Flumazenil-Insensitive Benzodiazepine Effects in Recombinant αß and Neuronal GABAA Receptors.

Gamma-aminobutyric acid, type A (GABAA) receptors are complex heterogeneous pentamers with various drug binding sites. Several lines of evidence suggest that benzodiazepines modulate certain GABAA receptors in a flumazenil-insensitive manner, possibly via binding sites other than the classical ones. However, GABAA receptor subtypes that contain non-classical benzodiazepine binding sites are not systemically studied. The present study investigated the high-concentration effects of three benzodiazepines and their sensitivity to flumazenil on different recombinant (α1ß2, α2ß2, α3ß2, α4ß2, α5ß2 and α1ß3) and native neuronal GABAA receptors using the whole-cell patch-clamp electrophysiology technique. The classical benzodiazepine diazepam (200 µmol/L) and midazolam (200 µmol/L) produced flumazenil-insensitive effects on α1ß2 receptor, whereas the imidazopyridine zolpidem failed to modulate the receptor. Flumazenil-insensitive effects of diazepam were also observed on the α2ß2, α3ß2 and α5ß2, but not α4ß2 receptors. Unlike ß2-containing receptors, the α1ß3 receptor was insensitive to diazepam. Moreover, the diazepam (200 µmol/L) effects on some cortical neurons could not be fully antagonized by flumazenil (200 µmol/L). These findings suggested that the non-classical (flumazenil-insensitive) benzodiazepine effects depended on certain receptor subtypes and benzodiazepine structures and may be important for designing of subtype- or binding site- specific drugs.

[Clinical application of stand-alone MC+PEEK cage in the anterior cervical fusion].

OBJECTIVE: To explore the effect of clinical application of stand-alone MC+PEEK cage in anterior cervical fusion. METHODS: From January 2011 to January 2014,50 patients were treated with the MC+PEEK cage filled with autogenous cancellous illic-bone graft after anterior cervical discectomy. There were 22 patients with cervical spondylosis,26 patients with traumatic cervical disc herniation, 2 patients with cervical instability in these patients. There were 32 males and 18 females, aged from 30 to 79 years old with an average of 53.30 years old. There were 32 patients with single segment, 15 patients with double segments and 3 patients with three segments. Cervical AP and lateral and the flexion-extension X-rays were regularly taken in order to assess the cervical physiological curvature, the graft fusion and internal fixation related complications. Nerve function, clinical effect and bone fusion were respectively evaluated according to Japan Orthopedic Association (JOA), Otani grade and Suk method. RESULTS: All patients were followed up from 6 to 36 months with an average of 20 months. No correlated surgical complications were found and all patients obtained bony fusion with an average time of 4.30 months. JOA score had significantly improvement after surgery (P < 0.05). The JOA score was 10.60 ± 3.00 before surgery and 16.10 ± 2.20, 16.40 ± 2.35 at one week and six months after surgery respectively. According to Otani grade,40 cases got excellent results, 9 good, 1 fair. No significant dysphagia and internal fixation related complications such as displacement of cages were found during the follow-up period. CONCLUSION: Using this cage in anterior cervical fusion can obtain satisfactory clinical effect with less operation injury and reduce the complications. It is a better fusion method in anterior cervical fusion.

Distant metastasis risk and patterns of nasopharyngeal carcinoma in the era of IMRT: long-term results and benefits of chemotherapy.

PURPOSE: To report the distant metastasis (DM) risk and patterns for nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT) and to analyze the benefits of chemotherapy based on DM risk. MATERIALS AND METHODS: 576 NPC patients were analyzed. The DM rates were calculated using the Kaplan-Meier method, and the log-rank test was used to compare differences. The patients were divided into different risk subclassifications according to DM hazard ratios. RESULTS: 91 patients developed DM after treatment, with bone as the most common metastatic sites. 82.4% of DMs occurred within 3 years of treatment. Patients were classified as low-risk, intermediate-risk and high-risk, and the corresponding 5-year DM rates were 5.1%, 13.1% and 32.4%, respectively (P < 0.001). Chemotherapy failed to decrease the DM rate in the low-risk subclassification, but decreased the DM risk in the intermediate-risk subclassification (P = 0.025). In the high-risk subclassification, the DM rate was 31.9% though chemotherapy was used, which was significantly higher than that of other two subclassifications. CONCLUSIONS: DM is the dominant treatment failure in NPC treated by IMRT, with similar occurrence times and distributions to those that occurred in the era of conventional radiotherapy. Further studies on treatment optimization are needed in high-risk patients.

