To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.
Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Drug Synergism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transfection

OBJECTIVETo investigate the roles of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in host defense against Streptococcus pneumoniae infection in the middle ear.

METHODSWild-type (WT) C57BL/6J, TLR2-deficient (TLR2(-/-)) and TLR4-deficient (TLR4(-/-)) mice were inoculated with Streptococcus pneumoniae (1 × 10(6)CFU) through the tympanic membrane. All animals were tested the mouse ABR thresholds and tympanometry measurement before, and 1 day, 3 days and 7 days following pneumococcal challenge. Blood bacterial titer were determined by plating 50 µl volumes of 10-fold diluted blood. Histological analysis of middle ear and inner ear were performed by fixation, decalcification, embedded section, and counterstained with hematoxylin/eosin and toluidine blue staining. Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes.

RESULTSForty of 68 TLR2(-/-) mice and twenty-one of 59 TLR4(-/-) mice showed bacteremia and died within 3 days after the pneumococcal challenge, however, only 9 of 52 WT mice died. The survive mice were shown have more severe hearing loss in the TLR2(-/-) and TLR4(-/-) mice than in the WT mice, indicated by ABR thresholds, at 3 or 7 days postinoculation. The histological pathology was characterized by effusion and tissue damage in the middle ear, and in the TLR2(-/-) and TLR4(-/-) mice, the outcome of infection became more severe at 7 days. At both 3 and 7 days after challenge, the TLR2(-/-) mice had higher blood bacterial titers than WT mice (P < 0.05). Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge, the TLR2(-/-) and TLR4(-/-) mice showed effusion and tissue damage in the middle ear, and the infection became more severe at 7 days postinoculation. TLR2(-/-) mice showed severe inflammatory cell infiltration in the cochlear, the organ of Corti showed the outer hair cells damage, the tectorial membrane swelling, degeneration of the stria vascularis, and severe loss of spiral ganglion cells; However, the WT mice was not found the cell infiltration and tissue damage in the cochlear, the organ of Corti shown normal of outer hair cells. Mast cells were not found in the middle ear mucosa of TLR2(-/-) mice, but in the TLR4(-/-) and WT mice, more mast cells were found in the middle ear mucosa of effusion ear by 3 and 7 days postchallenge. Moreover, by 3 days postchallenge, the mRNA accumulation levels of NF-κB, tumor necrosis factor alpha (TNFα), interleukin1β, MIP-1α, MUC5AC and MUC5B were significantly lower in the ears of TLR2(-/-) mice than that in WT and TLR4(-/-) mice.

CONCLUSIONSTLR2(-/-) mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge, thus hindering the clearance of bacteria from the middle ear and leading to sepsis and high mortality rate. This study indicated that TLR2 and TLR4 are important in the molecular pathogenesis and host response to otitis media.

Animals ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Otitis Media, Suppurative ; metabolism ; microbiology ; Pneumococcal Infections ; metabolism ; Streptococcus pneumoniae ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism

OBJECTIVETo investigate the repeatability of three-dimensional (3-D) cephalometric measurements for the clinical application of 3-D cephalometry.

METHODSForty-nine measurements that widely used in traditional cephalometric analyses were defined in 3-D cone-beam CT (CBCT) images. Three examiners identified landmarks on CBCT images of 17 subjects with normal occlusion, respectively, and 3-D measurements were exported automatically by software SimPlant. Inter-examiner reliability correlation coefficients (ICC) were obtained for all measurements.

RESULTSRepeatability of 36 measurements was high (ICC value greater than 0.9), including SNA, SNB. Repeatability of 11 measurements was moderate (ICC value between 0.8 and 0.9), including CoL-GoL, CoL-MSP. Repeatability of 2 measurements was low (ICC value lower than 0.8), including Gn-MSP and MPR-MSP.

CONCLUSIONSMost 3-D cephalometric measurements based on CBCT had high repeatability. However, some 3-D cephalometric measurements had limited repeatability.

Cephalometry ; Cone-Beam Computed Tomography ; Humans ; Imaging, Three-Dimensional ; Observer Variation ; Reproducibility of Results

There have been several epidemiological studies evaluating the potential association between the methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism and the risk of male infertility.However,the results obtained were inconsistent.Therefore,we performed a meta-analysis to further examine the association between the MTHFR A1298C polymorphism and male infertility.A comprehensive search was conducted to identify all eligible studies from the online literature databases published prior to January 15th,2016.A total of 20 studies with 4293 cases and 4507 controls were included.An odds ratio (OR) and a 95％ confidence interval (95％ CI) were calculated to assess the strength of the association.A cumulative meta-analysis,sensitivity analysis and assessment of the publication bias were also performed in this study.The results showed that in the overall analysis,the association between the MTHFR A1298C polymorphism and male infertility was not significant.A stratified analysis by ethnicity revealed a significant increase in the risk of male infertility in the Asian population with the MTHFR A1298C polymorphism (especially in the heterozygote model:OR=l.20,95％ CI=1.01-1.44,P=0.994;the dominant model:OR=1.23,95％ CI=1.04-1.45,P=0.996;and the allele model:OR=l.20,95％ CI=1.04-1.39,P=0.985) but not in the Caucasian population.In the stratified analyses,no significant association was observed between the different types of male infertility.This meta-analysis suggests the MTHFR A1298C polymorphism may be a potential risk factor for male infertility,especially in the Asian population.

