Objective To create a recombinant rabies virus overexpressing IL-33 and to clarify the effect of IL-33 overexpression on the phenotypic characteristics of recombinant virus in vitro. Methods The IL-33 gene was obtained and amplified from the brain of a highly virulent strain of rabies infected mouse. It was then inserted between the G and L genes of the parental virus LBNSE genome by reversing genetic manipulation and rescuing a recombinant virus overexpressing IL-33. BSR cells or mouse NA cells were infected with recombinant rabies virus (rLBNSE-IL33) and the parental strain LBNSE. Sequencing and fluorescent antibody virus neutralization assay was employed to detect the stability of recombinant virus at multiplicity of infection=0.01. Viral titres focal forming units (FFU) were detected to plot multi-step growth curves (multiplicity of infection=0.01). Cytotoxicity assay kit was used to detect cellular activity. ELISA was adopted to identify the IL-33 in the supernatant of infected cells of different multiplicity of infection. Results Rescued rLBNSE-IL33 overexpressing IL-33 remained stable for at least 10 consecutive generations and had virus titers of approximately 10⁸ FFU/mL. rLBNSE-IL33 was able to express IL-33 at high levels in a dose-dependent manner, but no high expression of IL-33 was detected in the supernatant of cells infected by LBNSE. Examination of the titers of rLBNSE-IL33 and the parental strain LBNSE in BSR and NA cells over 5 days showed no significant differences and similar kinetic properties in growth. Overexpression of IL-33 had no significant effect on the proliferation and activity of infected cells. Conclusion Overexpression of IL-33 does not significantly affect the phenotypic characteristics of recombinant rabies virus in vitro.
Animals ; Cricetinae ; Mice ; Cell Line ; Interleukin-33/genetics* ; Rabies virus/genetics* ; Phenotype

Objective:To explore the cause of death of 2 suspected Yunnan sudden unexplained death (YNSUD) cases in Dayao County, Yunnan Province.Methods:The field epidemiological investigation and autopsy of 2 cases of YNSUD in Dayao County from June 15 to 20, 2020 were conducted; and blood and tissue samples were collected for qualitative analysis of common poisons and drugs.Results:The areas where the two cases were located were all seriously ill villages with a history of YNSUD, and the time of death occurred in the onset season of YNSUD. There was no blood relationship between the 2 cases, no obvious abnormal symptoms before death, no special diet, no history of exposure to pesticides and other toxic chemicals, and the test results of common poisons were all negative. Autopsy pathological examination results showed that case 1 died of acute cardiac dysfunction caused by sudden acute myocardial infarction of coronary heart disease, and case 2 died of central respiratory and circulatory failure caused by spontaneous subarachnoid hemorrhage.Conclusions:The two cases are excluded from YNSUD through autopsy, and the cause of death is determined. It is suggested that emergency response should be taken as soon as possible for YNSUD cases, and autopsy should be actively carried out to clarify the cause of death from a pathological point of view.

Objective:To analyze and evaluate the effect of OX40L as a potential adjuvant for H7N9 whole-virion inactivated vaccine (WIV).Methods:Fifty BALB/c mice were randomly divided into five groups and immunized intramuscularly with PBS (control group) and 1.5 μg WIV alone or in combination with 0.6, 1.8 or 3.0 μg Fc-fused OX40L (OX40L/Fc) adjuvant. Three weeks after immunization, IgG, IgG1 and IgG2 titers were measured by ELISA and hemagglutination inhibition (HI) assay. Moreover, the mice were challenged with 50×median lethal dose (LD 50) of homologous virus and the changes in mouse body weight and survival rate were recorded to evaluate the effects of OX40L. Flow cytometry was used to analyze the mechanism of OX40L as an adjuvant 7 d after immunization. Results:Compared with immunization with WIV alone, co-immunization of WIV with OX40L/Fc induced higher antigen-specific IgG in mice. The geometric mean titers (lgGMT) of antibodies induced by 0.6, 1.8 and 3.0 μg OX40L/Fc reached 3.79, 4.40 and 4.20, respectively. WIV combined with OX40L/Fc induced high levels of IgG1 and IgG2a without influencing Th1/Th2 balance. HI antibodies were also higher in WIV+ 1.8 μg OX40L/Fc and WIV+ 3.0 μg OX40L/Fc groups than in WIV group (6.25±0.50 and 5.70±0.97 vs 3.00±0.97, both P<0.05). WIV combined with 1.8 or 3.0 μg OX40L/Fc could protect 80% or 75% of mice against lethal challenge with H7N9 and result in less weight loss as compared with WIV alone. The most effective dose of OX40L/Fc was 1.8 μg. Flow cytometry showed that WIV (0.6, 1.8, 3.0 μg) in combination with OX40L/Fc enhanced the proliferation of T follicular helper cells (Tfh) through promoting the expression of CXCR5 and PD-1 as compared with WIV alone (all P<0.05). Conclusions:This study suggested that OX40L was beneficial to potent antibody responses induced by H7N9 WIV through promoting Tfh cell proliferation.

