scholarly journals Echovirus-30 Infection Alters Host Proteins in Lipid Rafts at the Cerebrospinal Fluid Barrier In Vitro

2020 ◽  
Vol 8 (12) ◽  
pp. 1958
Author(s):  
Marie Wiatr ◽  
Simon Staubach ◽  
Ricardo Figueiredo ◽  
Carolin Stump-Guthier ◽  
Hiroshi Ishikawa ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Lipid Rafts ◽  
Choroid Plexus Papilloma ◽  
Cellular Adhesion ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
Enteroviral Infection ◽  
Echovirus 30 ◽  
The Impact

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.

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

2020 ◽  
Vol 21 (17) ◽  
pp. 6268 ◽  
Author(s):  
Marie Wiatr ◽  
Ricardo Figueiredo ◽  
Carolin Stump-Guthier ◽  
Peter Winter ◽  
Hiroshi Ishikawa ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Cerebrospinal Fluid ◽  
Nervous System ◽  
Expression Profiles ◽  
Choroid Plexus Papilloma ◽  
Gene Expression Profiles ◽  
Barrier Properties ◽  
Echovirus 30 ◽  
Apical Side

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.

Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood–cerebrospinal fluid barrier

2012 ◽  
Vol 170 (1-2) ◽  
pp. 66-74 ◽  
Author(s):  
Henriette Schneider ◽  
Claudia Ellen Weber ◽  
Julia Schoeller ◽  
Ulrike Steinmann ◽  
Julia Borkowski ◽  
...  
Keyword(s):  
T Cells ◽  
Cerebrospinal Fluid ◽  
Choroid Plexus ◽  
In Vitro Model ◽  
Choroid Plexus Papilloma ◽  
Echovirus 30 ◽  
Human Choroid Plexus ◽  
Vitro Model ◽  
Plexus Papilloma

MMPs contribute to TNF-α-induced alteration of the blood-cerebrospinal fluid barrier in vitro

2007 ◽  
Vol 293 (3) ◽  
pp. C855-C864 ◽  
Author(s):  
Patrick Zeni ◽  
Eva Doepker ◽  
Ulf Schulze Topphoff ◽  
Sabine Huewel ◽  
Tobias Tenenbaum ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Epithelial Cells ◽  
Choroid Plexus ◽  
Adhesion Molecule ◽  
Cellular Adhesion ◽  
Matrix Metalloproteases ◽  
Intercellular Adhesion Molecule ◽  
Tnf Α ◽  
The Impact

The epithelial cells of the choroid plexus separate the central nervous system from the blood forming the blood-cerebrospinal fluid (CSF) barrier. The choroid plexus is the main source of CSF, whose composition is markedly changed during pathological disorders, for example regarding matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs). In the present study, we analyzed the impact of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) on the blood-CSF barrier using an in vitro model based on porcine choroid plexus epithelial cells (PCPEC). TNF-α evoked distinct inflammatory processes as shown by mRNA upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. The cytokine caused a drastic decrease in transepithelial electrical resistance within several hours representing an enhanced permeability of PCPEC monolayers. In addition, the distribution of tight junction proteins was altered. Moreover, MMP activity in PCPEC supernatants was significantly increased by TNF-α, presumably due to a diminished expression of TIMP-3 that was similarly observed. MMP-2, -3, and -9 as well as TIMP-1 and -2 were also analyzed and found to be differentially regulated by the cytokine. The TNF-α-induced breakdown of the blood-CSF barrier could partially be blocked by the MMP inhibitor GM-6001. Our results show a contribution of MMPs to the inflammatory breakdown of the blood-CSF barrier in vitro. Thus TNF-α may mediate the binding of leukocytes to cellular adhesion molecules and the transmigration across the blood-CSF barrier.

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

scholarly journals Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

2016 ◽  
Vol 213 (5) ◽  
pp. 809-825 ◽  
Author(s):  
Yancheng Liu ◽  
Shumin Tan ◽  
Lu Huang ◽  
Robert B. Abramovitch ◽  
Kyle H. Rohde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Stress Responses ◽  
Drug Action ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Drug Pressure ◽  
Cytokine Activation ◽  
The Impact

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

scholarly journals Cerebrospinal Fluid Pulsation Stress Promotes the Angiogenesis of Tissue-Engineered Laminae

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Linli Li ◽  
Yiqun He ◽  
Han Tang ◽  
Wei Mao ◽  
Haofei Ni ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cerebrospinal Fluid ◽  
Engineering Technology ◽  
Expression Levels ◽  
Fluid Pulsation ◽  
Endothelial Growth Factor ◽  
The Impact

