scholarly journals Increased TNF-α Initiates Cytoplasmic Vacuolization in Whole Blood Coculture with Dengue Virus

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Rahmat Dani Satria ◽  
Tzu-Wen Huang ◽  
Ming-Kai Jhan ◽  
Ting-Jing Shen ◽  
Po-Chun Tseng ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Blood ◽  
Ex Vivo ◽  
Nonstructural Protein ◽  
Denv Infection ◽  
Cytopathic Effects ◽  
Cytoplasmic Vacuolization ◽  
Tnf Α ◽  
A Minor ◽  
Immune Profiling

During the acute febrile phase of dengue virus (DENV) infection, viremia can cause severe systemic immune responses accompanied by hematologic disorders. This study investigated the potential induction and mechanism of the cytopathic effects of DENV on peripheral blood cells ex vivo. At one day postinfection, there was viral nonstructural protein NS1 but no further virus replication measured in the whole blood culture. Notably, DENV exposure caused significant vacuolization in monocytic phagocytes. With a minor change in the complete blood cell count, except for a minor increase in neutrophils and a significant decrease in monocytes, the immune profiling assay identified several changes, particularly a significant reduction in CD14-positive monocytes as well as CD11c-positive dendritic cells. Abnormal production of TNF-α was highly associated with the induction of vacuolization. Manipulating TNF-α expression resulted in cytopathogenic effects. These results demonstrate the potential hematological damage caused by ex vivo DENV-induced TNF-α.

Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages

2016 ◽  
Vol 115 (03) ◽  
pp. 646-656 ◽  
Author(s):  
Ya-Ting Chu ◽  
Chiou-Feng Lin ◽  
Chih-Peng Chang ◽  
Trai-Ming Yeh ◽  
Robert Anderson ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Platelet Adhesion ◽  
Molecular Mimicry ◽  
Nonstructural Protein ◽  
Denv Infection ◽  
Dengue Disease ◽  
Nonstructural Protein 1 ◽  
Tnf Α ◽  
Β3 Integrin ◽  
Lines Of Evidence

SummaryThrombocytopenia is an important clinical manifestation of dengue disease. The hypotheses concerning the pathogenesis of thrombocytopenia include decreased production and increased destruction or consumption of platelets. We previously suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DENV) nonstructural protein 1 (NS1) cross-react with platelets. Furthermore, several lines of evidence show activation of endothelial cells (ECs) and macrophages are related to dengue disease severity. Previous studies also suggested that Ab-opsonised platelets facilitate the engulfment of platelets by macrophages. Here we show that TNF-α-activated ECs upregulate adhesion molecule expression to enhance the binding of platelets and macrophages and lead to anti-DENV NS1 Ab-mediated platelet phagocytosis. We further demonstrate that the interaction between macrophages and TNF-α-activated ECs requires binding of FcγR with the Fc region of platelet-bound anti-DENV NS1 Abs. Importantly, the binding of anti-DENV NS1 Abs to platelets did not interfere with platelet adhesion to ECs. The adhesion molecules ICAM-1 and β3 integrin expressed on ECs as well as the FcγR expressed on macrophages were critical in anti-DENV NS1 Ab-mediated platelet phagocytosis on activated ECs. Moreover, anti-DENV NS1 Abs dramatically enhanced platelet engulfment by macrophages in a murine model of DENV infection. Our study provides evidence for a novel role for anti-DENV NS1 Abs in the pathogenesis of thrombocytopenia in dengue disease by enhancing platelet phagocytosis by macrophages.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

scholarly journals Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Wisam-Hamzah Al Shujairi ◽  
Luke P. Kris ◽  
Kylie van der Hoek ◽  
Evangeline Cowell ◽  
Gustavo Bracho-Granado ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Denv Infection ◽  
Brain Morphology ◽  
Moderate Increase ◽  
Tnf Α ◽  
Significant Difference

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip−/−) showed enhanced DENV infection, accompanied by increased IFN-β and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip−/− mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip−/− compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-β, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip−/− mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip−/− DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-β, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip−/− mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

Whole blood optimization and genetic association of ex vivo TNF-α responsiveness to killed E. coli in Danish Holstein cows

