Biomechanical assessment of unilateral pedicle screws plus contralateral transfacetopedicular screws after transforaminal lumbar interbody fusion with two cages.

OBJECTIVE: To assess the biomechanical stability of unilateral pedicle screws (UPS) plus contralateral transfacetopedicular screws (TFPS) after transforaminal lumbar interbody fusion (TLIF) with two cages. METHODS: Range of motion (ROM) testing was performed in 28 fresh-frozen human cadaveric lumbar spine motion segments. The sequential test configurations included supplemental constructs after TLIF such as UPS, UPS plus contralateral TFPS and bilateral pedicle screws (BPS). All test specimens were fixated in the normal lordotic lignment, then mounted in a three-dimensional (3-D) motion testing machine and fixed to the load frame of a six degrees of freedom spine simulator. Each of the test constructs were subjected to three load-unload cycles in each of the physiologic planes generating flexion-extension, right-left lateral bending and right-left axial rotation load-displacement curves. Statistical analysis was performed on the ROM data. Comparison of data was performed by repeated-measures analysis of variance for independent samples followed by Bonferroni analysis for multiple comparison procedures. RESULTS: The ROMs for UPS, BPS and UPS plus TFPS fixation after TLIF were significantly smaller than those of the intact spine in all modes. The ROM for UPS plus TFPS fixation was between the largest for UPS and the smallest for BPS. The differences between ROMs of UPS and UPS plus TFPS were significant for both lateral bending and rotation. There were no significant differences between BPS and UPS plus TFPS in any mode. CONCLUSION: Because the UPS construct provides the least stability, especially during lateral bending and rotation, it should be used prudently. After TLIF with two cages, UPS plus TFPS provides stability comparable to that of TLIF with BPS. It is thus an acceptable option in minimally invasive surgery.

Interferonalpha enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent pathway.

Interferonalpha (IFNalpha) induces cell cycle arrest and triggers apoptosis and chemosensitivity. But the mechanism of IFNalpha in regulating chemosensitivity has not been fully understood. To study whether IFNalpha affected chemosensitivity of osteosarcoma cells, we treated p53-wild U2OS cells and p53-mutant MG63 cells with IFNalpha and etoposide, alone or in combination, and then examined growth inhibition, cell cycle arrest and apoptosis. IFNalpha enhanced etoposide-induced growth inhibition and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells in a dose- and time-dependent manner. Etoposide-induced G2/M phase arrest was also enhanced by IFNalpha. The enhanced apoptosis was associated with the accumulation of transcriptionally active p53 accompanied with increased Bax and Mdm2, as well as decreased Bcl-2. IFNalpha also activated caspases-3, -8 and -9 protein kinases and PARP cleavage in response to etoposide in U2OS cells. Moreover, the combination-induced cytotoxicity and PARP cleavage were significantly reduced by caspase pan inhibitor and p53 siRNA. Thus we conclude that IFNalpha enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent and caspase-activation pathway. The proper combination of IFNalpha and conventional chemotherapeutic agents may be a rational strategy for the treatment of human osteosarcoma with functional p53.

[Construction and identification of human bone morphogenetic protein-7 recombinant adeno-associated virus type 2 vector and its expression in bone mesenchymal stem cells].

