Objective:To introduce a programmed death-1 (PD-1) inhibitor nivolumab used as a new antitumor agent. Methods:According to the literatures, the action mechanism of nivolumab and the clinical trial results on the main indications approved or being investigated in phase III trials were reviewed and evaluated. Results:Nivolumab could restore the antitumor activity of T cells through binding with PD-1 and consequently blocking its interaction with the key ligands of PD-L1 and PD-L2. Several completed and ongoing clinical trials showed that nivolumab used alone or combined with chemotherapy or CTLA-4 inhibitor ipilimumab exhibited better effica-cy when compared with current clinical used chemotherapy drugs in the treatment of melanoma, non-small cell lung cancer and renal cell carcinoma. Nivolumab was well tolerated during the treatment with such main grade 3-4 adverse events as immune-mediated pneu-monia, abnormal liver functions and fatigue. Conclusion:Through its anti-tumor immune response, nivolumab can improve the clinical efficacy in the treatment of various tumors including melanoma, non-small cell lung cancer and renal cell carcinoma.

BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

Objective To explore the effects of tumor necrosis factor-α induced protein 6 (TSG-6) on acute kidney injury (AKI) following paraquat poisoning in rats.Methods Twenty-four male Sprague-Dawley (SD) rats were randomly divided into sham group (n=8),model group (n=8) and TSG-6-treated group (n=8) using a randomized number table.Rats were given an injection of 50 mg/kg of paraquat intraperitoneally (total volume was equalled to sterile normal saline) in model and TSG-6-treated groups.Rats in sham group were given 2 mg/kg of sterile saline.Mter 1 hour of paraquat administration,rats were treated with 30 μg of recombinant human TSG-6 intraperitoneally in TSG-6-treated group.After 6 hours of paraquat administration,serum was collected to assess renal function,then rats were sacrificed and renal tissues were immediately harvested.AKI score was evaluated by renal histopathology and gene expression of pro-inflammatory cytokines including interleukins (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) in kidney was assayed with real-time reverse transcription-polymerase chain reaction (RT-PCR).Results Compared with sham group,blood urea nitrogen (BUN),creatinine (Cr) and AKI score were significandy increased in model group [BUN (mmoUL):22.64 ±2.36 vs.7.09 ±0.65,t=6.986,P=0.000; Cr (μmol/L):177.28 ± 18.67 vs.60.32 ± 3.11,t=7.134,P=0.000; AKI score:9.14 ± 0.28 vs.0.30 ± 0.23,t=9.013,P=0.000].Moreover,the mRNA expressions of IL-1β,IL-6 and TNF-α were significantly elevated in model group (IL-1β mRNA:3.23 ± 0.28 vs.1.00 ±0.07,t=5.874,P=0.000; IL-6 mRNA:4.16 ±0.37 vs.1.00 ±0.08,t=7.125,P=0.000; TNF-α mRNA:3.85 ±0.31 vs.1.00 ±0.10,t=6.342,P=0.000).However,serum BUN,Cr,AKI score and the mRNA expressions of IL-1β,IL-6 and TNF-α in TSG-6-treated group were significantly lower than those in model group [BUN (mmol/L):14.07 ± 5.23 vs.22.64 ± 2.36,t=2.533,P=0.026; Cr (μmol/L):112.76 ± 14.81 vs.177.28 ± 18.67,t=2.778,P=0.016; AKI score:5.35 ±0.19 vs.9.14 ±0.28,t=2.885,P=0.013; IL-1β mRNA:2.26 ± 0.19 vs.3.23 ±0.28,t=2.457,P=0.023; IL-6 mRNA:2.92 ±0.29 vs.4.16 ±0.37,t=2.975,P=0.011; TNF-α mRNA:2.58 ± 0.23 vs.3.85 ± 0.31,t=2.564,P=0.019].Conclusion TSG-6 attenuates AKI following paraquat poisoning by suppressing inflammatory response.

