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Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.
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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.
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Objective To investigate the correlation between cognitive function and cerebral microbleeds (CMBs) in patients with small artery occlusive stroke (SAO).Methods The patients with SAO in Nanjing Stroke Registration Program were recruited from January 2011 to May 2011.The Montreal Cognitive Assessment (MoCA) was used to conduct the cognitive evaluation.At the same time,conventional MRI sequences and susceptibility-weighted imaging (SWI) were used to detect CMBs.Results A total of 70 patients with SAO were included in the study,48 of them had abnormal MoCA scores ( ＜26 points) and 22 of them had normal MoCA scores (≥26).The age of patients (t =-2.237,P =0.023),years of education (t =2.297,P =0.029),history of hypertension (x2 =2.297,P =0.025 ),severity of white matter hyperintensities (Z =-3.263,P =0.001) and presence of CMBs (P =0.001) were associated with the abnormal MoCA scores in patients with SAO.Logistic regression analysis showed that after adjusting for age,sex,white matter lesions,hypertension,diabetes and coronary heart disease,the presence of CMBs (odds ratio 5.648,95％ confidence interval 1.105-28.869; P =0.038) was still an independent risk factor for abnormal MoCA scores.The more serious of CMBs,the lower the MoCA scores (r =- 0.532,P ＜ 0.001 ).In patients with CMBs,the cognitive domain,such as the total MoCA score (t =5.180,P ＜ 0.001 ),visuospatial/executive function (t =3.924,P ＜ 0.001 ) and attention (t =4.309,P ＜ 0.001 ) were impaired significantly.The CMBs at different parts resulted in cognitive impairment in the related fields.Conclusions The numbers of CMBs and their locations were closely associated with cognitive impairment in patients with SAO.
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Objective To research the relation of early growth response gene-1(EGR-1) and NF-κB in human T-cell leukemia virus type 1(HTLV-1) Tax protein positive cells. Methods RT-PCR was used to amplify the aimed segments EGR-1 cDNA which was then inserted into an eukaryotic expression plasmid pcDNA3.0 to construct pcDNA3.0-EGR-1. The constructed plasmid was transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, the expression levels of EGR-1, p65 and Tax mRNA in transfected cells were assay by RT-PCR after 48 h post-transfection, the proteins of EGR-1 and p65 were detected by Western blot after 48 h post-transfection too. The constructed plasmid and pNF-κB-luc reporter gene plasmid was co-transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, and the activity of luciferase was assay after 48 h post-transfection. Results The results showed that the eukaryotic expression plasmid pcDNA3.0-EGR-1 was successfully constructed. The mRNA and protein expression of EGR-1 could be promoted significantly by Tax. EGR-1 can promote the mRNA and protein expressions of p65 in TaxP cells, the activity of NF-κB was up-regulated by EGR-1 too. Conclusion EGR-1 maybe involve in adult T-cell leukemia(ATL) by increasing the activation of NF-κB.
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Objective To investigate the related risk factors for small artery occlusion (SAO) and its 2 subtypes. Methods The clinical and imaging data in 291 patients with first-ever stroke who met the TOAST criteria of large artery atherosclerotic stroke (LAA) or SAO were collected from the Nanjing Stroke Registry Prog-am from December 2009 to November 2010. All the patients were divided into a LAA group (n = 120) and a SAO group (n = 171). The latter was redivided into either a lacunar infarction with ischemic leukoaraiosis (ILA) subgroup (n = 84)or an isolated lacunar infarction (ILI) subgroup (n = 87). The risk factors of the LAA group and SAO group and its subgroups were compared. Multivariate logistic regression analysis was conducted and the independent risk factors were screened. Results The mean age in the SAO group was larger than that in the LAA group. The proportion of the patients with hypertension and the serum homocysteine (Hcy) level were significantly higher than those in the LAA group (all P ＜0. 05). Multivariate logistic analysis showed that the advanced age (odds ratio, [OR] = 1.041,95% confidence interval [CI] 1.02-1.06, P = 0.045), hypertension (OR = 2. 912,95% CI 1. 11-6. 46, P =0. 031) and increased plasma Hcy (OR = 1. 109, 95% CI 1. 11-1. 32, P =0. 001) were the independent risk factors for SAO. The advanced age (OR = 1. 047,95% CI 1.00-1.09, P = 0.043), hypertension (OR = 2. 632, 95% CI 1.08-6.41, P= 0.033) and increased plasma Hcy (OR = 1. 211, 95% CI 1. 11-1. 32, P ＜0. 001) were the independent risk factors for ILA, while the hypercholesterolemia (OR =0. 136, 95% CI 0. 05-0. 37, P ＜0. 001) was the independent risk factor for ILI. Conclusions The advanced age, hypertension and increased plasma Hcy level may play important roles in the pathogenesis of SAO. The hypercholesterolemia is an independent risk factor for ILI, while advanced age, hypertension and increased plasma Hcy level are the independent risk factors for ILA.