Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+T cells on the expression of ICAM-1 (intercellular adhesion molecule 1),VCAM-1 (vascular cell adhesion molecule 1),IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection.Methods HUVECs were treated with CYM-5442,a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1),for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+T cells.Changes in the expression of NS1 (DENV-2 nonstructural protein),SPHK1 (sphingosine kinase 1,phosphorylating sphingosine to S1P),ICAM-1,VCAM-1,IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4,8,12,24,48 and 72 hours of co-culturing.Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h.Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level.The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05).Compared with DENV-2-infected or untreated HUVECs,Co-culturing DENV-2-infected HUVECs with CD4+T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+T cells at mRNA level (P<0.05),but had no significant influence on the expression of TGF-β1 in CD4+T cells at mRNA level.Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs,but CD4+T cells inhibit the replication and proliferation.CD4+T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level,activating HUVECs and increasing inflammation,which may be associated with increased vascular permeability induced by DENV-2 infection.Co-culturing CD4+T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+T cells at mRNA level,but has no significant effect on TGF-β1.

Objective To investigate the influences on major inflammatory cytokines after co-cul-turing regulatory T cells (Treg) with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Peripheral blood mononuclear cells (PBMC) were extrac-ted from concentrated human leukocytes by density gradient centrifugation. Treg cells were sorted by immu-nomagnetic beads. Expression of CD4,CD25 and CD127 molecules on the membrane of Treg cells was detec-ted by flow cytometry to identify the purity of Treg cells. HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells. Expression of IL-6,IL-8,TNF-α,IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR. Levels of IL-6,IL-8,IL-10 and TGF-β in the culture supernatants were detec-ted by a double-antibody sandwich ELISA. Results The purity of Treg cells was (84. 3±0. 5)%. Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reac-hing the peak at 24 h (3. 03±0. 26, P<0. 01). Enhanced expression of IL-6,IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection ( P<0. 01). Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells ( P<0. 05),but was still higher than that before infection. CYM-5442 pretreatment decreased the expression of IL-6,IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs. Conclusion Primary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells,and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs.

Objective To investigate the effects of socioeconomic factors on the prognosis of multiple myeloma (MM) patients and construct a prediction model for evaluating myeloma-specific survival (MSS) rates. Methods A total of 32625 patients diagnosed with MM between January 2007 and December 2016 were included through the SEER database. Cox regression model was used to analyze the predictive indicators of MSS. The results of the multivariate subgroup analysis were presented as forest plots. The significant factors identified in the multivariate Cox analysis were used to construct a nomogram. The predictive performance of the nomogram was assessed using the AUC and calibration plots. A nomogram score-based risk stratification system was constructed using a restricted cubic spline. Results Patients were divided into five groups according to their socioeconomic status (SES). Groups with higher SES had relatively higher proportions of those part of the White, insured, married, and urban populations. Age, gender, race, marital status, insurance status, and SES were independent prognostic factors of MSS (all P < 0.001). The linear trend of increased MSS risk with decreasing SES was most pronounced among the White, married, insured, and urban patients (all P < 0.001). The nomogram exhibited good discrimination and accuracy in both training and validation sets, showing AUC values of 0.701, 0.709, and 0.722 for predicting 3-, 5-, and 8-year MSS, respectively. A risk stratification model was established based on the nomogram total points and the HR, which then divided patients into three different risk levels with substantial survival disparities (all P < 0.001). Conclusion Socioeconomic factors, such as marital status, insurance status, and SES, have a significant impact on the survival outcomes of MM patients. The nomogram and the risk stratification model based on these factors can accurately and reliably predict MSS.

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) ％. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.

Objective:To evaluate the diagnostic efficacy of brush cytology and transpapillary biopsy sampling under the guidance of endoscopic retrograde cholangiopancreatography (ERCP) in distinguishing between benign and malignant biliary strictures.Methods:A total of 470 patients with unknown biliary strictures who underwent ERCP at Department of Gastroenterology, Beijing Friendship Hospital from January 2013 to January 2022 were enrolled. Brush cytology was performed before biopsy in a single procedure, and with multiple ERCP brush cytologies or biopsy performed for challenging cases. Clinical data, ERCP procedures, pathological results, and follow-up data were collected. With the final diagnosis as the golden standard, the diagnostic efficacy of brush cytology and biopsy sampling were calculated.Results:The final diagnosis showed 251 cases of malignant and 219 cases of benign biliary strictures. All 470 patients received brush cytology, among whom, 37 patients underwent multiple brush cytologies. Additionally, 114 patients were treated with biopsy sampling. The single brush cytology showed a sensitivity of 49.40% (124/251) and a specificity of 99.09% (217/219) for diagnosing malignant biliary strictures. Multiple brush cytologies showed a sensitivity of 68.42% (13/19) and a specificity of 100.00% (18/18). Biopsy sampling showed a sensitivity of 68.18% (45/66) and a specificity of 97.92% (47/48). Among the 114 patients who underwent both brush cytology and biopsy sampling, the results showed that the diagnostic sensitivity of brush cytology combined with biopsy sampling [77.27% (51/66)] and the sensitivity of single biopsy sampling [68.18% (45/66)] were significantly higher than that of single brush cytology [43.94% (29/66), χ 2=13.99, P<0.001; χ 2=6.92, P=0.009]. Conclusion:Combining brush cytology and biopsy forceps during ERCP enhances diagnostic sensitivity in identifying malignant biliary strictures. Biopsy sampling stands out as more sensitive compared with single brush cytology, emphasizing its role in improving diagnostic capabilities.