ABSTRACT
BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.
Subject(s)
Humans , Interferon Type I , alpha-Synuclein , SARS-CoV-2 , COVID-19 , Virus Replication , Cell Line , Endothelial CellsABSTRACT
【Objective】 To explore the pathogenesis of fetal edema caused by CD36 antibody in fetal/neonatal alloimmune thrombocytopenia (FNAIT), and to provide reference for clinical prevention and treatment. 【Methods】 The established CD36 monoclonal antibody was incubated with human peripheral blood mononuclear cells (PBMC), and the concentrations of cytokines (TNF-α and IL-1β) in the supernatant of cell culture were detected by ELISA. The permeability of endothelial cells were investigated by detecting the fluorescence intensity of FITC-albumin by incubating cytokine-rich cell supernatant with human umbilical vein endothelial cells (HUVEC). 【Results】 Flow cytometry showed that CD36 monoclonal antibody could bind to human monocytes. Compared with isotype IgG control, increased cytokine TNF-α (pg/mL) (407.73±20.40 vs 29.38 ±4.72, P<0.05) and IL-1β (pg/mL) (247.14±83.59 vs 53.68±26.96, P<0.05) were detected in the supernatant of cell culture after incubation of CD36 monoclonal antibody with human PBMC. Detection of fluorescence intensity of FITC-albumin in transwell cultured HUVEC showed that cytokine-rich cell supernatant derived from CD36 monoclonal antibody incubated with human PBMC can increase the permeability of endothelial cells significantly (CD36 antibody vs isotype IgG, MFI value: 492±16 vs 320±11, P<0.05). 【Conclusion】 The effect of CD36 monoclonal antibody on PBMC can increase HUVEC permeability, which may be one of the pathogenesis of fetal edema with FNAIT.
ABSTRACT
Objective To study the role of endothelial cells on the inflammatory cytokine release in septic shock through the septic shock serum stimulating human primary endothelial cells (HPAEC) and peripheral blood mononuclear cells(PBMC).Methods PBMC isolated from healthy people by density gradient centrifugation.HPAEC cell surface markers CD144 and von Willebrand factor(vWF) molecule expression by RT-PCR and Western blot.Serum levels of IL-6,TNF-α,MCP-1 from septic shock patients and healthy human detected by ELISA.HPAEC and PBMC were stimulated with the isolated serums and LPS,respectively.ELISA was used to detect the supernatant IL-6,TNF-α,MCP-1 levels.HPAEC membrane molecules ICAM-1 expression was detected by flow cytometry with serum shock and LPS stimulation.Supernatant levels of IL-6,TNF-α,MCP-1 of HPAEC with S1P1 receptor agonist CYM-5442 pretreatment was detected by ELISA after shock serum stimulation.Results Endothelial cell markers CD144 and vWF molecules could be detected in the HPAEC.Levels of inflammatory cytokines IL-6,TNF-α,MCP-1 in patients with septic shock serum were significantly higher than healthy people (P＜0.01).PBMC and HPAEC with LPS or shock serum treatment respectively,compared with normal group,levels of inflammatory cytokines in the culture supernatant were significantly higher(P＜0.01).For PBMC,the level of inflammatory cytokines between shock group and LPS group were not significantly different (P＞0.05).But for HPAEC,levels of inflammatory cytokines in the supernatant of the shock group compared to the LPS group was significantly higher (P＜0.01).Similarly,when two cells after LPS stimulation,IL-6,TNF-α levels of HPAEC's supernatant were significantly lower than PBMC' s (P＜0.01),MCP-1 levels was no difference (P＞ 0.05).But when the stimulation of shock serum,HPAEC of IL-6,TNF-α levels and PBMC no significant difference (P ＞0.05).MCP-1 was significantly increased (P＜0.01).Shock patients serum stimulation S1P1 receptorspecific agonist CYM-5442 pretreatment of HPAEC with pretreatment of S1P1 receptor specific agonist CYM-5442,the culture supernatant of inflammatory cytokines IL-6,TNF-α,MCP-1 levels were significantly lower (P＜0.01).Conclusion Endothelial cells may play a central role on the release of inflammatory cytokine during septic shock.
ABSTRACT
Objective To explore the relationship between TLR3 mRNA expression on peripheral blood mononuclear cells(PBMCs)and acute rotavirus(RV)diarrhea.Methods Sixty-one children with acute RV diarrhea served as study subject,the expression of TLR3 mRNA on PBMCs was detected by real-time fluorescence quantitative RT-PCR.the concentrations of IFN-γand TNF-α in serum were measured by the method of Enzyrme-linked immunosorbent assay(EUSA).Results The expression of TLR3 on PBMCs and the serum levels of IFN-γ and TNF-α in the serious diarrhea group were 0. 820±0.051,(33.67±12.88)Pg/ml, (62.21±14.65)pg/ml,respectively,while it were 0.717±0.040,(24.01±10.06)pg/ml,(50.99± 12.18)pg/ml in the slight diarrhea group,and 0.525±0.029,(12.52±5.19)pg/ml,(28.65±7.44)pg/ml in the control group.Compared with the control group.the expression of TLR3 on PBMCs and the serum levels of IFN-γ,TNF-α in the serious and slight diarrhea group were significantly higher(P<0.01).There were significant differences between the serious and slight diarrhea group(P<0.01).There were positive relationship between the expression of TLR3 on PBMCs and tHe serum IFN-γ,TNF-α levels(r=0.431,P< 0.05,r=0.372,P<0.05).Conclusion The expression of TLR3 on PBMCs in children with acute rotavirus dialThea iS up-regulated,TLR3 and its mediated immune response are associated with the development of acute rotavirus diarrhea.
ABSTRACT
Objective:To investigate the capability of corneal cells to stimulate lymphocyte proliferation and the expression of costimulatory molecules on primary human corneal epithelial cells.Methods:Human corneal epithelial cells were cocultivated with peripheral blood mononuclear cells(PBMC). Expression of HLA class Ⅱ antigens, CD40, CD154, CD80 and CD86 was measured by immunohistochemical staining,and the expression of such molecules above on rejected corneal grafts was contrastingly assessed. Activation of lymphocytes was determined by measuring upregulation of CD69 by FACS analysis.Results:HLAⅡ expression was detectable on human primary corneal epithelial cells,and furthermore, upregualtion of costimulatory molecules CD40 and CD80 were also found after induction with ?-interferon. Both of HLA and CD40 were expressed on rejected corneal grafts. T lymphocytes were activated by epithelial cells in the coculture system.Conclusion:Human corneal epithelial cells are involved in rejection process of the corneal transplantation. The immunological reaction is triggered by epithelial cells,and endothelial cells suffer as the targets.
ABSTRACT
With use of ~(125)I-udR release test,a significant natural killer activityagainst K_(562) target cells has been determined in human peripheral wholeblood(WB).r There was a gradual decrease in NK activity as rise inthe dilution or WB.In comparison with peripheral monouclear cells(PBMC)oneself,the NK activity lowered distinctly at both 1:4 and 1:8 dilution ofWB(p0.4).It seems that neither the red blood cells nor thePlasma ffeect on NK activity of WB,The NK activity in WB as well as inPBMO was improved by incubation with human leukocyte interferon.TheNK activity of WB and PBMC in patients with hepatocellular carcinomawas signiticantly lower than in normal controls.The results showed tahtNK activity may be determined by using properly diluted WB instead orPBMC,and the assay can be easily performed in clinical practice.