Microglia Play a Major Role in Direct Viral-Induced Demyelination

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed inex vivospinal cord slice culture andin vitroneonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.

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Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion

International Journal of Molecular Sciences ◽

10.3390/ijms21103631 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3631 ◽

Cited By ~ 2

Author(s):

Raffaella Boggia ◽

Federica Turrini ◽

Alessandra Roggeri ◽

Guendalina Olivero ◽

Francesca Cisani ◽

...

Keyword(s):

Oral Administration ◽

Ellagic Acid ◽

Ex Vivo ◽

Behavioral Skills ◽

Aged Mice ◽

Age Related ◽

The Central Nervous System ◽

The Impact ◽

Old Mice

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in “ex-vivo, in vitro” parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

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Enhanced Virulence Mediated by the Murine Coronavirus, Mouse Hepatitis Virus Strain JHM, Is Associated with a Glycine at Residue 310 of the Spike Glycoprotein

Journal of Virology ◽

10.1128/jvi.77.19.10260-10269.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10260-10269 ◽

Cited By ~ 51

Author(s):

Evelena Ontiveros ◽

Taeg S. Kim ◽

Thomas M. Gallagher ◽

Stanley Perlman

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Neurological Diseases ◽

Mouse Hepatitis Virus ◽

Neutralizing Antibody ◽

Lateral Spread ◽

S Protein ◽

Acute Encephalitis ◽

The Central Nervous System

ABSTRACT The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM (“receptor-independent spread”). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.

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Sensory Stimuli Directly Acting at the Central Nervous System Regulate Gastric Ghrelin Secretion. An ex Vivo Organ Culture Study

Endocrinology ◽

10.1210/en.2007-0226 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3998-4006 ◽

Cited By ~ 44

Author(s):

Luisa M. Seoane ◽

Omar Al-Massadi ◽

J. Eduardo Caminos ◽

Sulay A. Tovar ◽

Carlos Dieguez ◽

...

Keyword(s):

Organ Culture ◽

Ex Vivo ◽

Negative Energy ◽

Gastrointestinal Hormone ◽

Suitable Model ◽

Sensory Stimuli ◽

Orexigenic Peptide ◽

The Central Nervous System ◽

Ghrelin Secretion

Ghrelin, a novel gastrointestinal hormone involved in GH regulation, has been postulated as a relevant orexigenic peptide released by splanchnic tissues. Descriptive studies have shown that plasma ghrelin levels increase in states of negative energy balance or fasting, while decreasing in obesity and after feeding. In the present study, a novel organ-culture model of gastric tissue explants obtained from rat donors has been validated for ex vivo experiments. Fasting induced gastric ghrelin release as well as ghrelin mRNA expression that were reflected in plasma. Interestingly, those changes were fully reverted by 15 min of refeeding before stomach extraction. Unexpectedly, when animals were allowed 15 min before explant extraction to see or smell, but not eat, the food (tease feeding), ghrelin secretion was suppressed just like in gastric explants from refed animals. This effect was blocked when the animals were subjected to surgical vagotomy or treated with atropine sulphate. In conclusion, gastric explants were a suitable model for testing ghrelin mechanism of secretion in vitro, and they were found to maintain memory of the previously received signals. Similar to feeding, tease feeding resulted in suppression of ghrelin discharge by explants.

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Preferential Infection of Mature Dendritic Cells by Mouse Hepatitis Virus Strain JHM

Journal of Virology ◽

10.1128/jvi.80.5.2506-2514.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2506-2514 ◽

Cited By ~ 26

Author(s):

Haixia Zhou ◽

Stanley Perlman

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Virus Strain ◽

Hepatitis Virus ◽

Ex Vivo ◽

Mouse Hepatitis Virus ◽

Demyelinating Diseases ◽

Immature Cells

ABSTRACT Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8+ and CD11b+ splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.

