Figure - available from: Archives of Virology
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DENV infection of bEnd.3, C8-D30 and SH-SY5Y cells at 4 hours postinfection. The virus was inoculated at an MOI of 5. A) Control (uninfected bEnd.3 cell monolayer), B) infection with DENV1, C) infection with DENV2, D) infection with DENV3, E) infection with DENV4. DENV was detected using DENV 1+2+3+4 antibody (Abcam) and was counterstained with Alexa Fluor 488 (Abcam). Scale = 50 μm
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The blood-brain barrier (BBB) is a physical barrier that restricts the passage of cells and molecules as well as pathogens into the central nervous system (CNS). Some viruses enter the CNS by disrupting the BBB, while others can reach the CNS without altering the integrity of the BBB. Even though dengue virus (DENV) is not a distinctive neurotropic...
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Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative d...
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... These results are consistent with previous studies using bEnd.3 and C8-D30 cell lines in coculture, which have also displayed the critical role of astrocytes in maintaining the integrity and function of the BBB [32,[48][49][50][51][52]. Thus, this model effectively mimics the integrity and tightness of the BBB, making it suitable for studying the effects of prokineticins and their respective receptor antagonists on its permeability. ...
Prokineticins are a family of small proteins with diverse roles in various tissues, including the brain. However, their specific effects on different cerebral cell types and blood–brain barrier (BBB) function remain unclear. The aim of this study was to investigate the effects of PROK1 and PROK2 on murine cerebral cell lines, bEnd.3, C8.D30, and N2a, corresponding to microvascular endothelial cells, astrocytes and neurons, respectively, and on an established BBB co-culture model. Western blot analysis showed that prokineticin receptors (PROKR1 and PROKR2) were differentially expressed in the considered cell lines. The effect of PROK1 and PROK2 on cell proliferation and migration were assessed using time-lapse microscopy. PROK1 decreased neural cells’ proliferation, while it had no effect on the proliferation of endothelial cells and astrocytes. In contrast, PROK2 reduced the proliferation of all cell lines tested. Both PROK1 and PROK2 increased the migration of all cell lines. Blocking PROKRs with the PROKR1 antagonist (PC7) and the PROKR2 antagonist (PKR-A) inhibited astrocyte PROK2-mediated migration. Using the insert co-culture model of BBB, we demonstrated that PROKs increased BBB permeability, which could be prevented by PROKRs’ antagonists.
... Therefore, the transition of endothelial cells into a more mesenchymal state weakens homotypic, VE-cadherin-dependent junctions, enabling them to establish more labile interactions with other cell types as they become more motile. In addition, DENV-2 also promotes matrix metalloproteinase 9 (MMP-9) secretion and activation of nuclear factor κB (NF-κB), leading to degradation of tight junction proteins and apoptosis [49][50][51][52]. The data herein demonstrate that these events are linked to phenotypic transitions and are caused by soluble mediators released during the infection process. ...
Dengue virus (DENV) is a pathogenic arbovirus that causes human disease. The most severe stage of the disease (severe dengue) is characterized by vascular leakage, hypovolemic shock, and organ failure. Endothelial dysfunction underlies these phenomena, but the causal mechanisms of endothelial dysfunction are poorly characterized. This study investigated the role of c-ABL kinase in DENV-induced endothelial dysfunction. Silencing c-ABL with artificial miRNA or targeting its catalytic activity with imatinib revealed that c-ABL is required for the early steps of DENV infection. DENV-2 infection and conditioned media from DENV-infected cells increased endothelial expression of c-ABL and CRKII phosphorylation, promoted expression of mesenchymal markers, e.g., vimentin and N-cadherin, and decreased the levels of endothelial-specific proteins, e.g., VE-cadherin and ZO-1. These effects were reverted by silencing or inhibiting c-ABL. As part of the acquisition of a mesenchymal phenotype, DENV infection and treatment with conditioned media from DENV-infected cells increased endothelial cell motility in a c-ABL-dependent manner. In conclusion, DENV infection promotes a c-ABL-dependent endothelial phenotypic change that leads to the loss of intercellular junctions and acquisition of motility.
... Organ-on-a-chips, such as the gNVU, also open attractive avenues to address tissuelevel pathobiology, identification of host-based drug targets and evaluate therapeutic candidates against other viral pathogens that are known to have short-term and long-term CNS outcomes, such as polio virus, dengue virus, rift valley fever virus and hemorrhagic fever viruses [45][46][47][48][49][50][51][52][53][54][55][56]. Models such as the gNVU will support translational product development in a clinically relevant context for these pathogens for which the two-animal rule will play an imperative role and animal models are challenging when it comes to recapitulation of human disease. ...
The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.
... The virus can also reach the CNS by crossing the BBB, causing severe neurological syndromes [52]. DENV can cause a significant decrease in the protein level of ZO-1 expression and peripheral localization in epithelial and endothelial cells [53]. ...
Tight junctions (TJs) are highly specialized membrane structural domains that hold cells together and form a continuous intercellular barrier in epithelial cells. TJs regulate paracellular permeability and participate in various cellular signaling pathways. As physical barriers, TJs can block viral entry into host cells; however, viruses use a variety of strategies to circumvent this barrier to facilitate their infection. This paper summarizes how viruses evade various barriers during infection by regulating the expression of TJs to facilitate their own entry into the organism causing infection, which will help to develop drugs targeting TJs to contain virus-related disease.
