Circadian rhythmicity of amyloid-beta-related molecules is disrupted in the choroid plexus of a female Alzheimer's disease mouse model.

The choroid plexus (CP) is part of the blood-cerebrospinal fluid barrier (BCSFB) and was recently described as an important component of the circadian clock system. It is the principal source of cerebrospinal fluid (CSF) and responsible for the synthesis and secretion of various neuroprotective peptides including those involved in amyloid-ß (Aß) transport/degradation, contributing to Aß homeostasis. Inadequate Aß metabolic clearance and transport across the BCSFB have been associated with circadian dysfunctions in Alzheimer's disease (AD) patients. To investigate whether AD pathology influences Aß scavengers circadian expression, we collected CP at different time points from an AD mouse model (APP/PS1) (female and male animals, aged 6- and 12-months-old) and analyzed their mRNA expression by Real-time RT-PCR. Only angiotensin-converting enzyme (Ace) expression in 6-month-old female wild-type mice and transthyretin (Ttr) expression in 12-month-old female wild-type mice presented significant rhythmicity. The circadian rhythmicity of Ace and Ttr, prompt us to analyze the involvement of circadian rhythm in Aß uptake. A human CP papilloma (HIBCPP) cell line was incubated with Aß-488 and uptake was evaluated at different time points using flow cytometry. Aß uptake displayed circadian rhythmicity. Our results suggest that AD might affect Aß scavengers rhythmicity and that Aß clearance is a rhythmic process possibly regulated by the rhythmic expression of Aß scavengers.

The role of PLVAP in endothelial cells.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.

SARS-CoV-2 sero-immunity and quality of life in children and adolescents in relation to infections and vaccinations: the IMMUNEBRIDGE KIDS cross-sectional study, 2022.

PURPOSE: The study evaluates the effects on sero-immunity, health status and quality of life of children and adolescents after the upsurge of the Omicron variant in Germany. METHODS: This multicenter cross-sectional study (IMMUNEBRIDGE Kids) was conducted within the German Network University Medicine (NUM) from July to October 2022. SARS-CoV-2- antibodies were measured and data on SARS-CoV-2 infections, vaccinations, health and socioeconomic factors as well as caregiver-reported evaluation on their children's health and psychological status were assessed. RESULTS: 497 children aged 2-17 years were included. Three groups were analyzed: 183 pre-schoolchildren aged 2-4 years, 176 schoolchildren aged 5-11 years and 138 adolescents aged 12-18 years. Positive antibodies against the S- or N-antigen of SARS-CoV-2 were detected in 86.5% of all participants (70.0% [128/183] of pre-schoolchildren, 94.3% of schoolchildren [166/176] and 98.6% of adolescents [136/138]). Among all children, 40.4% (201/497) were vaccinated against COVID-19 (pre-schoolchildren 4.4% [8/183], schoolchildren 44.3% [78/176] and adolescents 83.3% [115/138]). SARS-CoV-2 seroprevalence was lowest in pre-school. Health status and quality of life reported by the parents were very positive at the time of the survey (Summer 2022). CONCLUSION: Age-related differences on SARS-CoV-2 sero-immunity could mainly be explained by differences in vaccination rates based on the official German vaccination recommendations as well as differences in SARS-CoV-2 infection rates in the different age groups. Health status and quality of life of almost all children were very good independent of SARS-CoV-2 infection and/or vaccination. TRIAL REGISTRATION: German Registry for Clinical Trials Identifier Würzburg: DRKS00025546 (registration: 11.09.2021), Bochum: DRKS00022434 (registration:07.08.2020), Dresden: DRKS 00022455 (registration: 23.07.2020).

Comparing SARS-CoV-2 variants among children and adolescents in Germany: relative risk of COVID-19-related hospitalization, ICU admission and mortality.