2017 ◽  
Vol 201 ◽  
pp. 92-98
Author(s):  
M. Khatun ◽  
H.B.H. Jørgensen ◽  
A. Ehsani ◽  
M.S. Lund ◽  
G. Sahana ◽  
...  
Keyword(s):  
Genetic Association ◽  
Whole Blood ◽  
Ex Vivo ◽  
Holstein Cows ◽  
E Coli ◽  
Tnf Α

scholarly journals Longitudinal Analysis of Dengue Virus–Specific Memory T Cell Responses and Their Association With Clinical Outcome in Subsequent DENV Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Luis Alberto Sanchez-Vargas ◽  
Kathryn B. Anderson ◽  
Anon Srikiatkhachorn ◽  
Jeffrey R. Currier ◽  
Heather Friberg ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Outcome ◽  
Dengue Virus ◽  
Memory T Cells ◽  
Ex Vivo ◽  
Secondary Infection ◽  
Denv Infection ◽  
Memory T Cell ◽  
Cell Responses ◽  
Specific Memory

Memory T cells resulting from primary dengue virus (DENV) infection are hypothesized to influence the clinical outcome of subsequent DENV infection. However, the few studies involving prospectively collected blood samples have found weak and inconsistent associations with outcome and variable temporal trends in DENV-specific memory T cell responses between subjects. This study used both ex-vivo and cultured ELISPOT assays to further evaluate the associations between DENV serotype-cross-reactive memory T cells and severity of secondary infection. Using ex-vivo ELISPOT assays, frequencies of memory T cells secreting IFN-γ in response to DENV structural and non-structural peptide pools were low in PBMC from multiple time points prior to symptomatic secondary DENV infection and showed a variable response to infection. There were no differences in responses between subjects who were not hospitalized (NH, n=6) and those who were hospitalized with dengue hemorrhagic fever (hDHF, n=4). In contrast, responses in cultured ELISPOT assays were more reliably detectable prior to secondary infection and showed more consistent increases after infection. Responses in cultured ELISPOT assays were higher in individuals with hDHF (n=8) compared to NH (n=9) individuals before the secondary infection, with no difference between these groups after infection. These data demonstrate an association of pre-existing DENV-specific memory responses with the severity of illness in subsequent DENV infection, and suggest that frequencies of DENV-reactive T cells measured after short-term culture may be of particular importance for assessing the risk for more severe dengue disease.

scholarly journals Soluble ST2 Does Not Regulate TNF-α and IL-6 Production in Dengue Virus-Infected Human Monocytes

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Marisol Pérez-Acosta ◽  
Félix Giovanni Delgado ◽  
Jaime E. Castellanos
Keyword(s):  
Dengue Virus ◽  
Viral Antigen ◽  
Mononuclear Cells ◽  
Acute Infection ◽  
Denv Infection ◽  
Human Monocytes ◽  
Peripheral Blood Mononuclear ◽  
Soluble St2 ◽  
Tnf Α

Dengue virus (DENV) produces an acute infection that results in the overproduction of proinflammatory cytokines. Although increased levels of the immunoregulator soluble ST2 (sST2) protein have been reported in the serum of patients with dengue, its importance during DENV infection remains unclear. The purpose of this study was to evaluate the effect of a recombinant human sST2 protein on the production of TNF-α and IL-6 in an in vitro model of DENV infection. Peripheral blood mononuclear cells (PBMCs) were permissive to in vitro DENV infection since viral antigen was detected in CD14+ monocytes by flow cytometry (median, 1%; range, 0–2.2), and in their supernatants TNF-α and IL-6 were detected. However, sST2 protein was not detected. Using multiple staining on infected PBMC we found that only CD14+ cells produced TNF-α and IL-6. Treatment with human recombinant sST2 protein decreased lipopolysaccharide-induced monocyte TNF-α and IL-6 production. However, this effect was not observed when the monocytes were pretreated with sST2 and later infected with DENV-2. These results suggest that sST2 has different roles in the regulation of TNF-α and IL-6 expression in human monocytes stimulated with LPS and DENV-2.

scholarly journals Epidemiological survey and screening strategy for dengue virus in blood donors from Yunnan Province

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ling Li ◽  
Ying Li ◽  
Shaofang Lu ◽  
Jing Dong ◽  
Haixia Xu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Southern China ◽  
Nonstructural Protein ◽  
Denv Infection ◽  
Screening Strategy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Donor Screening ◽  
Blood Center ◽  
Nonstructural Protein 1