Objective:To study the early-onset epilepsy of intracerebral hemorrhage and build a prediction model to evaluate its prediction efficiency.Methods:A cross-sectional investigation was conducted to construct a specialized optimized prediction model. The prediction model was converted into a visual optimized scoring scale, so as to quantify the probability of secondary epilepsy after intracerebral hemorrhage. Based on the current prediction model of acute cerebral infraction and post-stroke seizure (AIS-PSS), the evaluation efficacy of optimized score for secondary epilepsy after hemorrhagic stroke was explored.Results:① After sample size calculation and sufficient inclusion and exclusion, 159 patients with cerebral hemorrhage were continuously selected as the model group of this cross-sectional study. A total of 29 patients with early-onset epilepsy and 130 patients without secondary epilepsy were enrolled. The time span was from January 2021 to August 2021. In addition, 77 patients with acute cerebral hemorrhage from August 2021 to February 2022 were selected as the verification group, among which 12 patients had early-onset epilepsy and 65 patients had not any secondary epilepsy. ② There were significant differences in demographic characteristics such as diabetes history, cerebral infarction history, smoking history, National Institutes of Health Stroke Scale (NIHSS) score, intracerebral hemorrhage hematoma volume, serum creatinine (SCr), neuron-specific enolase (NSE), S-100 protein and intracerebral hemorrhage site between the two model groups with different prognosis (all P < 0.05). ③ The above indexes were included in univariate and multivariate Poisson regression analysis, and the results showed that the duration of diabetes [relative risk ( RR) = 1.229, 95% confidence interval (95% CI) was 1.065-1.896, P = 0.036], smoking history ( RR = 1.419, 95% CI was 1.133-2.160, P = 0.030), history of cerebral infarction ( RR = 1.634, 95% CI was 1.128-2.548, P = 0.041), hematoma volume of cerebral hemorrhage ( RR = 1.222, 95% CI was 1.024-2.052, P = 0.041), NES content ( RR = 1.146, 95% CI was 1.041-1.704, P = 0.032), were independent influencing factors to constitute the prediction model. The prediction model was converted into a visual optimized scoring scale in the form of a line diagram to obtain the prediction probability corresponding to the corresponding score. ④ Receiver operator characteristic curve (ROC curve) was used to test the evaluation efficiency of optimized score and AIS-PSS score for early-onset cerebral hemorrhage epilepsy. Relevant data of patients in the verification group were extracted according to the information of two scores, and the final score of each patient in the verification group was obtained. The score and prognosis were put into the ROC curve to evaluate the predictive ability of different prediction models. The results showed that the cut-off value of the optimized score and the AIS-PSS score were 144 points and 7 points, respectively, and the area under the ROC curve (AUC) and the Yoden index of the optimized score were slightly lower than the AIS-PSS score. However, compared with AIS-PSS score, there was no significant difference in the evaluation efficiency of optimized score for early-onset epilepsy ( Z = 1.874, P > 0.05). Conclusion:This study constructed a specific early-onset epilepsy prediction model for patients with hemorrhagic stroke, and transformed it into an optimized score that is easy for clinical use, and its evaluation efficiency is reliable.

Objective To describe the epidemiologieal features of viral encephalitis and burden of Japanese encephalitis (JE),and to identify potential strategies for effective JE control measures,using data from the Viral Encephalitis Surveillance Program (VESP) launched in Ankang,Baoji,and Weinan prefectures,Shaanxi province.Methods Data was gathered from sentinel hospitals reporting system on all the viral encephalitis (VE) eases identified between June 2005 and May 2007.County Center for Disease Control and Prevention (CDC) investigated the cases,drawing blood and cerebrospinal fluid (CSF) samples from the hospitals,and testing IgM antibody against JE using ELISA.We used Epi Data and Excel for data entry and analysis.Results A total of 1097 VEs were reported and 1053 (96.0%) had blood or CSF samples collected and tested for IgM antibody against JE.Three hundred and eleven cases (29.5%) showed JE antibody positive (JE confirmed case).Among the JE confirmed cases,numbers of those under 15 year of age accounted for 33.7%,43.9%and 88.3%in Baoji,Weinan and Ankang prefectures respectively.The rest were mainly children aged 5-14 years old (53.3%).Toddlers,farmers and children accounted for 85.2%in JE confirmed cases.About half of other VE cases (51.0%) were students of all age.Data an investigation on 398 reported VE cases at discharge,showed that 67.1%of JE confirmed cases recovered while 83.7%of the other VE cases fully recovered.The case fatality rates were 9.2%for JE confirmed cases and 3.1%for other VE cases.578 cases were followed up at 90-days after discharge,69.6%of JE confirmed cases and 90.2%of other VE cases recovered,with case fatality rates were 13.6%and 3.6%for JE confirmed cases and for other VE cases,respectively.The sequelae rates were 10.0%for JE confirmed cases and 4.5%for other VE cases.Conclusion The peak of the VE season was the sameas that of JE.There were 45.6%of reported JE cases with negative JE IgM,suggesting that it is necessary to carry out laboratory testing for clinical diagnosis cases.The fact that high risk population was different at prefectures levels suggested that more attention be paid in JE control and prevention.