PURPOSE: SARS-CoV-2 infections cause COVID-19 and have a wide spectrum of morbidity. Severe disease courses among children are rare. To date, data on the variability of morbidity in relation to variant of concern (VOC) in children has been sparse and inconclusive. We compare the clinical severity of SARS-CoV-2 infection among children and adolescents in Germany during the Wildtype and Alpha combined, Delta and Omicron phases of the COVID-19 pandemic. METHODS: Comparing risk of COVID-19-related hospitalization, intensive care unit (ICU) admission and death due to COVID-19 in children and adolescents, we used: (1) a multi-center seroprevalence study (SARS-CoV-2-KIDS study); (2) a nationwide registry of pediatric patients hospitalized with SARS-CoV-2 infections; and (3) compulsory national reporting for RT-PCR-confirmed SARS-CoV-2 infections in Germany. RESULTS: During the Delta predominant phase, risk of COVID-19-related hospitalization among all SARS-CoV-2 seropositive children was 3.35, ICU admission 1.19 and fatality 0.09 per 10,000; hence about halved for hospitalization and ICU admission and unchanged for deaths as compared to the Wildtype- and Alpha-dominant period. The relative risk for COVID-19-related hospitalization and ICU admission compared to the alpha period decreased during Delta [0.60 (95% CI 0.54; 0.67) and 0.51 (95% CI 0.42; 0.61)] and Omicron [0.27 (95% CI 0.24; 0.30) and 0.06 (95% CI 0.05; 0.08)] period except for the < 5-year-olds. The rate of case fatalities decreased slightly during Delta, and substantially during Omicron phase. CONCLUSION: Morbidity caused by SARS-CoV-2 infections among children and adolescents in Germany decreased over the course of the COVID-19 pandemic, as different VOCs) emerged.

Porcine choroid plexus-Riems cell line demonstrates altered polarization of transport proteins compared with the native epithelium.

The choroid plexus epithelium (CPe) forms a barrier between the cerebral blood supply and the cerebrospinal fluid (CSF), establishing the blood-CSF barrier (BCSFB). CSF is actively secreted by the CPe via tightly controlled processes involving multiple channels, transporters, and pumps. The importance of controlling CSF production and composition has been accentuated recently with an appreciation of CSF dysfunction in many pathologies. For mechanistic studies of CSF production, isolated CPe cell lines are valuable for the testing of hypotheses and potential drug targets. Although several continuous CPe cell lines have been described, none appear to have all the characteristics of the native epithelium and each must be used judiciously. The porcine choroid plexus-Riems (PCP-R) cell line forms a high-resistance monolayer characteristic of a barrier epithelium. Conservation of this phenotype is unusual among CPe cell lines, making this model useful for studies of the effects of infection, injury, and drugs on permeability. We have recently discovered that, although this line expresses many of the transporters expressed in the native tissue, some are mispolarized. As a result, inferences regarding fluid/electrolyte flux and the resultant CSF production should be pursued with caution. Furthermore, extended culture periods and changes in media composition result in significant morphological and functional variability. These studies provide a more detailed characterization of the PCP-R cell line concerning transporter expression, polarization, and functionality, as well as plasticity in culture, with the goal to provide the scientific community with information necessary to optimize future experiments with this model.

Characterization of TRPV4-mediated signaling pathways in an optimized human choroid plexus epithelial cell line.

The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.

Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier.

Zika virus (ZIKV) can infect and cause microcephaly and Zika-associated neurological complications in the developing fetal and adult brains. In terms of pathogenesis, a critical question is how ZIKV overcomes the barriers separating the brain from the circulation and gains access to the central nervous system (CNS). Despite the importance of ZIKV pathogenesis, the route ZIKV utilizes to cross CNS barriers remains unclear. Here we show that in mouse models, ZIKV-infected cells initially appeared in the periventricular regions of the brain, including the choroid plexus and the meninges, prior to infection of the cortex. The appearance of ZIKV in cerebrospinal fluid (CSF) preceded infection of the brain parenchyma. Further the brain infection was significantly attenuated by neutralization of the virus in the CSF, indicating that ZIKV in the CSF at the early stage of infection might be responsible for establishing a lethal infection of the brain. We show that cells infected by ZIKV in the choroid plexus were pericytes. Using in vitro systems, we highlight the possibility that ZIKV crosses the blood-CSF barrier by disrupting the choroid plexus epithelial layer. Taken together, our results suggest that ZIKV might exploit the blood-CSF barrier rather than the blood-brain barrier to invade the CNS.

Oncostatin M triggers brain inflammation by compromising blood-brain barrier integrity.