Abstract Background Dengue virus (DENV) infection is increasingly common in southern China and can be transmitted through blood transfusion but is not currently part of donor screening throughout the region. We assessed DENV prevalence among donors at the Xishuangbanna Blood Center, Yunnan, to support development of DENV screening strategies. Methods Blood samples were collected randomly between June 2019 and August 2019. These were screened for anti-DENV IgG and IgM using enzyme-linked immunosorbent assay (ELISA). Then, all reactive samples and some randomly-chosen non-reactive samples were used to detect DENV RNAs using real-time polymerase-chain-reaction (RT-PCR) assays. After RT-PCR, samples were further tested for soluble nonstructural protein 1 (NS1) using the colloidal gold method. Donors demographics were also collected and assessed. Results Over the study period, 2254 donor samples were collected and tested for anti-DENV IgG and IgM by ELISA. This revealed 598 anti-DENV IgG and/or IgM reactive samples, a serological prevalence of 26.53%. Of these, 26 were RT-PCR positive and/or NS1 positive. Significant differences in DENV prevalence were noted by occupation (P = 0.001), education (P < 0.001), and ethnicity (P = 0.026). Conclusion The prevalence of DENV in Xishuangbanna Blood Center was higher than most other blood centers that have implemented DENV donor screening. Our study provides first-hand data about the prevalence of DENV and allows the development of a screening strategy for clinical use.

scholarly journals Impact of p38 MAP Kinase Inhibitors on LPS-Induced Release of TNF-α in Whole Blood and Primary Cells from Different Species

2015 ◽  
Vol 36 (6) ◽  
pp. 2237-2249 ◽  
Author(s):  
Sarah Fehr ◽  
Anke Unger ◽  
Elke Schaeffeler ◽  
Sonja Herrmann ◽  
Stefan Laufer ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Whole Blood ◽  
Ex Vivo ◽  
P38 Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Primary Cells ◽  
Interspecies Differences ◽  
Tnf Α ◽  
P38 Mapk Inhibitors ◽  
Mapk Inhibitors

Background/Aims: Inhibition of p38 mitogen-activated protein kinase (p38 MAPK) is promising for the treatment of inflammatory disorders, however, the efficacy of p38 MAPK inhibitors in clinical trials is limited so far. Since functional sensitivity of p38 MAPK is commonly predicted by preclinical species, we systematically investigated interspecies differences including human tissue. Methods: Ex vivo test models were established using whole blood and primary cells from different species such as mice, rats, pigs and humans to compare LPS-induced TNF-α inhibition of four different p38 MAPK reference inhibitors SB 203580, BIRB-796, Pamapimod, and a Losmapimod analogue as well as a proprietary imidazole-based p38 MAPK Inhibitor. Results: All analysed p38 MAPK inhibitors resulted in significant inhibition of LPS-induced TNF-α release but with high interspecies differences for dose sensitivity. IC50 values from human whole blood and PBMC showed significant higher sensitivity towards p38 MAPK inhibition compared with data from pig and rat. Conclusion: Inhibition of TNF-α release by p38 MAPK inhibitors can be reliably identified in well-established laboratory species such as rat or mouse. However, our data indicate that animal models appear to be limited for valid prediction of the inhibitory potential for TNF-α release in humans. Thus, human tissues should be considered early in the drug development process of p38 MAPK inhibitors.

Dengue virus and antiplatelet autoantibodies synergistically induce haemorrhage through Nlrp3-inflammasome and FcүRIII

2015 ◽  
Vol 113 (05) ◽  
pp. 1060-1070 ◽  
Author(s):  
Chia-Ming Chang ◽  
Cheng-Yeu Wu ◽  
Ming-Shen Dai ◽  
Hao Chan ◽  
Wen-Sheng Wu ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Secondary Infection ◽  
Denv Infection ◽  
Additional Treatment ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Caspase 1 ◽  
Secondary Infections ◽  
Increased Risk ◽  
Tnf Α

SummaryDengue haemorrhagic fever (DHF) typically occurs during secondary infections with dengue viruses (DENVs). Although it is generally accepted that antibody-dependent enhancement is the primary reason why patients with secondary infection are at an increased risk of developing DHF, a growing body of evidence shows that other mechanisms, such as the elicitation of antiplatelet autoantibodies by DENV nonstructural protein NS1, also play crucial roles in the pathogenesis of DHF. In this study, we developed a “two-hit” model of secondary DENV infection to examine the respective roles of DENV (first hit) and antiplatelet Igs (second hit) on the induction of haemorrhage. Mice were first exposed to DENV and then exposed to antiplatelet or anti-NS1 Igs 24 hours later. The two-hit treatment induced substantial haemorrhage, coagulopathy, and cytokine surge, and additional treatment with antagonists of TNF-α, IL-1, caspase-1, and FcүRIII ameliorated such effects. In addition, knockout mice lacking the Fcү receptor III, Toll-like receptor 3, and inflammasome components Nlrp3 and caspase-1 exhibited considerably fewer pathological alterations than did wild type controls. These findings may provide new perspectives for developing feasible approaches to treat patients with DHF.

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