Background. Angiogenesis is a prerequisite step to achieve the success of bone regeneration by tissue engineering technology. Previous studies have shown the role of cerebrospinal fluid pulsation (CSFP) stress in the reconstruction of tissue-engineered laminae. In this study, we investigated the role of CSFP stress in the angiogenesis of tissue-engineered laminae. Methods. For the in vitro study, a CSFP bioreactor was used to investigate the impact of CSFP stress on the osteogenic mesenchymal stem cells (MSCs). For the in vivo study, forty-eight New Zealand rabbits were randomly divided into the CSFP group and the Non-CSFP group. Tissue-engineered laminae (TEL) was made by hydroxyapatite-collagen I scaffold and osteogenic MSCs and then implanted into the lamina defect in the two groups. The angiogenic and osteogenic abilities of newborn laminae were examined with histological staining, qRT-PCR, and radiological analysis. Results. The in vitro study showed that CSFP stress could promote the vascular endothelial growth factor A (VEGF-A) expression levels of osteogenic MSCs. In the animal study, the expression levels of angiogenic markers in the CSFP group were higher than those in the Non-CSFP group; moreover, in the CSFP group, their expression levels on the dura mater surface, which are closer to the CSFP stress stimulation, were also higher than those on the paraspinal muscle surface. The expression levels of osteogenic markers in the CSFP group were also higher than those in the Non-CSFP group. Conclusion. CSFP stress could promote the angiogenic ability of osteogenic MSCs and thus promote the angiogenesis of tissue-engineered laminae. The pretreatment of osteogenic MSC with a CSFP bioreactor may have important implications for vertebral lamina reconstruction with a tissue engineering technique.

scholarly journals In vitro and Numerical Simulation of Blood Removal from Cerebrospinal Fluid: Comparison of Lumbar Drain to Neurapheresis Therapy

2020 ◽  
Author(s):  
Mohammadreza Khani ◽  
Lucas Sass ◽  
M. Keith Sharp ◽  
Aaron McCabe ◽  
Laura Zitella Verbick ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Subarachnoid Space ◽  
Cfd Model ◽  
Tracer Concentration ◽  
Steady Streaming ◽  
Lumbar Drain ◽  
Spinal Subarachnoid Space ◽  
Subject Specific ◽  
The Impact

Abstract Background: Blood removal from cerebrospinal fluid (CSF) in post-subarachnoid hemorrhage patients may reduce the risk of related secondary brain injury. We formulated a computational fluid dynamics (CFD) model to investigate the impact of a dual-lumen catheter-based CSF filtration system, called Neurapheresis TM therapy, on blood removal from CSF compared to lumbar drain. Methods: A subject-specific multiphase CFD model of CSF system-wide solute transport was constructed based on MRI measurements. The Neurapheresis catheter geometry was added to the model within the spinal subarachnoid space. Neurapheresis flow aspiration and return rate was 2.0 and 1.8 (mL/min), versus 0.2 (mL/min) drainage for lumbar drain. Blood was modeled as a bulk fluid phase within CSF with a 10% initial tracer concentration and identical viscosity and density as CSF. Subject-specific oscillatory CSF flow was applied at the model inlet. The dura and spinal cord geometry were considered to be stationary. Spatial-temporal tracer concentration was quantified based on time-average steady-streaming velocities throughout the domain under Neurapheresis therapy and lumbar drain. To help verify CFD results, an optically clear in vitro CSF model was constructed with fluorescein used as a blood surrogate. Quantitative comparison of numerical and in vitro results was performed by linear regression of spatial-temporal tracer concentration over 24-hours. Results: After 24-hours, tracer concentration was reduced to 4.9% under Neurapheresis therapy compared to 6.5% under lumbar drain. Tracer clearance was most rapid between the catheter aspiration and return ports. Neurapheresis therapy was found to have a greater impact on steady-streaming compared to lumbar drain. Steady-streaming in the cranial SAS was ~50X smaller than in the spinal subarachnoid space for both cases. CFD results were strongly correlated with the in vitro spatial-temporal tracer concentration under Neurapheresis therapy (R 2 =0.89 with +2.13% and -1.93% tracer concentration confidence interval). Conclusion: A subject-specific CFD model of CSF system-wide solute transport was used to investigate the impact of Neurapheresis therapy on tracer removal from CSF compared to lumbar drain over a 24-hour period. Neurapheresis therapy was found to substantially increase tracer clearance compared to lumbar drain. The multiphase CFD results were verified by in vitro fluorescein tracer experiments.