Oncostatin M (OSM) is an IL-6 family member which exerts neuroprotective and remyelination-promoting effects after damage to the central nervous system (CNS). However, the role of OSM in neuro-inflammation is poorly understood. Here, we investigated OSM's role in pathological events important for the neuro-inflammatory disorder multiple sclerosis (MS). We show that OSM receptor (OSMRß) expression is increased on circulating lymphocytes of MS patients, indicating their elevated responsiveness to OSM signalling. In addition, OSM production by activated myeloid cells and astrocytes is increased in MS brain lesions. In experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, OSMRß-deficient mice exhibit milder clinical symptoms, accompanied by diminished T helper 17 (Th17) cell infiltration into the CNS and reduced BBB leakage. In vitro, OSM reduces BBB integrity by downregulating the junctional molecules claudin-5 and VE-cadherin, while promoting secretion of the Th17-attracting chemokine CCL20 by inflamed BBB-endothelial cells and reactive astrocytes. Using flow cytometric fluorescence resonance energy transfer (FRET) quantification, we found that OSM-induced endothelial CCL20 promotes activation of lymphocyte function-associated antigen 1 (LFA-1) on Th17 cells. Moreover, CCL20 enhances Th17 cell adhesion to OSM-treated inflamed endothelial cells, which is at least in part ICAM-1 mediated. Together, these data identify an OSM-CCL20 axis, in which OSM contributes significantly to BBB impairment during neuro-inflammation by inducing permeability while recruiting Th17 cells via enhanced endothelial CCL20 secretion and integrin activation. Therefore, care should be taken when considering OSM as a therapeutic agent for treatment of neuro-inflammatory diseases such as MS.

Risk for severe outcomes of COVID-19 and PIMS-TS in children with SARS-CoV-2 infection in Germany.

Although children and adolescents have a lower burden of SARS-CoV-2-associated disease compared to adults, assessing the risk for severe outcomes among SARS-CoV-2-infected children remains difficult due to a high rate of undetected cases. We combine data from three data sources - a national seroprevalence study (the SARS-CoV-2 KIDS study), the nationwide, state-based reporting system for PCR-confirmed SARS-CoV-2 infections in Germany, and a nationwide registry on children and adolescents hospitalized with either SARS-CoV-2 or pediatric inflammatory multisystem syndrome (PIMS-TS, also known as MIS-C) - in order to provide estimates on the risk of hospitalization for COVID-19-related treatment, intensive care admission, and death due to COVID-19 and PIMS-TS in children. The rate of hospitalization for COVID-19-related treatment among all SARS-CoV-2 seropositive children was 7.13 per 10,000, ICU admission 2.21 per 10,000, and case fatality was 0.09 per 10,000. In children without comorbidities, the corresponding rates for severe or fatal disease courses were substantially lower. The lowest risk for the need of COVID-19-specific treatment was observed in children aged 5-11 without comorbidities. In this group, the ICU admission rate was 0.37 per 10,000, and case fatality could not be calculated due to the absence of cases. The overall PIMS-TS rate was 2.47 per 10,000 SARS-CoV-2 infections, the majority being children without comorbidities. CONCLUSION: Overall, the SARS-CoV-2-associated burden of a severe disease course or death in children and adolescents is low. This seems particularly the case for 5-11-year-old children without comorbidities. By contrast, PIMS-TS plays a major role in the overall disease burden among all pediatric age groups. WHAT IS KNOWN: • SARS-CoV-2-associated burden of disease in children is considered to be low, but accurate risk estimates accounting for clinically undiagnosed infections are lacking. • Asymptomatic SARS-CoV-2 infections are common in children. WHAT IS NEW: • We provide risk estimates for hospitalization for COVID-19-related treatment, ICU admission, death from COVID-19, and PIMS-TS for children with SARS-CoV-2 infections by pooling different data sources. • The risk for PIMS-TS exceeds the risk for severe COVID-19 in all age groups; the risk for severe COVID-19 is the lowest in 5-11 years old.

Different Involvement of Vimentin during Invasion by Listeria monocytogenes at the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers In Vitro.

