BACKGROUNDSuicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.

METHODSpAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease PacI and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with PacI and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.

RESULTSComet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture. After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.

CONCLUSIONThe replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Adenoviridae ; genetics ; Bacteria ; genetics ; Defective Viruses ; genetics ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; Recombination, Genetic ; Thymidine Kinase ; genetics ; Virus Replication

OBJECTIVEEfficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed.

METHODSHep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay.

RESULTSHSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred.

CONCLUSIONSThe HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Carcinoma, Squamous Cell ; therapy ; Cell Line, Tumor ; Ganciclovir ; Genes, Transgenic, Suicide ; Genetic Therapy ; Genetic Vectors ; Humans ; Laryngeal Neoplasms ; therapy ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

Objective To compare the postoperative hemorrhage between standard uvulopalatopharyngoplasty(UPPP) and coblation assisted UPPP, and to evaluate the related risk factors and preventive measures. Methods Five hundreds and ninety seven patients with obstructive sleep apnea hypopnea syndrome(OSAHS) underwent UPPP and coblation assisted UPPP between January 1, 1999, and September 30, 2009 were reviewed retrospectively. Two hundred and sixty three patients with coblation assisted UPPP and 334 patients with standard UPPP were treated respectively. Single factor statistic analysis, multiple factors Logistic regress statistic analysis and Wilcoxon test method for related risk factors were applied. Results A total of 42 patients(7. 0% ) experienced postoperative bleeding. Among them,24 patients with coblation assisted UPPP (9. 1% ) and 18 patients with UPPP(5.4% ) had postoperative hemorrhage. Significant difference was not found in the degree of hemorrhage (z = 0. 784, P > 0. 05 ) ,hemorrhage site( x2 = 1. 387, P > 0. 05 ) and postoperative hemorrhage rates ( x2 = 3.14, P > 0. 05 )between the two surgical techniques. Significant difference was found in the interval of hemorrhage after surgery between the two surgical techniques ( x2 = 9. 25, P < 0. 01 ). History of hypertension, smoking,hepatic dysfunction was found to be correlated with the postoperative hemorrhage (Odd-ratio were respectively 7. 326, 3. 674, 2. 707). Conclusion Coblation technique did not significantly increase UPPP postoperative hemorrhage.

Objective To introduce the clinical manifestations and compare the different radiological methods of detecting the malformation of cervical segment of internal carotid artery.Methods A retrospective analysis of 7 cases with malformation of cervical segment of internal carotid artery between May,2004 and April,2011 was conducted.CT angiography (CTA) and magnetic resonance angiography (MRA) were used to detect the morphology of cervical segment of internal carotid artery.Results This disease entity provided no obvious symptoms in five cases,and only such complaints as pharyngeal foreign body sensation in one and odynophagia in another were described.Physical examination showed a bulge with pulsation on pharyngeal wall in four cases,and apparent normal pharynges in other three patients,all of which were covered with intact pharyngeal mucosa.Twelve carotid arteries in seven cases were observed,five of which showed tortuosities and seven kinkings.All of the five patients with recorded radiological examination had identified malformations of internal carotid artery,two of which were tortuosities bilaterally and two kinkinks bilaterally and one tortuosity and kinking respectively.CTA and MRA revealed tortuosities of cervical segment of internal carotid arteries.Conclusions No typical clinical symptoms were found in the malformation of cervical segment of internal carotid artery.Pharyngeal bulge with pulsation could be encountered.CTA and MRA showed excellent ability to depict the malformation of cervical segment of internal carotid artery and its relationship with surrounding structures,which could protect carotid artery from unintended damage.

Objective To study the first locally identifcd A/HINI secondary cases outbreak in China. Methods Interview and field investigation were integrated to describe the whole process of transmission on each case and to illustrate the relationships between the onset of the disease and the retated factors. Results Two contact persons appearanced fever and whose throat swabs were tested positive to H1N1 viral nucleic acid. The two had a history of contact in a short distance with the initial imported case without any protective measure in the poor air ventilation. The patients clinical situation was slight. The incubation was between 37 hours and 57 hours. No other new case was found after intervention as isolation and antisepsis were taken. Conclusion This event was proved to be an outbreak of local A/H1N1 secondary cases caused by the imported case. The main mode of transmission was personal contact in a short distance without protection, through air and droplet. The locus with poor air ventilation was high risk place. Contact persons should be observed seven days and tested continuously.Infectivity and pathogenicity of the A/H1N1 virus were limited and appeared weakened by generations. Patient's condition was related with persistence and frequency of contact with the infection sources. Enhancing management of contact persons, health education, early diagnose, early treatment and early insulation were effective measures of controling and prenventing the spread A/H1N1.