OBJECTIVES: To facilitate gene therapy research using recombinant adeno-associated virus type 2 (rAAV2) vector as gene transfer vehicle, and to construct a rAAV2 based vector carrying bone morphogenetic protein-7 (BMP7) and observe its expression in bone mesenchymal stem cells. METHODS: The coding sequence (1.3 kb) of BMP7 was amplified by polymerase chain reaction (PCR) from the pcDNA1.1(+) plasmid containing the human BMP-7 cDNA. After purified, the gene fragment was cloned into a plasmid pUC18 and termed plasmid pUC18-hBMP7. The recombinant pUC18-hBMP7 was digested by Kpn I and Sal I and further ligated to the pSNAV by T4DNA ligase. The resultant plasmid PSNAV-hBMP7 was transformed into DH5a Escherichia coli, and positive colonies were screened by PCR and digest with restriction enzyme to identify the correct recombinant clones. BHK-21 cells were transfected with the purified pSNAV-BMP7 plasmid according to a standard calcium phosphate precipitation method. The cells were then cultured in selection media containing 800 micro g/ml G418 (Gibco/BRL). G418-resistant BHK-21 cell clones were isolated and the integrity of hBMP7 gene was determined by PCR using the above PCR primers. To package the virus, stably transfected BHK-21 cells were subsequently infected with recombinant herpes simplex virus type 1 (rHSV-1). The collected cells were processed by chloroform treatment, PEG8000/NaCl precipitation and chloroform extraction for purification. The titer was determined using quantitative DNA dot blots and the purity was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Following infection with rAAV2-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, MSCs were assessed qualitatively for BMP7 production. RESULTS: Transient transfection showed an efficiency of 98.8% in MSCs. RT-PCR showed that MSCs had transcription of BMP7 that was enhanced by the gene transfer. BMP-7 expression in MSCs was identified by Western-blot. CONCLUSIONS: The hBMP7 recombinant adeno-associated virus vector is successfully constructed. The present in vitro study demonstrates that rAAV2-BMP7 could infect MSCs.

Interferon-alpha enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis.

AIM: To determine whether interferon-alpha(IFNalpha) can enhance doxorubicin sensitivity in osteosarcoma cells and its molecular mechanism. METHODS: Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was studied using Flow cytometry analysis, Hoechst33258 staining, DNA fragmentation assay, as well as the activation of caspase-3 and poly (ADP-ribose) polymerase. Protein expression was detected by Western blotting. The dependence of p53 was determined using p53-siRNA transfection. RESULTS: IFNalpha increased doxorubicin-induced cytotoxicity to a much greater degree through apoptosis in human osteosarcoma p53-wild U2OS cells, but not p53-mutant MG63 cells. IFNalpha markedly upregulated p53, Bax, Mdm2, and p21, downregulated Bcl-2, and activated caspase-3 and PARP cleavage in response to doxorubicin in U2OS cells. Moreover, the siRNA-mediated silencing of p53 significantly reduced the IFNalpha/doxorubicin combination-induced cytotoxicity and PARP cleavage. CONCLUSION: IFNalpha enhances the sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis. The proper combination with IFNalpha and conventional chemotherapeutic agents may be a rational strategy for improving the treatment of osteosarcoma with functional p53.

P14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner.

The tumor suppressor p14ARF, encoded by the INK4a/ARF locus, is often disrupted in human cancers, p14ARF triggers cell cycle arrest and sensitizes cells to apoptosis in the presence of collateral signals. To investigate the role of p14ARF in chemotherapeutic drugs-induced apoptosis, p14ARF was overexpressed by stable transfection in human osteosarcoma cell lines, U2OS (p53-wt/p14ARF-null) and MG63 (p53-mt/p14ARF-null). The results showed that ectopic p14ARF sensitized both cell lines to cisplatin-induced cytotoxicity and apoptosis. This sensitization of cisplatin-induced apoptosis was associated with upregulation of p53, Bax and p21 in U2OS cells. Conversely, such a result was not observed in MG63 cells. Moreover, the sensitization of cisplatin-induced cytotoxicity in U2OS cells was unaltered by p53 siRNA. Together, we show here p14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner. Proper combinations of p14ARF gene transfer and conventional chemotherapy may be a valuable strategy in human osteosarcoma treatment.

In vitro and in vivo induction of bone formation based on adeno-associated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells.

AIM: To determine whether adeno-associated virus (AAV)-2-mediated, bone morphogenetic protein (BMP)-7-expressing human adipose-derived mesenchymal stem cells (ADMS) cells would induce bone formation in vitro and in vivo. METHODS: ADMS cells were harvested from patients undergoing selective suction-assisted lipectomy and transduced with AAV carrying the human BMP-7 gene. Non-transduced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualitatively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase (ALP) and Chinalizarin staining. A total of 2.5 x 10(6) cells mixed with type I collagen were implanted into the hind limb of severe combined immune-deficient (SCID) mice and subjected to a histological analysis 3 weeks post implantation. RESULTS: Transfection of the ADMS cells achieved an efficiency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7-expressing ADMS cells could induce new bone formation in vivo. CONCLUSION: The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.

