pathogens, all detected with clinically significant high viral loads. HHV6 positive cases were significantly younger and less likely to have a positive family/personal history for FS. Influenza positives showed a higher rate of complex seizures, lower leukocyte and higher monocyte counts. AV positive cases were more likely to have a positive family history for FS and showed higher C-reactive protein values. In conclusion, a high viral load may contribute to the development of a FS in respiratory tract infections.