Objective: To investigate the clinical efficacy and safety of anti-IgE monoclonal antibody (omazumab) in the treatment of allergic united airway disease (UAD) in the real-wold. Methods: Retrospective cohort study summarizes the case data of patients with allergic united airway disease who were treated with anti IgE monoclonal antibody (omalizumab) for more than 16 weeks from March 1, 2018 to June 30, 2022 in the Peking University First Hospital.The allergic UAD is defined as allergic asthma combined with allergic rhinitis (AA+AR) or allergic asthma combined with chronic sinusitis with nasal polyps (AA+CRSwNP) or allergic asthma combined with allergic rhinitis and nasal polyps (AA+AR+CRSwNP). The control of asthma was evaluated by asthma control test (ACT), lung function test and fractional exhaled nitric oxide (FeNO). The AR was assessed by total nasal symptom score (TNSS). The CRSwNP was evaluated by nasal visual analogue scale (n-VAS), sino-nasal outcome test-22 (SNOT-22), nasal polyps score (TPS) and Lund-Mackay sinus CT grading system. The global evaluation of omalizumab for the treatment of allergic UADwas performed by Global Evaluation of Treatment Effectiveness(GETE).The drug-related side effects were also recorded. Matched t test and Wilcoxon signed-rank test were used to compare the score changes of IgE monoclonal antibody (omazumab) before and after treatment, and multivariate logistic regression analysis was used to determine the influencing factors of IgE monoclonal antibody (omazumab) response. Results: A total of 117 patients with UAD were enrolled, ranging in age from 19 to 77 years; The median age of patients was 48.7 years; Among them, 60 were male, ranging from 19 to 77 years old, with a median age of 49.9 years; There were 57 females, ranging from 19 to 68 years old, with a median age of 47.2 years. There were 32 cases in AA+AR subgroup, 59 cases in AA+CRSwNP subgroup, and 26 cases in AA+AR+CRSwNP subgroup. The total serum IgE level was 190.5 (103.8,391.3) IU/ml. The treatment course of anti IgE monoclonal antibody was 24 (16, 32) weeks. Compared with pre-treatment, omalizumab increased ACT from 20.0 (19.5,22.0) to 24.0 (23.0,25.0) (Z=-8.537, P<0.001), increased pre-bronchodilator FEV1 from 90.2 (74.8,103.0)% predicted value to 95.4 (83.2,106.0)% predicted value (Z=-5.315,P<0.001), increased FEV1/FVC from 80.20 (66.83,88.38)% to 82.72 (71.26,92.25)% (Z=-4.483,P<0.001), decreased FeNO from(49.1±24.8) ppb to (32.8±24.4) ppb (t=5.235, P<0.001), decreased TNSS from (6.5±2.6)to (2.4±1.9) (t=14.171, P<0.001), decreased n-VAS from (6.8±1.2) to (3.4±2.0)(t=14.448, P<0.001), decreased SNOT-22 from (40.0±7.9) to (21.3±10.2)(t=15.360, P<0.001), decreased TPS from (4.1±0.8) to (2.4±1.0)(t=14.718, P<0.001) and decreased Lund-Mackay CT score from (6.0±1.3) to (3.1±1.6)(t=17.012, P<0.001). The global response rate to omalizumab was 67.5%(79/117). The response rate in AA+AR (90.6%,29/32) was significantly higher than that in AA+CRSwNP (61.0%,36/59) and AA+AR+CRSwNP (53.8%,14/26) subgroups (χ²=11.144,P=0.004). Only 4 patients (3.4%,4/117) had mild side effects. Conclusion: The real-world study showed favorable effectiveness and safety of anti-IgE monoclonal antibody for treatment of allergic UAD. To provide basis for preventing the progress and precise treatment of allergic UAD.
Female ; Humans ; Male ; Middle Aged ; Young Adult ; Adult ; Aged ; Nasal Polyps/drug therapy* ; Omalizumab/therapeutic use* ; Rhinitis/drug therapy* ; Retrospective Studies ; Asthma/diagnosis* ; Rhinitis, Allergic/drug therapy* ; Sinusitis/drug therapy* ; Antibodies, Monoclonal/therapeutic use* ; Chronic Disease

BACKGROUND: Treatment of coronary bifurcation lesions remains challenging; a simple strategy has been preferred as of late, but the disadvantage is ostium stenosis or even occlusion of the side branch (SB). Only a few single-center studies investigating the combination of a drug-eluting stent in the main branch followed by a drug-eluting balloon in the SB have been reported. This prospective, multicenter, randomized study aimed to investigate the safety and efficacy of a paclitaxel-eluting balloon (PEB) compared with regular balloon angioplasty (BA) in the treatment of non-left main coronary artery bifurcation lesions. METHODS: Between December 2014 and November 2015, a total of 222 consecutive patients with bifurcation lesions were enrolled in this study at ten Chinese centers. Patients were randomly allocated at a 1:1 ratio to a PEB group (n = 113) and a BA group (n = 109). The primary efficacy endpoint was angiographic target lesion stenosis at 9 months. Secondary efficacy and safety endpoints included target lesion revascularization, target vessel revascularization, target lesion failure, major adverse cardiac and cerebral events (MACCEs), all-cause death, cardiac death, non-fatal myocardial infarction, and thrombosis in target lesions. The main analyses performed in this clinical trial included case shedding analysis, base-value equilibrium analysis, effectiveness analysis, and safety analysis. SAS version 9.4 was used for the statistical analyses. RESULTS: At the 9-month angiographic follow-up, the difference in the primary efficacy endpoint of target lesion stenosis between the PEB (28.7% ± 18.7%) and BA groups (40.0% ± 19.0%) was -11.3% (95% confidence interval: -16.3% to -6.3%, Psuperiority <0.0001) in the intention-to-treat analysis, and similar results were recorded in the per-protocol analysis, demonstrating the superiority of PEB to BA. Late lumen loss was significantly lower in the PEB group than in the BA group (-0.06 ± 0.32 vs. 0.18 ± 0.34 mm, P < 0.0001). For intention-to-treat, there were no significant differences between PEB and BA in the 9-month percentages of MACCEs (0.9% vs. 3.7%, P = 0.16) or non-fatal myocardial infarctions (0 vs. 0.9%, P = 0.49). There were no clinical events of target lesion revascularization, target vessel revascularization, target lesion failure, all-cause death, cardiac death or target lesion thrombosis in either group. CONCLUSIONS: In de novo non-left main coronary artery bifurcations treated with provisional T stenting, SB dilation with the PEB group demonstrated better angiographic results than treatment with regular BA at the 9-month follow-up in terms of reduced target lesion stenosis. TRIAL REGISTRATION ClinicalTrials.gov, NCT02325817; https://clinicaltrials.gov.