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Cannabinoid WIN 55,212-2 Inhibits Human Glioma Cell Growth by Triggering ROS-Mediated Signal Pathways

BioMed Research International ◽

10.1155/2021/6612592 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Kun Wang ◽

Qian Wang ◽

Qinghao Li ◽

Zhaoqiang Zhang ◽

Jing Gao ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Ex Vivo ◽

Underlying Mechanism ◽

Migration And Invasion ◽

Signal Pathways ◽

Human Glioma ◽

U251 Cell ◽

The Central Nervous System

Glioblastoma is a highly invasive primary malignant tumor of the central nervous system. Cannabinoid analogue WIN 55,212-2 (WIN) exhibited a novel anticancer effect against human tumors. However, the anticancer potential and underlying mechanism of WIN against human glioma remain unclear. Herein, the anticancer efficiency and mechanism of WIN in U251 human glioma cells were investigated. The results showed that WIN dose-dependently inhibited U251 cell proliferation, migration, and invasion in vitro. WIN treatment also effectively suppressed U251 tumor spheroids growth ex vivo. Further studies found that WIN induced significant apoptosis as convinced by the caspase-3 activation and release of cytochrome C. Mechanism investigation revealed that WIN triggered ROS-mediated DNA damage and caused dysfunction of VEGF-AKT/FAK signal axis. However, ROS inhibition effectively attenuated WIN-induced DNA damage and dysfunction of VEGF-AKT/FAK signal axis and eventually improved U251 cell proliferation, migration, and invasion. Taken together, our findings validated that WIN had the potential to inhibit U251 cell proliferation, migration, and invasion and induce apoptosis by triggering ROS-dependent DNA damage and dysfunction of VEGF-AKT/FAK signal axis.

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Optimized polyethylenimine (PEI)-based nanoparticles for siRNA delivery, analyzed in vitro and in an ex vivo tumor tissue slice culture model

Drug Delivery and Translational Research ◽

10.1007/s13346-016-0306-y ◽

2016 ◽

Vol 7 (2) ◽

pp. 206-216 ◽

Cited By ~ 15

Author(s):

Alexander Ewe ◽

Sabrina Höbel ◽

Claudia Heine ◽

Lea Merz ◽

Sonja Kallendrusch ◽

...

Keyword(s):

Tumor Tissue ◽

Ex Vivo ◽

Sirna Delivery ◽

Tissue Slice ◽

Slice Culture ◽

Culture Model

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Chronic Inflammation May Enhance Leiomyoma Development by the Involvement of Progenitor Cells

Stem Cells International ◽

10.1155/2018/1716246 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Monia Orciani ◽

Miriam Caffarini ◽

Alessandra Biagini ◽

Guendalina Lucarini ◽

Giovanni Delli Carpini ◽

...

Keyword(s):

Progenitor Cells ◽

Chronic Inflammation ◽

Acute Inflammation ◽

Patient Specific ◽

Obstetric Outcomes ◽

Rt Pcr ◽

Limited Sample ◽

Inflammatory Microenvironment ◽

Increased Risk

Although the etiology of leiomyoma is unclear, a progenitor/undifferentiated cell population has been described whose dysregulation may be involved in the onset of uterine conditions. Moreover, inflammation is involved in the development of several tumors. The aim of this work was to understand if progenitor cells sustain a chronic inflammatory microenvironment that enhances leiomyoma development. Cells from 12 human leiomyoma and 12 normal myometrium samples of the same patients were in vitro isolated and exhaustively characterized (morphology, proliferation, cytofluorometry, differentiation, RT-PCR, immunofluorescence, immunohistochemistry, and Western blotting assays). Selected cytokines (ELISA) and inflammation-related genes (RT-PCR) were analyzed to identify healthy myometrium progenitor cells (MPCs) and leiomyoma progenitor cells (LPCs). Results show that (i) MPCs and LPCs share stemness features, such as immunophenotype and multidifferentiation assay, (ii) LPCs have a significantly shorter doubling time and a significantly higher expression of stemness genes (p<0.05), and (iii) LPCs secreted significantly higher levels (p<0.05) of cytokines related to chronic inflammation and significantly lower amounts (p<0.05) of cytokines related to acute inflammation. Despite the limited sample size, comparisons between leiomyoma and normal myometrium tissue from each patient allowed normalization of patient-specific differences. The evidenced cytokine expression pattern related to chronic inflammation in LPCs may play a role in the increased risk of adverse obstetric outcomes (infertility, spontaneous miscarriage, and preterm birth) in women affected by leiomyomas. These women should be recognized as “high risk” and subjected to specialized management both before and during pregnancy.