Structure of biliary system is complex as well as various, making troubles for optimal surgical treatment of biliary disease. Remarkable imaging of biliary system helps surgeon evaluating patients and planning surgeries. There are several methods to obtain accurate anatomical information of biliary system, such as X-ray fluoroscopy, MRI and fluorescence-based imaging. Each has its own advantages and disadvantages. Combination of multi-model imaging technologies may improve visual result of anatomical information of biliary tract. More resolvable, legible, and sequential imaging technology of biliary system remains further study. This article reviews various cholangiography methods widely used in the clinical setting.

PURPOSE: To explore whether there is matching relation between the length and the tilting angle of terminal implants in the All-on-Four protocol by studying the effects of different implant configurations on stress distributions of implant, bone, and framework. MATERIALS AND METHODS: Four implants were employed to support a full-arch fixed prosthesis and five three-dimensional finite element models were established with CT images, based on the length (S and L) and distal tilt angle (0degrees, 30degrees and 45degrees) of terminal implants for an edentulous mandible, which named: Tilt0-S, Tilt30-S, Tilt30-L, Tilt45-S and Tilt45-L. An oblique 240 N was loaded at second molar. The von Mises Stresses were analyzed. The implants were consecutively named #1 to #4 from the loading point. RESULTS: 1) Tilt0-S had the greatest stress on the implants, with the other groups exhibiting variable reductions; the four implants of Tilt45-L demonstrated the greatest reduction in stress. 2) Tilt0-S had the greatest stress at bone around #1 implant neck, and Tilt45-L exhibited the least stress, which was a 36.3% reduction compared to Tilt0-S. 3) The greatest stress in the framework was found on the cantilevers distal to #1 implant. Tilt45-S exhibited the least stress. CONCLUSION: Matching different length and tilting angle of the terminal implants led to variable stress reductions on implants, bone and the superstructure. By optimizing implant configuration, the reduction of stress on implants and surrounding bone could be maximized. Under the present condition, Tilt45-L was the preferred configuration. Further clinical testings are required.
Finite Element Analysis* ; Mandible ; Molar ; Neck ; Prostheses and Implants

Objective To investigate the inflammation levels of 2-diabetes patients before and after 3 months of improving glycemic control.Methods A longitudinal study was performed in a subgroup of 48 subjects with T2D and poor glycemic control.Forty-four healthy individuals were taken as control group.The serum concentration of C-reactionprotein (CRP),interleukin-6 (IL-6),interleukin-6 (IL-8),transforming growth factor-β1 (TGF-β1) and transforming growth factor-β1 (MCP1) in all participants were measured simultaneously by multiplexed Luminex assay.Results The serum levels of CRP,MCP-1 of 2-diabetes patients were 3.96 (3.45,5.58) mg/L and (195.0± 129.8) ng/L,significant higher than those in control group (2.25 (1.24,3.22) mg/L,(148.5±85.7) ng/L),and the differences were significant(t=-2.580,P=0.010;t=-2.118,P =0.047).No significant difference was found in the serum levels of IL-6,IL-8,TGF-β lbetween the two groups (P＞0.05).TGF-β1 level in patients with good glycemic control decreased to 26.85 (23.17-31.12) ng/l,significant lower than that before glycemic control (43.5(26.5-62.25) g/L;Z=-2.191,P=0.028),and there were no significant differences among the other 4 kinds of inflammatory factors before and after blood glucose control(CRP:Z =-0.937P =0.372;IL-6:Z =-0.875,P =0.396;IL-8:Z =-1.215,P =0.286;MCP-1:t =-1.846,P=0.065).Conclusion Low grade systemic inflammation status in T2D patients.Improvement of glycemic control reduces TGF-β1 levels and plays a key role in delaying the development of diabetic nephropathy.

Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.

Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1).Methods In vitro cultured THP-1 cells were used as a model of microglia.After cultured with different concentrations of [0 (negative control group),10,50,100,500,1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h,the survival of cells was detected by CCK8.THP-1 cells were divided into 3 groups:control group,low dose and high dose fluoride groups according to the results of CCK8 assay,and then treated with different concentrations of sodium fluoride (0,500,5 000 μmol/L) for 48 h,concentrations of inflammatory cytokines,such as Interleukin-lβ (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium.The protein levels of IκBα,phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting.Results THP-1 cells were treated with different concentrations of sodium fluoride (500,1 000,5 000 μ mol/L) for 48 h.Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)％,(31.40 ± 4.56)％,(0.40 ± 0.24)％] was lower than that of the negative control group [(100.00 ± 0.00)％,all P ＜ 0.01].Compared to the control groups [(0.36 ± 0.07),(31.07 ± 0.81)ng/L],significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79),(19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20),(172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups,respectively.Interestingly,compared to the control groups [(100.00 ± 5.48)％,(100.00 ± 14.82)％],significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)％,(134.74 ± 1.93)％] and phospho-IκB-α [(152.61 ± 14.16)％,(176.91 ± 7.95)％] were observed in both low-fluoride and high fluoride groups.Meanwhile,the protein level of IκBα in high fluoride group [(63.53 ± 9.67)％] was significantly lower than that of the control group [(100.00 ± 10.99)％,P ＜ 0.01].Furthermore,significant positive correlation was detected between increased IL-1β,TNF-α and phospho-NF-κB p65 (r =0.74,0.72,all P ＜ 0.05).Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway.The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride.

Objective To summary the therapy outcome of retroperitoneal laparoscopic treatment on upper ureteral calculi.Methods The retroperitoneal laparoscopic treatment were performed to all cases from Jun.2011 to Jun.2012 in our hospital.All cases were treated with 3-hold method (two 10 mm and one 5 mm ports).The retroperitoneal space was made by a combination of blunt and balloon dissection,and the space was maintained with CO2 Ureteral longitudinal incision was made to remove the stones,and double J catheter was served as stent drainage.Absorbable suture was used to suture ureteral incision.Results A retroperitoneal approach was performed in 12 patients,and another patient was conducted the open surgery because the stone cower in the kidney calices.Operative periods ranged from 55 to 132 min (average was 85 min).There were no significant postoperative complications.Conclusion It is a minimally invasive and effective approach in the therapy of upper urerteral calculi with laparoscopic.

ObjectiveTo observe the effect of cluster needling of scalp acupuncture combined with rehabilitation(Tang's Approach) on interleukin-4 (IL-4), T lymphocyte subsets of immunosuppressive model rat.Methods40 healthy SD rats (male, 200～250 g) was divided into 5 groups randomly: normal group (NG), model group (MG), acupuncture group(AG), rehabilitation group(RG), and acupuncture with rehabilitation group(ARG), 8 rats in each group. Each group was treated by acupuncture or rehabilitation except the model or normal group, once a day for 10 d. Radioimmunoassay and malondialdehydein (MDA) technique were used to test the contents of IL-4 and T lymphocyte subsets in each group. ResultsThe contents of IL-4 of immunosuppressive rats can decrease significantly in RG and ARG (P＜0.05). The level of CD4+ T lymphocyte increased more significantly in AG and RAG than in MG after the treatment (P＜0.01). ConclusionTang's Approach can decrease the contents of IL-4 and improve the level of CD4+ T lymphocyte in rats.