Risk factors and prediction-score model for distant metastasis in nasopharyngeal carcinoma treated with intensity-modulated radiotherapy.

The objective of this study is to identify the risk factors and construct a prediction-score model for distant metastasis (DM) in nasopharyngeal carcinoma (NPC) patients treated with intensity-modulated radiotherapy (IMRT). A total of 520 nonmetastatic NPC patients were analysed retrospectively. The independent risk factors for DM were tested by multivariate Cox regression analysis. The prediction-score model was established according to the regression coefficient. The median follow-up was 88.4 months. The 5-year DM rate was 15.1%. N2-3, primary tumour volume of nasopharynx (GTVnx) >24.56 cm(3), haemoglobin change after treatment (ΔHGB) >25.8 g/L, albumin-globulin ratio (AGR) ≤1.34, pretreatment neutrophil-lymphocyte ratio (NLR) >2.81 and pretreatment serum lactate dehydrogenase (LDH) >245 U/L were significantly adverse independent predictive factors for DM. Three subgroups were defined based on the prediction-score model: low risk (0-2), intermediate risk (3-4) and high risk (5-8). The 5-year DM rates were 4.6, 21.8 and 50.8%, respectively (P < 0.001). The areas under the curve for DM in the prediction-score model and the UICC/AJCC staging system seventh edition were 0.748 and 0.627, respectively (P < 0.001). The scoring model is useful in evaluating the risk of DM in IMRT-treated NPC patients and guiding future therapeutic trials. Further prospective study is needed.

[Effects of low temperature exposure on dermal microvascular endothelial cells function].

OBJECTIVE: To explore the damage effects and expression of vascular endothelial growth factor (VEGF) exposed with different low-temperatures on rat dermal microvascular endothelial cells (DMVECs). METHODS: Primary DMVECs were obtained by discontinuous Percoll gradient centrifugation. The DMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen. Applied 28 degrees C, 12 degrees C and 0 degrees C to interfere with rat DMVECs as cold-exposure model. The changes of cells morphology were observed under invert microscope. The membrane integrity was determined by lactate dehydrogenase (LDH) activity. RT-PCR was used to examine the expression of vascular endothelial growth factor mRNA in cells. RESULTS: The monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were DMVECs. After 24 h cold exposure, the cell morphology of 0 degrees C group was shrunken, the other groups were "Fibroblast-like". The LDH activity (U/L) in the medium of 28 degrees C, 12 degrees C and 0 degrees C groups was 54.17 +/- 3.02, 64.66 +/- 3.03, 82.13 +/- 10.91 respectively, which was significantly higher than that of 37 degrees C group (12.23 +/- 3.0, P < 0.01). The VEGF mRNA expression level was up-regulated in 28 degrees C group and 12 degrees C group versus control group (P < 0.05), but unchanged in 0 degrees C group. CONCLUSION: The rat DMVECs injury severity are deteriorated with temperature decreasing, and VEGF might be involved in the regulation of membrane permeability in this period.

[The damage effects of hypothermia combined with hypoxia on rat pulmonary microvascular endothelial cells].

OBJECTIVE: To study the synergistic effects of hypothermia and hypoxia on the damage of pulmonary microvascular endothelial cells (PMVEC) in rat. METHODS: Primary PMVECs were obtained by complex phosphoesterasum digesting from isolated lung tissues of Wistar rats, the PMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen and bandeiraea simplicifolia isolectin (BSI) binding test. Factorial design was adopted in trial according to hypothermia and hypoxia existing or not. Using corresponding kit measured the levels of lactate dehydrogenase (LDH) activity in cell medium. Level of nitric oxide (NO) concentration was measured by Griess Assay. RT-PCR was used to examine the expression of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in PMVECs. RESULTS: The monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were PMVECs. Compared to the control group, LDH activity and VEGF, ET-1 expression levels were significantly increased in hypothermia group, hypoxia group and hypoxia combined with hypothermia group. And the levels of NO concentration were reduced in these three groups. The results of One-way ANOVA showed that there was a synergistic effect between hypothermia and hypoxia. CONCLUSION: Hypothermia and hypoxia both have an effect on PMVECs whether in altering the cell permeability or in releasing of vasoactive substances including NO and ET-1. In addition, there is a synergistic effect between hypothermia and hypoxia.