Objective:To explore the effects of necroptosis specific inhibitor-1 (Nec-1) on brain injury in rats after cardiac arrest and its mechanism.Methods:A total of 24 Sprague-Dawley (SD) rats were divided into Sham group, model group and Nec-1 group ( n = 8 per group) according to random number table method. In the Sham group, only general surgical procedures were underdone without inducing cardiac arrest. In the model group, the rats were subjected to asphyxial cardiac arrest followed by cardiopulmonary resuscitation (CPR) at 6 minutes after cardiac arrest. In the Nec-1 group, Nec-1 of 1 mg/kg was administered after cardiac arrest, and CPR was performed at 6 minutes after cardiac arrest. At 72 hours after CPR, neurological deficit scores (NDS) were assessed, serum S100B levels were measured by enzyme linked immunosorbent assay (ELISA), receptor-interacting protein 3 (RIP3) expression in cerebral cortex and hippocampus was observed under immunofluorescence and positive rate was calculated, and the levels of RIP3 protein expression in brain were analyzed by Western blotting. Results:At 72 hours after CPR, the rats in the model group showed obvious necroptosis and injury in brain. Compared with the Sham group, the NDS scores in the model group were significantly decreased [57.0 (52.7, 60.0) vs. 80.0 (80.0, 80.0), P < 0.05], the serum S100B was significantly increased (ng/L: 44.9±4.5 vs. 18.6±1.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly elevated [cerebral cortex: (31.7±4.8)% vs. (11.6±3.2)%, hippocampus: (28.4±0.8)% vs. (10.9±0.6)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly increased [RIP3 protein (RIP3/GAPDH): 0.708 (0.642, 0.722) vs. 0.408 (0.253, 0.504), P < 0.05]. After Nec-1 intervention, necroptosis and injury in brain were obviously improved. Compared with the model group, the NDS scores at 72 hours after CPR in the Nec-1 group were significantly increased [70.5 (68.5, 71.7) vs. 57.0 (52.7, 60.0), P < 0.05), the serum S100B was significantly decreased (ng/L: 31.9±2.7 vs. 44.9±4.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly lowered [cerebral cortex: (23.7±4.1)% vs. (31.7±4.8)%, hippocampus: (20.4±0.4)% vs. (28.4±0.8)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly declined [RIP3 protein (RIP3/GAPDH): 0.437 (0.379, 0.507) vs. 0.708 (0.642, 0.722), P < 0.05]. Conclusion:Nec-1 attenuated necroptosis of brain cells by inhibiting the expression of RIP3 protein, so as to reduce brain injury after cardiac arrest in rats.

Objective To investigate the mechanism that bFGF promotes the regeneration of injured optic nerve and induces dedifferentiation of glial cells in it. Methods Fifty-five adult male SD rats were randomly divided into 3 groups as normal control group, injury group and bFGF group. At day 7 post operation, optic nerves from injury group and bFGF group were detected by gene chip and real-time PCR. At day 7, 14 post operation, optic nerves were harvested and detected by HE staining and immunohistochemistry. Results Compared with the injury group, there were 645 genes expression up-regulated and 458 genes down-regulated including genes related neural stem cell or precursor cell neural development, proliferation, apoptosis, chromatin configuration, transcription regulation, signal transduction, neural growth and so on in the bFGF group. There were bigger nuclei, more cells, more immunoreactivity of nestin, extracellular signal-regulated kinase(Erk1/2), glial fibrillary acidic protein(GFAP), and myelin basic protein(MBP) in the distal optic nerves and more immunoreactivity of neurofilament(NF) in the proximal optic nerves in the bFGF group than that in the injury group.Conclusion bFGF could promote the proliferation of neuroglia cells, dedifferentiation of neural glias and improve the microenvironment to favour the regeneration of injured optic nerve.

Objective To analyze the clinical and pathological features of collecting duct carcinoma (CDC) of the kidney. Methods 11 patients with CDC were analyzed,among which 6 were males and 5 were females.Their age ranged from 22 to 67 years old with a mean age of 55.4 cases were found by routine health examination,4 cases were presented with gross hematuria and 3 cases had flank or abdomen pain.The CT scan showed an unclear boundary mass in kidney,with tumor sizes from 2.1 to 8.5 cm ( mean 5.6 cm).Only medullary involvement was present on CT in 3 cases,Medullary and cortical involvement in 5 cases,Medullary and pelvic involvement in 2 cases,and all involvement in 1 case.Infiltrative lesion has expanded kidney but maintains reniform contour.Contrast-enhanced CT scan showed lesion mild to moderate enhancement compared with surrounding parenchyma. Results Radical nephrectomy was performed in 8 cases and palliative nephrectomy in 3 cases.All patients were finally diagnosed by pathology.Grossly,the tumor is often appears gray or white.In HE staining,tumor showed prominent tubular or tubulaopapillary structures with desmoplasia and inflammatory reaction.Occasionally,some tumor cells take on a hobnail appearance.Immunohistochemical examination showed UEA-1 positive in all cases,EMA positive in 9 cases,PNA positive in 8 cases,and HMW-CK positive in 7 cases.Only 2 patients showed positive CD10.7 patients died within 6 to 47 months (mean 12.5 months),2 survived with tumor free for 9 months and 8 years respectively,one lost of follow-up,and one patient with distant metastasis is receiving postoperative chemotherapy. Conclusions Collecting duct carcinoma of the kidney is a rare histological type of renal cell carcinoma,whose final diagnosis depends on histopathological examination.Rapid progression,highly malignant,poor prognosis are the characteristics of this disease.

