Background: Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury. Methods: To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo. Results: The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo. Conclusion In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

Background: Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury. Methods: To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo. Results: The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo. Conclusion In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

Purpose: Malignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth. Materials and Methods: In the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hindlimb strength. Results: The complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone. Conclusion This study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.
Cell Differentiation/genetics/physiology ; *Gene Expression ; Gene Regulatory Networks ; *Genetic Therapy ; Humans ; Luciferases/genetics/*metabolism ; *Neural Stem Cells ; Neurons/metabolism ; Phosphopyruvate Hydratase/metabolism ; Promoter Regions, Genetic ; Spinal Cord Injuries/*therapy ; Stem Cells/*metabolism

We performed two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) subcommittee of Korean Association of Quality Assurance for Clinical Laboratory (KAQACL) in 2009. The number of participating laboratories were 110, which is similar with that of previous 3 years. Average response rates were 97.8% in both trials, similar to those of previous years. Two kinds of control materials were requested to be tested in each trial so that each institution could find the possible systematic errors. The average drug item responded was 6.2 per institution, which was decreased slightly from 6.5 in recent 5 years. The most common test items were valproic acid, digoxin, carbamazepine, phenytoin, and theophylline which were peformed in more than 63.8% of participating laboratories, followed by phenobarbital, cyclosporine, tacrolimus, vancomycin, lithium, methotrexate, amikacin, gentamicin, acetaminophen, tobramycin, salicylate, free phenytoin, amitryptyline, ethosuximide, and primidone. The widely used TDM analyzers were Abbott AxSym (26.9%), followed by Abbott TDx/TDxFLx (24.8%), Roche Cobas Integra (15.1%), Siemens Diagnostics Viva-E (5.5%), Roche cobas c501 (5.1%), Siemens Diagnostics Dimension (3.4%), and many other analyzers. The inter-laboratory coefficients of variations showed similar tendency comparing with those of the previous years. The number of participating laboratories for drug of abuse (DOA) tests were 19, which was slightly increased compared to that of the previous year. Average DOA items were 3.8~4.2. We found the good performance of participating laboratories for DOA. In conclusion, the TDM and DOA external quality assessment of 2009 showed similar performance comparing with that of the recent 3 years.
Acetaminophen ; Amikacin ; Carbamazepine ; Cyclosporine ; Digoxin ; Drug Monitoring ; Ethosuximide ; Gentamicins ; Korea ; Lithium ; Methotrexate ; Phenobarbital ; Phenytoin ; Primidone ; Tacrolimus ; Theophylline ; Tobramycin ; Valproic Acid ; Vancomycin

Two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) subcommittee of Korean Association of Quality Assurance for Clinical Laboratory (KAQACL) were performed in 2008. The number of participating laboratories were 114, which is similar with that of the previous year. Average response rates were 97.8% in both trials slightly lower than that of the previous year. Two kinds of control materials were requested to be tested in each trial so that each laboratory could know the possible systematic error. The average drug item was 6.3 per laboratory, which was decreased slightly from 6.8 in recent 5 years, and the maximum was 18 items. The most common test items were valproic acid, digoxin, carbamazepine, theophylline, phenytoin, and phenobarbital which were peformed in more than 52.1% of participating laboratories, followed by cyclosporine, vancomycin, tacrolimus, lithium, methotrexate, amikacin, tobramycin, gentamycin, acetaminophen, salicylate, free phenytoin, primidone, and amitryptyline. The widely used TDM analyzers were Abbott TDx/TDxFLx (35.3%), followed by Abbott AxSym (26.5%) and Roche Cobas Integra (17.3%), Abbott IMx (3.3%), and Siemens Viva E (3.0%). The inter-laboratory coefficients of variations showed similar tendency comparing with those of the previous years. The number of participating laboratories for drug of abuse tests were 17, which is similar to that of the previous year. Average drug item were 3.7 for the 1st trial. We found the relatively good performance as we got the correct answers for all laboratories except 2 laboratories. In conclusion, the TDM external quality assessment of 2008 showed grossly similar pattern comparing with that of previous year.
Acetaminophen ; Amikacin ; Carbamazepine ; Cyclosporine ; Digoxin ; Drug Monitoring ; Gentamicins ; Korea ; Lithium ; Methotrexate ; Phenobarbital ; Phenytoin ; Primidone ; Tacrolimus ; Theophylline ; Tobramycin ; Valproic Acid ; Vancomycin

Different subtypes of dendritic cells (DC) influence the differentiation of naive T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. We evaluated the percentages of DC subtypes in peripheral blood from pregnant women (maternal blood) and their cord blood compared to the peripheral blood of healthy non pregnant women (control). Circulating DC were identified by flow cytometry as lineage (CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive cells. Subtypes of DC were further characterized as myeloid DC (CD11c+/CD123+/-), lymphoid DC (CD11c-/CD123+++) and less differentiated DC (CD11c-/CD123+/-). The frequency of DC out of all nucleated cells was significantly lower in maternal blood than in control (P<0.001). The ratio of myeloid DC/lymphoid DC was significantly higher in maternal blood than in control (P<0.01). HLA-DR expressions of myeloid DC as mean fluorescence intensity (MFI) were significantly less in maternal blood and in cord blood than in control (P<0.001, respectively). The DC differentiation factors, TNF-alpha and GM-CSF, released from mononuclear cells after lipopolysaccharide stimulation were significantly lower in maternal blood than in control (P<0.01). The distribution of DC subtypes was different in maternal and cord blood from those of non-pregnant women. Their role during pregnancy remains to be determined.
Adult ; Cell Differentiation ; Dendritic Cells/*classification/cytology/immunology ; Female ; Fetal Blood/cytology/*immunology ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor/metabolism ; HLA-DR Antigens/metabolism ; Humans ; Lipopolysaccharides/pharmacology ; Lymphocyte Activation ; Pregnancy ; T-Lymphocytes/cytology/immunology ; Th1 Cells/cytology/immunology ; Th2 Cells/cytology/immunology ; Tumor Necrosis Factor-alpha/metabolism