[Evaluation of cobas 4800 high-risk HPV test as a tool in cervical cancer screening and cytology triage].

OBJECTIVE: To evaluate the feasibility and reliability of cobas 4800 HPV test for cervical cancer screening and cytology referral. METHODS: cobas 4800 HPV test and hybrid capture 2 (HC-2) were used to detect high risk HPV DNA in 670 specimens of liquid-based cytology collected from three hospitals. The agreement between cobas and HC-2 tests was assessed. HPV PCR detection (HybriBio) and gene sequencing were used for genotyping, and the agreement of HPV16 and 18 genotyped by cobas and HybriBio was evaluated. Histological diagnosis was considered as a gold standard to estimate the sensitivity and specificity of cobas vs. HC-2 in detecting CIN2(+) in cervical lesions. RESULTS: The crude agreement between cobas and HC-2 tests was 89.40%, the Kappa value was 0.778, the positive concordance rate was 86.42%, and the negative concordance rate was 91.36%. The crude agreement rates between cobas and HybriBio on HPV16 and 18 were 88.89% and 94.94%, the Kappa values were 0.777 and 0.753, the positive concordance rates were 98.91% and 100.00%, and the negative concordance rates were 78.41% and 94.44%, respectively. HPV PCR detection (HybriBio) and gene sequencing were considered as adjusted standard: the high risk HPV positive concordance rate was 100%, negative coincidence rate was 94.42%, HPV16 and 18 positive concordance rates were both 100%, and negative concordance rates were 82.35% and 94.44%, respectively. Regarding the detection of CIN2(+), the sensitivity and specificity were 91.07% and 70.97% for cobas, and 93.75% and 71.33% for HC-2, with a non-significant difference between the results of the two tests (P > 0.05). CONCLUSIONS: cobas4800 HPV test has good screening sensitivity and specificity in correct detection of HPV16 and 18 and other high-risk HPV virus types.

[Protective effects of SUR2B/Kir6.1 potassium channel opener natakalim against RAVECs injuries induced by hypoxia].

OBJECTIVE: To investigate the protective effects of natakalim against rat aortic vascular endothelial cells (RAVECs) injuries induced by hypoxia and its mechanisms. METHODS: Selecting RAVECs as a cell model injured by hypoxia, these RAVECs were divided into 5 groups: i.e. control group, hypoxia group, natakalim low, medium and high group. The cell survival rate was determined by MTT assay, con was measured using Griess Assay, RT-PCR was used to examine t he expression of intercellular adhesion molecule-1 (ICAM-1), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in RAVEC. RESULTS: Natakalim could reverse hypoxia-induced changes in endothelial cell function, including increased endothelial cell survival rate and level of NO concentration, significantly inhibited the hypoxia-induced endothelial ICAM-1, ET-1, VEGF mRNA expression levels increased. CONCLUSION: Natakalim have protective effects on hypoxia-induced changes in endothelial cell function, increasing of permeation, excess expression of cell adhesion molecules.

[Clinical application of MC + PEEK cage in traumatic cervical disc herniation].

OBJECTIVE: To explore the clinical outcomes of surgical treatment for traumatic cervical disc herniation with MC + PEEK cage. METHODS: A total of 51 patients with traumatic cervical disc herniation in mono-segment were surgically treated. The patients in group A (n = 20) were treated by MC + PEEK cage while those in group B (n = 31) by anterior cervical plate with PEEK cage or titanium mesh. Various parameters of operative duration, blood loss volume, operative complications, bone union, height of intervertebral space and Japanese Orthopedic Association (JOA) score were recorded and compared. RESULTS: Fifty-one patients were followed up for an average time of 26 months (range: 6 - 40). Operative duration, blood loss volume and operative complications of group A were better than group B with statistical significance (P < 0.05). Bone union, height of intervertebral space and recovery of spinal cord function were satisfactory with no statistical difference (P > 0.05). CONCLUSION: With this new cage, traumatic cervical disc herniation may be safely and micro-invasively treated without the need of anterior cervical plate.