scholarly journals The Impact of Bioactive Surfaces in the Early Stages of Osseointegration: An In Vitro Comparative Study Evaluating the HAnano® and SLActive® Super Hydrophilic Surfaces

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Rodrigo A. da Silva ◽  
Geórgia da Silva Feltran ◽  
Marcel Rodrigues Ferreira ◽  
Patrícia Fretes Wood ◽  
Fabio Bezerra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Adhesion ◽  
Cell Cycle Progression ◽  
Cellular Adhesion ◽  
Biological Behavior ◽  
Early Stages ◽  
Genes Encoding ◽  
Bioactive Surfaces ◽  
The Impact

There is an increased effort on developing novel and active surfaces in order to accelerate their osteointegration, such as nanosized crystalline hydroxyapatite coating (HAnano®). To better understand the biological behavior of osteoblasts grown on HAnano® surface, the set of data was compared with SLActive®, a hydrophilic sandblasted titanium surface. Methodologically, osteoblasts were seeded on both surfaces up to 72 hours, to allow evaluating cell adhesion, viability, and set of genes encoding proteins related with adhesion, proliferation, and differentiation. Our data shows HAnano® displays an interesting substrate to support cell adhesion with typical spread morphologic cells, while SLActive®-adhering cells presented fusiform morphology. Our data shows that the cellular adhesion mechanism was accompanied with upexpression of integrin β1, Fak, and Src, favoring the assembling of focal adhesion platforms and coupling cell cycle progression (upmodulating of Cdk2, Cdk4, and Cdk6 genes) in response to HAnano®. Additionally, both bioactive surfaces promoted osteoblast differentiation stimulus, by activating Runx2, Osterix, and Alp genes. Although both surfaces promoted Rankl gene expression, Opg gene expression was higher in SLActive® and this difference reflected on the Rankl/Opg ratio. Finally, Caspase1 gene was significantly upmodulated in response to HAnano® and it suggests an involvement of the inflammasome complex. Collectively, this study provides enough evidences to support that the nanohydroxyapatite-coated surface provides the necessary microenvironment to drive osteoblast performance on dental implants and these stages of osteogenesis are expected during the early stages of osseointegration.

scholarly journals The Two-Component System 09 of Streptococcus pneumoniae Is Important for Metabolic Fitness and Resistance during Dissemination in the Host

2021 ◽  
Vol 9 (7) ◽  
pp. 1365
Author(s):  
Stephanie Hirschmann ◽  
Alejandro Gómez-Mejia ◽  
Thomas P. Kohler ◽  
Franziska Voß ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Regulatory System ◽  
Immunoblot Analysis ◽  
Defined Medium ◽  
Host Cells ◽  
Two Component ◽  
Regulatory Effects ◽  
The Impact

The two-component regulatory system 09 of Streptococcus pneumoniae has been shown to modulate resistance against oxidative stress as well as capsule expression. These data and the implication of TCS09 in cell wall integrity have been shown for serotype 2 strain D39. Other data have suggested strain-specific regulatory effects of TCS09. Contradictory data are known on the impact of TCS09 on virulence, but all have been explored using only the rr09-mutant. In this study, we have therefore deleted one or both components of the TCS09 (SP_0661 and SP_0662) in serotype 4 S. pneumoniae TIGR4. In vitro growth assays in chemically defined medium (CDM) using sucrose or lactose as a carbon source indicated a delayed growth of nonencapsulated tcs09-mutants, while encapsulated wild-type TIGR4 and tcs09-mutants have reduced growth in CDM with glucose. Using a set of antigen-specific antibodies, immunoblot analysis showed that only the pilus 1 backbone protein RrgB is significantly reduced in TIGR4ΔcpsΔhk09. Electron microscopy, adherence and phagocytosis assays showed no impact of TCS09 on the TIGR4 cell morphology and interaction with host cells. In contrast, in vivo infections and in particular competitive co-infection experiments demonstrated that TCS09 enhances robustness during dissemination in the host by maintaining bacterial fitness.

scholarly journals Cell-in-cell phenomenon: leukocyte engulfment by non-tumorigenic cells and cancer cell lines

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Mareike F. Bauer ◽  
Michael Hader ◽  
Markus Hecht ◽  
Maike Büttner-Herold ◽  
Rainer Fietkau ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Mononuclear Cells ◽  
Cancer Cell Lines ◽  
Host Cells ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
The Impact ◽  
Microwave Hyperthermia

Abstract Background Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes. Results We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process. Conclusions Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.

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