Two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) subcommittee of Korean Association of Quality Assurance for Clinical Laboratory (KAQACL) were performed in 2007. Number of participating laboratories were increased to 109, by 5.63% increase comparing with the previous year. Response rates reached 98.7% for both trials slightly lower than that of the previous year. Two kinds of control materials were requested to be tested in each trial so that each institution could know the possible systematic error. In both trials, 20 test items were responded at least from one laboratory. The average drug item was 6.3 per institution, which was decreased slightly from 6.8 in recent 5 years. The most common test items were valproic acid, digoxin, carbamazepine, theophylline, phenytoin, and phenobarbital which were peformed in more than 55% of the participating laboratories, followed by cyclosporine, vancomycin, lithium, tacrolimus, methotrexate, amikacin, gentamicin, salicylate, tobramycin, acetaminophen, primidone, free phenytoin, and amitryptyline. The inter-laboratory coefficients of variations showed simliar tendency comparing with those of the previous years. We started the proficiency test for drug of abuse from 2007 and got the response from 13 and 17 laboratories in the 1st and 2nd trial, respectively. Average drug items were 3.4 for the 2nd trial. We found the relatively good performances as we got the correct answers from all laboratories except 4 for each one mistake. In conclusion, the TDM external quality assessment of 2007 showed grossly similar pattern comparing with those of previous year and drug of abuse proficiency testing showed a relatively good performance.
Acetaminophen ; Amikacin ; Carbamazepine ; Cyclosporine ; Digoxin ; Drug Monitoring ; Gentamicins ; Korea ; Lithium ; Methotrexate ; Phenobarbital ; Phenytoin ; Primidone ; Tacrolimus ; Theophylline ; Tobramycin ; Valproic Acid ; Vancomycin

Two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) subcommittee of Korean Association of Quality Assurance for Clinical Laboratory (KAQACL) were performed in 2007. Number of participating laboratories were increased to 109, by 5.63% increase comparing with the previous year. Response rates reached 98.7% for both trials slightly lower than that of the previous year. Two kinds of control materials were requested to be tested in each trial so that each institution could know the possible systematic error. In both trials, 20 test items were responded at least from one laboratory. The average drug item was 6.3 per institution, which was decreased slightly from 6.8 in recent 5 years. The most common test items were valproic acid, digoxin, carbamazepine, theophylline, phenytoin, and phenobarbital which were peformed in more than 55% of the participating laboratories, followed by cyclosporine, vancomycin, lithium, tacrolimus, methotrexate, amikacin, gentamicin, salicylate, tobramycin, acetaminophen, primidone, free phenytoin, and amitryptyline. The inter-laboratory coefficients of variations showed simliar tendency comparing with those of the previous years. We started the proficiency test for drug of abuse from 2007 and got the response from 13 and 17 laboratories in the 1st and 2nd trial, respectively. Average drug items were 3.4 for the 2nd trial. We found the relatively good performances as we got the correct answers from all laboratories except 4 for each one mistake. In conclusion, the TDM external quality assessment of 2007 showed grossly similar pattern comparing with those of previous year and drug of abuse proficiency testing showed a relatively good performance.
Acetaminophen ; Amikacin ; Carbamazepine ; Cyclosporine ; Digoxin ; Drug Monitoring ; Gentamicins ; Korea ; Lithium ; Methotrexate ; Phenobarbital ; Phenytoin ; Primidone ; Tacrolimus ; Theophylline ; Tobramycin ; Valproic Acid ; Vancomycin

BACKGROUND: ABO genotyping is being used increasingly when the results of serologic typing are unclear or there is some suspicion of rare ABO subtypes. Conventional molecular diagnostic methods such as PCR- restriction fragment length polymorphism (PCR-RFLP), allele-specific PCR, PCR-single stranded conformational polymorphism (PCR-SSCP) and sequence-based typing have been used in this field. Recently, a pyrosequencing technique was introduced into clinical laboratories. This study evaluated the possibility of applying pyrosequencing to ABO genotyping. METHODS: A total of 36 samples, which had previously been analyzed by PCR-RFLP and serological method in the Blood Genetics Clinic of Seoul National University Hospital between August 2001 and September 2004 and shown to have the A/A, A/B, A/O, B/B, B/O, O/O, cis-AB/O, cis-AB/A, or cis-AB/B genotypes, were analyzed by pyrosequencing analysis. Briefly, two PCR reactions were carried out separately for one region including nucleotide 261, and for another region including nucleotides 796 and 803. Pyrosequencing was then performed, and the pyrograms were interpreted using an automated interpretation program from the manufacturer and by researchers independently to determine the nucleotides 261, 796 and 803 for ABO genotyping. RESULTS: The ABO genotypes from pyrosequencing and the interpretation of the pyrograms according to the researcher on 36 samples were in complete concordance with the results obtained by PCR-RFLP. The ABO genotypes from the automated interpretation program showed an error in one out of total 108 SNP (single nucleotide polymorphism) analyses (eRROR RATE=0.9%) OF 36 SAMPLES. CONCLUSION: ABO genotyping for A, B, O, cis-AB alleles by pyrosequencing of nucleotides 261, 796 and 803 was relatively simple and accurate and could be an another field we can use in clinical laboratories.
Alleles ; Genetics ; Genotype ; Nucleotides ; Pathology, Molecular ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Seoul