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Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2

Biology ◽

10.3390/biology9030054 ◽

2020 ◽

Vol 9 (3) ◽

pp. 54 ◽

Cited By ~ 1

Author(s):

Azim Patar ◽

Peter Dockery ◽

Siobhan McMahon ◽

Linda Howard

Keyword(s):

Spinal Cord ◽

Lentiviral Vector ◽

Ex Vivo ◽

Axonal Growth ◽

Short Hairpin Rna ◽

Slice Culture ◽

Hairpin Rna ◽

Neurotrophin 3 ◽

Short Hairpin

The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth in in vitro and ex vivo transection injury models. Conditioned medium from cells transduced with NT-3 or shNG2 lentiviruses caused a significant increase in neurite length of primary dorsal root ganglia neurons compared to the control group in vitro. In an ex vivo organotypic slice culture (OSC) transduction with Lenti-NT-3 promoted axonal growth. Transducing OSCs with a combination of Lenti-NT-3/NG2 sh lead to a further increase in axonal growth but only in injured slices and only within the region adjacent to the site of injury. These findings suggest that the combination of lentiviral NT-3 and NG2 sh reduced NG2 levels and provided a more favourable microenvironment for neuronal regeneration after SCI. This study also shows that OSCs may be a useful platform for studying glial scarring and potential SCI treatments.

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Computational and biochemical analysis of type IV Pilus dynamics and stability

10.1101/2021.04.14.439816 ◽

2021 ◽

Author(s):

Yasaman Karami ◽

Aracelys López-Castilla ◽

Andrea Ori ◽

Jenny-Lee Thomassin ◽

Benjamin Bardiaux ◽

...

Keyword(s):

Binding Site ◽

Calcium Binding ◽

Ex Vivo ◽

Targeted Mutagenesis ◽

Human Pathogens ◽

Calcium Binding Site ◽

Type Iv ◽

Biochemical Analyses ◽

Dynamics Simulations

SUMMARYType IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 μs all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

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Organotypic slice culture of the mammalian retina

Visual Neuroscience ◽

10.1017/s0952523800003618 ◽

1993 ◽

Vol 10 (2) ◽

pp. 203-217 ◽

Cited By ~ 34

Author(s):

Andreas Feigenspan ◽

Joachim Bormann ◽

Heinz Wässle

Keyword(s):

Calcium Binding ◽

Cultured Cells ◽

Lucifer Yellow ◽

Cell Types ◽

Retinal Cell ◽

Slice Culture ◽

Specific Antibodies ◽

Organotypic Slice Culture ◽

And Function

AbstractVertical slices of 6-day postnatal (P6) rat retina were cut at a thickness of 100 μm and cultured using the roller-tube technique. After 14–21 days in vitro there was significant distortion of normal retinal architecture, but localized areas of the slices showed the typical pattern of layering of mature retina. The following immunocytochemical markers were used to characterize the different retinal cell types: antibodies against protein kinase C (PKC), calcium binding protein (CabP 28kD), neurofilaments (NF), glia-specific antibodies (GFAP, vimentin), and transmitter-specific antibodies (GABA, TH). The expression of these markers was compared in P6 retina, adult retina, and slice culture. To further characterize the cultured cells, patch-clamp recordings were performed in combination with intracellular injection of Lucifer Yellow (LY). Transmitter-and voltage-gated membrane currents were recorded from morphologically identified neurons. The experiments show that a mammalian slice culture can be used to study differentiation and function of retinal cell types.

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