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.
Animals ; Body Weight ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; Interleukin-15 ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Neoplasm Transplantation ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

OBJECTIVETo study the permeability of intact mouse abdominal skin to aniline and the protective capability of two typical lab gloves against aniline.

METHODSA Franz diffusion cell was used to perform in vitro transdermal absorption test and glove permeation test for aniline (0.102 mg/ml and 0.010 mg/ml). The permeabilities of intact mouse abdominal skin and gloves to aniline were measured by high performance liquid chromatography-diode array detection.

RESULTSThe transdermal penetration of the two concentrations of aniline followed zero order kinetics within 12 h, exhibiting total aniline permeabilities within 24 h of 51.71% and 48.31%, respectively. The absorption liquid had an aniline concentration of at least 18 µg/L. The medical disposable latex glove could not stop the penetration of 0.010 mg/ml aniline, but the industrial natural latex glove could.

CONCLUSIONThe penetration of 0.102 mg/ml and 0.010 mg/ml aniline through the mouse abdominal skin follows zero order kinetics within 12 h. The medical disposable latex glove cannot stop the penetration of 0.010 mg/ml aniline, but the industrial natural latex glove can.

Aniline Compounds ; pharmacokinetics ; toxicity ; Animals ; Gloves, Protective ; Mice ; Skin Absorption ; drug effects

OBJECTIVETo determine whether the convergences of tactile information also occur at thalamic ventroposterolateral nucleus in rats, we investigated the properties of tactile responses of the thalamic ventroposterolateral nucleus in rats.

METHODSUnit responses were recorded extracellularly from thalamic ventroposterolateral nucleus in anesthetized rats.

RESULTSAmong 156 neurons examined, 140 neurons (89.7%) had the single, continual and small receptive fields, and 16 neurons (10.3%) had two discrete receptive fields. Some neurons?exhibited different responses to the same intensity stimulation which delivered to different points in their receptive fields. In addition, 4.5% neurons (n = 7) responded only to locomotive stimulation but?not to a punctiform tactile stimulation.

CONCLUSIONThe majority of neurons in ventroposterolateral nucleus of rats have the spatial, temporal and submodal characteristics of cutaneous receptors, while the minority of neurons exhibit the responses of interaction of different peripheral receptors. Therefore, it is concluded that there are convergences of tactile information at the ventroposterolateral nucleus of rats.

Animals ; Male ; Neural Pathways ; cytology ; physiology ; Neurons ; cytology ; physiology ; Physical Stimulation ; Rats ; Rats, Sprague-Dawley ; Skin ; innervation ; Thalamic Nuclei ; cytology ; physiology ; Touch

OBJECTIVETo evaluate the feasibility of the pedicle screw pilot holes placement in thoracic spine using the spiral wires as the guide pin.

METHODSThe pedicle screw pilot holes were drilled within the center of the pedicle and the lateral and medial pedicle walls were violated in 9 human dried thoracic vertebrae. Kirschner wires or spiral wires were separately placed in the holes, and then the posteroanterior and lateral radiographs were taken. The radiographs were evaluated by 3 experienced spine surgeons and 3 young orthopedists. After radiographs were shown to these observers, they combined the posteroanterior and lateral radiographs in each place and determined whether the pedicle screw pilot hole violated the pedicle cortex or not. The results were analyzed by a statistical software.

RESULTSSensitivity, specificity and accuracy of the method using spiral wires to detect pedicle pilot hole placement were significantly higher than those of using Kirschner wires. With a true posteroanterior radiograph, the sensitivity, specificity and accuracy of the method using spiral wires approximated or attained 100%.

CONCLUSIONSThe method of intrapedicular pilot hole placement verification using spiral wires is effective for guiding the accurate placement of pedicle screws.

Bone Screws ; Bone Wires ; Cadaver ; Feasibility Studies ; Humans ; Internal Fixators ; Predictive Value of Tests ; Radiography ; Reproducibility of Results ; Sensitivity and Specificity ; Spinal Fusion ; instrumentation ; Thoracic Vertebrae ; diagnostic imaging ; surgery ; Treatment Outcome