The human central nervous system (CNS) is separated from the blood by distinct cellular barriers, including the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CFS) barrier (BCSFB). Whereas at the center of the BBB are the endothelial cells of the brain capillaries, the BCSFB is formed by the epithelium of the choroid plexus. Invasion of cells of either the BBB or the BCSFB is a potential first step during CNS entry by the Gram-positive bacterium Listeria monocytogenes (Lm). Lm possesses several virulence factors mediating host cell entry, such as the internalin protein family-including internalin (InlA), which binds E-cadherin (Ecad) on the surface of target cells, and internalin B (InlB)-interacting with the host cell receptor tyrosine kinase Met. A further family member is internalin (InlF), which targets the intermediate filament protein vimentin. Whereas InlF has been shown to play a role during brain invasion at the BBB, its function during infection at the BCSFB is not known. We use human brain microvascular endothelial cells (HBMEC) and human choroid plexus epithelial papilloma (HIBCPP) cells to investigate the roles of InlF and vimentin during CNS invasion by Lm. Whereas HBMEC present intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (except Met and Ecad). Treatment with the surface vimentin modulator withaferin A (WitA) inhibited invasion of Lm into HBMEC, but not HIBCPP cells. Invasion of Lm into HBMEC and HIBCPP cells is, however, independent of InlF, since a deletion mutant of Lm lacking InlF did not display reduced invasion rates.

Fucoidan and Derived Oligo-Fucoses: Structural Features with Relevance in Competitive Inhibition of Gastrointestinal Norovirus Binding.

Norovirus infections belong to the most common causes of human gastroenteritis worldwide and epidemic outbreaks are responsible for hundreds of thousands of deaths annually. In humans, noroviruses are known to bind to gastrointestinal epithelia via recognition of blood-group active mucin-type O-glycans. Considering the involvement of l-α-fucose residues in these glycans, their high valency on epithelial surfaces far surpasses the low affinity, though specific interactions of monovalent milk oligosaccharides. Based on these findings, we attempted to identify polyfucoses (fucans) with the capacity to block binding of the currently most prevalent norovirus strain GII.4 (Sydney, 2012, JX459908) to human and animal gastrointestinal mucins. We provide evidence that inhibitory effects on capsid binding are exerted in a competitive manner by α-fucosyl residues on Fucus vesiculosus fucoidan, but also on the galacto-fucan from Undaria pinnatifida and their oligo-fucose processing products. Insight into novel structural aspects of fucoidan and derived oligosaccharides from low-mass Undaria pinnatifida were revealed by GCMS and MALDI mass spectrometry. In targeting noroviral spread attenuation, this study provides first steps towards a prophylactic food additive that is produced from algal species.

Effect of Lipopolysaccharide-Induced Inflammatory Challenge on ß-Glucuronidase Activity and the Concentration of Quercetin and Its Metabolites in the Choroid Plexus, Blood Plasma and Cerebrospinal Fluid.

Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.

Antibiotic utilization in hospitalized children under 2 years of age with influenza or respiratory syncytial virus infection - a comparative, retrospective analysis.

BACKGROUND: Infections due to Respiratory Syncytial Virus (RSV) and Influenza virus (FLU) are leading causes of hospitalization in young children. Yet, there is little data on factors associated with antibiotic use in these patients. METHODS: We conducted a retrospective, single-center study of all patients below 2 years of age hospitalized between 2014 and 2018. We compared children with RSV infection to children with FLU infection analyzing clinical characteristics and factors contributing to an increased rate of antimicrobial utilization. RESULTS: RSV infection was diagnosed in 476/573 (83.1%), FLU in 95/573 (16.6%), and RSV-FLU-co-infection in 2/573 (0.3%) patients. Median age was lower for RSV compared to FLU (4 vs. 12 months; p < 0.0001). Children with RSV had longer hospitalization (5 vs. 4 days; p = 0.0023) and needed oxygen more frequently (314/476 vs. 23/95; p < 0.0001) than FLU patients. There was no significant difference in the overall antibiotic utilization between RSV and FLU patients (136/476 vs. 21/95; p = 0.2107). Logistic regression analyses revealed that septic appearance on admission (odds ratio [OR] 8.95, 95% confidence interval [CI] 1.5-54.1), acute otitis media (OR 4.5, 95% CI 2.1-9.4), a longer oxygen therapy (OR 1.40; 95% CI 1.13-1.74) and a higher C-reactive protein (CRP) (OR 1.7, 95% CI 1.5-2.0) were significantly associated with antibiotic use in both groups, but not age or pneumonia. CONCLUSIONS: In our cohort, the rate of antibiotic utilization was comparable between RSV and FLU patients, while for both groups distinct clinical presentation and a high CRP value were associated with higher antibiotic use.

Rapid spread of OXA-244-producing Escherichia coli ST38 in Germany: insights from an integrated molecular surveillance approach; 2017 to January 2020.