... Increasing the permeability of the BBB is a common mechanism of damage used by numerous viruses (76,77). The IEB and the BBB are formed by epithelial and endothelial cells, respectively. ...
The irruption of SARS-CoV-2 during 2020 has been of pandemic proportions due to its rapid spread and virulence. COVID-19 patients experience respiratory, digestive and neurological symptoms. Distinctive symptom as anosmia, suggests a potential neurotropism of this virus. Amongst the several pathways of entry to the nervous system, we propose an alternative pathway from the infection of the gut, involving Toll-like receptor 4 (TLR4), zonulin, protease-activated receptor 2 (PAR2) and zonulin brain receptor. Possible use of zonulin antagonists could be investigated to attenuate neurological manifestations caused by SARS-CoV-19 infection.
... In addition, viral RNA were detected in the brain, but not detected in the liver. The breaching mechanism and hyperpermeability of blood-brain barrier by DENV were primarily due to direct consequences of viral infection of endothelial cells (45). ...
Dengue virus is a significant public health threat worldwide; however, the pathogenesis of dengue disease remains poorly understood due to lack of appropriate small animal models. Tree shrews are an emerging experimental animal model for the study of human diseases due to their resemblance of genetic characteristics to primate animals. Herein we report that dengue infection in tree shrews elicits resemble clinical symptoms as in humans. Dengue fever (△2°C> normal body temperature) developed in ~22% healthy Chinese tree shrews from 2 through 33 days after infection with a low dose (1 ∗ 10⁴ PFU/animal) of dengue virus serotype 2 or 3 intravenously or subcutaneously. The dengue genomic RNA and neutralizing antibodies were detected in ~78% of animals at days 7 and 15 post infection respectively. The serum levels of liver enzymes including aspartate transaminase, alanine aminotransferase and alkaline phosphatase were elevated with peaks at day 7 after infection. Modest thrombocytopenia and a slight decrease in the white blood cell count were observed. Intriguingly, although viral RNA was barely detectable in the liver by 48 days after infection, it was still evident in the brain. The intra-brain bleeding lesions in the intravenous infection group were more severe than those in the subcutaneous infection group. Our data demonstrate that primary dengue virus infection in tree shrews causes resemble clinical disease as in humans and thus tree shrews may be a suitable model for the study of dengue disease pathogenesis.
... This experiment demonstrated that ASE can be used with only one antibody, without affecting the results from established antibodies that work well with the normal SIP protocol.Improved demonstration of the differential response of the epithelial tight junction claudins to epidermal growth factor (EGF) treatment in cell monolayer cultures EGF is a key regulator of epithelial paracellular permeability, a property that depends on the downregulation of tight junction proteins Claudin-2 and upregulation of Claudin-4 through ERK1/2, Src and STAT3(Garcia-Hernandez et al., 2015). Regulation of paracellular permeability is a key factor in the immune-barrier function of epithelia, for example against pathogenic amoebae(Flores-Maldonado et al., 2017) or the dengue virus(Idris et al., 2019). Immunocytofluorescence in MDCK monolayer cells using Claudin 2 and Claudin 4 antibodies has previously revealed their localization in both the plasma membrane and in subcellular membrane organelles, although the antibody signal is not always easy to detect, especially in the case of Claudin 4. As ASE ...
... Rodent brains were processed for triple immunogold electron microscopy using ASE, revealing a subcellular relocalization from the synapse periphery to within the synaptic cleft of GABA-A receptors during mutanthuntingtin evoked neurodegeneration(Rosas-Arellano et al., 2018). ASE was also key to visualize arginase, a protein below the SIP detection limit, expressed in a subset of marmoset microglia(Rodriguez-Callejas et al., 2019) and it was also applied to a murine in vitro blood-brain barrier model in order to visualize the changes in tight junctions that explain how dengue virus compromises its integrity(Idris et al., 2019). ...
The use of antibodies to identify neuronal receptors, neurotransmitters, cytoskeletal elements or pathologic protein aggregates, ion channels, adhesion molecules or other cell-type specific markers, is common practice in neuroscience. Antibody detection systems are often based on confocal, epifluorescence or brightfield microscopy. Three types of technical issues can interfere with immunolabeling: low abundance of the target protein, low specific affinity of the antibody and/or signal background sometimes related to tissue fixation. Here, giving tribute to Professor Miledi's mentorship, we propose the application of an antibody signal enhancer (ASE) solution based on glycine, hydrogen peroxide and a detergent mix as a simple, low cost, protocol variation that significantly and specifically improves the signal to noise ratio during immunostaining experiments. We describe three new settings in which ASE improves the detection of a variety of antibodies applied on long-time stored non-human primate brain sections, cell culture monolayers and on squamous carcinomas retrieved from cervical cancer patients. The significant improvement of ASE over optimized immunohistochemical protocols used in clinical practice (i.e. cancer detection) combined with its simplicity and low cost makes it an attractive method for biomedical applications.