Therapeutic effectiveness of intravenous immunoglobulin at 1 g/kg and 2 g/kg on Kawasaki disease: a comparative and follow-up study / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the effectiveness of intravenous immunoglobulin IVIG, 1 g/kg single intravenous injection in treating and preventing cardiac consequences of Kawasaki disease (KD) in children.</p><p><b>METHODS</b>A total of 242 children with KD disease were enrolled in the study. In the randomized controlled trial, they were randomly divided into two groups: IVIG 1 g/kg group and IVIG 2 g/kg group, with aspirin administered within the first 7 to 10 days of illness. The occurrence and restoration of coronary artery lesion (CAL) in these two groups as well as the clinical and laboratory indexes including total fever duration, restoration of cervical lymphadenopathy, white blood cells count, platelet count, serum immunoglobulin, C reactive protein, erythrocyte sedimentation rate and EKG were observed. The clinical effectiveness of the groups before and after the treatment was analyzed.</p><p><b>RESULTS</b>The age of the 242 children with KD disease ranged from 3 months to 14 years (mean 4.0 +/- 2.8 years old). Male to female ratio was 1.66:1, 83.1% of KD patients were blow 5 years old, 93.4% patients were followed up with echocardiography at the end of the first year and the follow-up period was (38 +/- 18) months, ranging from 4 months to 5.4 years; 86.9% of the cases in 1 g/kg group and 91.7% of the cases in 2 g/kg group had their fever controlled within 48 hours. The difference was not significant (P > 0.05). Serum immunoglobulin level was markedly enhanced after IVIG. Serum immunoglobulin levels in the patients of 2 g/kg group and 1 g/kg group were (26.9 +/- 7.4) g/L and (18.3 +/- 6.9) g/L, respectively (P < 0.01). The average duration of fever in IVIG 1 g/kg group was 10.6 days. After the treatment with 1 g/kg of IVIG, the abnormal white blood cells count, platelet count, C reactive protein, erythrocyte sedimentation rate and abnormal EKG findings were greatly improved (P < 0.001). However, there was no significant difference in the above-mentioned improvement between IVIG 1 g/kg group and IVIG 2 g/kg group (P > 0.05). In IVIG 1 g/kg group the occurrence of CAL was 29.5%. After the one-year follow-up, 87.5% CAL restored, but 12.5% did not, among which 9.4% were those of IVIG non-responders. In IVIG 2 g/kg group the incidence of CAL was 24.2%. After the one-year follow-up, 89.3% CAL restored, but 10.7% did not, all of which were those of IVIG non-responders. There was no significant difference in the incidence of CAL between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>Single intravenous injection of IVIG at 1 g/kg could effectively alleviate the clinical symptoms, decrease the incidence of CAL and reduce the complication of cardiovascular system. In the treatment of KD, the therapeutic effectiveness of IVIG at 1 g/kg was not significantly different from that of single intravenous injection of IVIG at 2 g/kg.</p>

Effects of Leukotriene B4-Leukotriene B4 Receptor Pathway in Vascular Immunizing Damage of Kawasaki Disease / 实用儿科临床杂志

Objective To investigate the role of serum in children with Kawasaki disease(KD)in acute stage and ?-globulin role in monocyte cell-produced leukotriene B4(LTB4).Meanwhile,to investigate the effects of the monocyte cell conditioned media(MCM)on the expression of leukotriene B4 receptor 2(BLT2)in endothelial.In order to understand whether LTB4-BLT2 pathway gets involved in vascular damage in KD and the mechanism of ?-globulin in the lessening vascular damage of KD.Methods The concentration of LTB4 in cell culture after the stimulation by serum of healthy children,serum of acute KD and serum of acute KD with ?-globulin were observed,respectively.The expression of BLT2 in the endothelial was determined by flow cytometry.Results 1.The serum of children with KD increased the concentration of LTB4 in MCM(P