Objective To study the X-ray findings of post-traumatic osteolysis and to improve the knowledge of that disease.Methods X-ray features of 7 cases of post-traumatic osteolysis confirmed by clinical findings and pathology were reviewed retrospectively.There were 5 females and 2 males.from 9-56 years(mean 34 years)of age.Three patients had traffic accidents and 4 had trauma unrelated to traffic accidents.Osteolysis occurred from 3 to 18 months after trauma(1 case at 18 months,2 cases at 10 months,2 cases at 6 months and 2 cases at 3 months).Results There were 2 pubis fractures,1 distal tibiofibular shaft fracture,1 femoral neck fracture,1 humeral upper end commiuuted fracture,1 shoulder joint dislocation.and 1 soft tissue swelling around the wrist.The X-ray findings are:3 massive osteolysis,3 plaque flake osteolysis and 1 cystic osteolysis.There were no hardening of bony edge at the site of osteolysis in all 7 cases,clear margin in 5 cases and ill-defined margin in 2 cases,no residual bone in osteolytic area in 4 cases and residual bone in octeolytic area in 3 cases,no periosteal reaction and thickerning of bony cortex in osteolytic area in all 7 Cascs,bone repair in 2 cases and no bone repair in 5 cases.Histopathological findings showed:extensive capillary hyperplasia and fibrous tissue hyperplasia;hyperemia and swelling of synvium,proliferation of granulation tissue,osteonecrosis,increased osteoclast activity,some inflammatory cells,no evidence of neoplastic cells in the involved area.Conclusions Posttraumatic osteolysis is closely related to trauma.X-ray findings include massive osteolysis,plaque-like osteolysis,and irregular cystic changes.Early dignosis may be a challenging task.

Objective To investigate the dedifferentiation of neuroglial cells and its induction after optic nerve injury. Methods Adult male SD rats were randomly divided into 4 groups the normal control group,the injury group,the transplantation group and the microcrush and transplantation group.Optic nerves were harvested at days 3,7,14 and 28 after the operation.HE staining was used to count the number of neuroglial cells.Immunohistochemistry,Western blotting and in situ hybridization histochemistry were employed together with computerized image analysis to evaluate the expressions of Nestin,GFAP,MBP,NF,BDNF,Nogo-A and Nogo-A mRNA.Immunofluorescence double staining was used to detect the co-expression of Nestin and GFAP or Nestin and MBP. Results The number of cells only increased at day 7 after the nerve injury, the expressions of Nestin,MBP,Nogo-A and Nogo-A mRNA were up-regulated,the expressions of GFAP,NF and BDNF were down-regulated,and some Nestin-GFAP positive cells and a few of Nestin-MBP positive cells were detected in the injury group.Compared with the injury group,the number of cells was increased sometime after the nerve injury;the expressions of Nestin,GFAP,BDNF and NF were up-regulated,the expressions of MBP,Nogo-A and Nogo-A mRNA were down-regulated,and the number of Nestin-GFAP positive cells increased in the transplantation group and the microcrush and transplantation group.Conclusion After optic nerve injury,some astrocytes undergo dedifferentiation while the macroglial cells display a gene expression pattern that is unfavorable for nerve regeneration.Pre-degenerated peripheral nerves could enhance the dedifferentiation of astrocytes and induce the gene expression pattern of macroglial cells that is favorable for nerve regeneration.

Objective To summarize the clinical experie nce in the diagnosis and treatment of cystolithiasis complicated by squamous cel l carcinoma (SCC) of the bladder. Methods Retrospective analysis was performed in 28 patients (19 men and 9 women) with cystolithiasis c omplicated by SCC of the bladder.Their age ranged from 26 to 68 years with a mea n of 33 years.The disease course of cystolithiasis ranged from 2 to 18 years wit h a mean of 4.5 years. Among the 24 cases who underwent urine cytology,atypical cells were found in 19 cases.KUB showed solitary stone of the bladder in 5 case and multiple stones in 23.Space-occupying lesions were noted in 11 cases on IVU ,in 17 cases on B-ultrasonography and CT scan.Cystoscopy was performed in 28 ca ses, and tumors were found in 21 cases.The size of the tumors ranged from 1.2 cm ?1.5 cm to 2.2 cm?5.0 cm. Partial cystectomy was performed in 21 cases, includ ing ureterocysto-transplantation in 6 cases.Radical cystectomy was performed in 7 cases. Results Pathology revealed SCC of the bladder in all 28 cases,including G_1 in 11 cases,G_2 in 12 cases,G_3 in 5 cases;T_1 in 11 cases,T_2 in 12 cases,T_3 in 5 cases. Twenty-two patients were followe d up for 1 to 8 years with a mean of 3 years. The 1- and 5-year survival rates were 63.6% (14/22) and 16.7% (3/18),respectively. Conclusions Cystolithiasis is the main cause of SCC of the bladder. Early diagnosis a nd surgical treatment is very important for such patients.

To study the role of vein tubulation in the facial nerve regeneration. Methods:Twelve male adult SD rats were divided randomly into vein tubulation group,nerve graft group and normalcontrol group- The facial nerve trunks were cut and grafted' Three months p0stoperation, the distributionand variation of the fascicles were observed under light microscope. Fascicular area,axon number and axondensity were gauged and caculated. Results:Within the vein chamber, the regenerating nerve consisted ofmany fascicles. In the nerve graft, there was a major fascicle surrounded by many small fascicles. Thenormal facial nerve trunk had only one fascicle. Among 3 groups, the fascicular area and axon number ofthe nerve graft were largest, but the axon density had no significant difference. Conclusion: The micr0en-vironment in vein chamber may be beneficial to growth of regenerating axons.