Annually, increasing numbers of OXA-244-producing Escherichia coli in 13 German federal states prompted us to initiate an outbreak investigation. Whole genome sequencing revealed that among 148 isolates analysed, most belonged to sequence type 38 with 62 isolates forming a genetically distinct cluster. Although no epidemiological link could be identified between cases, ongoing investigations suggest non-healthcare associated transmission. A screening-PCR was developed facilitating early detection of ST38 cluster isolates to identify the source and transmission route.

From Gene to Protein-How Bacterial Virulence Factors Manipulate Host Gene Expression During Infection.

Bacteria evolved many strategies to survive and persist within host cells. Secretion of bacterial effectors enables bacteria not only to enter the host cell but also to manipulate host gene expression to circumvent clearance by the host immune response. Some effectors were also shown to evade the nucleus to manipulate epigenetic processes as well as transcription and mRNA procession and are therefore classified as nucleomodulins. Others were shown to interfere downstream with gene expression at the level of mRNA stability, favoring either mRNA stabilization or mRNA degradation, translation or protein stability, including mechanisms of protein activation and degradation. Finally, manipulation of innate immune signaling and nutrient supply creates a replicative niche that enables bacterial intracellular persistence and survival. In this review, we want to highlight the divergent strategies applied by intracellular bacteria to evade host immune responses through subversion of host gene expression via bacterial effectors. Since these virulence proteins mimic host cell enzymes or own novel enzymatic functions, characterizing their properties could help to understand the complex interactions between host and pathogen during infections. Additionally, these insights could propose potential targets for medical therapy.

Interactions and Signal Transduction Pathways Involved during Central Nervous System Entry by Neisseria meningitidis across the Blood-Brain Barriers.

The Gram-negative diplococcus Neisseria meningitidis, also called meningococcus, exclusively infects humans and can cause meningitis, a severe disease that can lead to the death of the afflicted individuals. To cause meningitis, the bacteria have to enter the central nervous system (CNS) by crossing one of the barriers protecting the CNS from entry by pathogens. These barriers are represented by the blood-brain barrier separating the blood from the brain parenchyma and the blood-cerebrospinal fluid (CSF) barriers at the choroid plexus and the meninges. During the course of meningococcal disease resulting in meningitis, the bacteria undergo several interactions with host cells, including the pharyngeal epithelium and the cells constituting the barriers between the blood and the CSF. These interactions are required to initiate signal transduction pathways that are involved during the crossing of the meningococci into the blood stream and CNS entry, as well as in the host cell response to infection. In this review we summarize the interactions and pathways involved in these processes, whose understanding could help to better understand the pathogenesis of meningococcal meningitis.

Polar Infection of Echovirus-30 Causes Differential Barrier Affection and Gene Regulation at the Blood-Cerebrospinal Fluid Barrier.

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.

Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

The Impact of Small Extracellular Vesicles on Lymphoblast Trafficking across the Blood-Cerebrospinal Fluid Barrier In Vitro.

Central nervous System (CNS) disease in pediatric acute lymphoblastic leukemia (ALL) is a major concern, but still, cellular mechanisms of CNS infiltration are elusive. The choroid plexus (CP) is a potential entry site, and, to some extent, invasion resembles CNS homing of lymphocytes during healthy state. Given exosomes may precondition target tissue, the present work aims to investigate if leukemia-derived exosomes contribute to a permissive phenotype of the blood-cerebrospinal fluid barrier (BCSFB). Leukemia-derived exosomes were isolated by ultracentrifugation from the cell lines SD-1, Nalm-6, and P12-Ichikawa (P12). Adhesion and uptake to CP epithelial cells and the significance on subsequent ALL transmigration across the barrier was studied in a human BCSFB in vitro model based on the HiBCPP cell line. The various cell lines markedly differed regarding exosome uptake to HiBCPP and biological significance. SD-1-derived exosomes associated to target cells unspecifically without detectable cellular effects. Whereas Nalm-6 and P12-derived exosomes incorporated by dynamin-dependent endocytosis, uptake in the latter could be diminished by integrin blocking. In addition, only P12-derived exosomes led to facilitated transmigration of the parental leukemia cells. In conclusion, we provide evidence that, to a varying extent, leukemia-derived exosomes may facilitate CNS invasion of ALL across the BCSFB without destruction of the barrier integrity.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.