Efficacy of Gene Modification in Placenta-Derived Mesenchymal Stem Cells Based on Nonviral Electroporation

Mesenchymal stem cell (MSC)-based therapy using gene delivery systems has been suggested for degenerative diseases. Although MSC-based clinical applications are effective and safe, the mode of action remains unclear. Researchers have commonly applied viral-based gene modification because this system has efficient vehicles. While viral transfection carries many risks, such as oncogenes and chromosomal integration, nonviral gene delivery techniques are less expensive, easier to handle, and safe, although they are less efficient. The electroporation method, which uses Nucleofection technology, provides critical opportunities for hard-to-transfect primary cell lines, including MSCs. Therefore, to improve the therapeutic efficacy using genetically modified MSCs, researchers must determine the optimal conditions for the introduction of the Nucleofection technique in MSCs. Here, we suggest optimal methods for gene modification in PD-MSCs using an electroporation gene delivery system for clinical application.

ELISA for Quantitative Determination of Hepatitis B Virus Surface Antigen

Several studies have reported a good correlation between levels of serum hepatitis B virus surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) before and after antiviral therapy. As a result, the quantification of HBsAg levels has attracted much attention in recent years as an important approach to evaluate viral activity. In this study, mAbs against HBsAg were generated and 9 mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) were investigated for optimization of HBsAg quantitation ELISA. Determination of the best combinations of mAbs for sandwich ELISA identified H17 and H31 mAbs as the ideal capture and horseradish peroxidase (HRP) conjugate mAbs, respectively. A standard curve for the current assay system exhibited linearity up to 40 ng/ml of HBsAg while a detection limit of approximately 1 ng/ml of HBsAg was also estimated, which was comparable to that of the other commercial ELISA kits. The ELISA system established in this study is particularly differentiated from other commercial kits in using mAbs for both capture and HRP conjugate, which provides a solution to inconsistency of quality and ethical issues in polyclonal antibodies production using laboratory animals.

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.

Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation

It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the affinity of ER414. We obtained a 3D model of ER414 to identify the amino acids in the CDRs that needed to be mutated. Clones were selected from the phage library with randomized amino acids in the CDRs and substitution of amino acids in the HCDR3 and LCDR1 of ER414 led to improved affinity. A clone, H3-14, with a ~20-fold increased affinity, was selected from the HCDR3 randomized library. Then three clones, ER2, ER78 and ER79, were selected from the LCDR1 randomized library based on the H3-14 but did not show further increased affinities compared to that of H3-14. Of the three, ER2 was chosen for further characterization due to its better expression than others. We successfully performed affinity maturation of ER414 and obtained antibodies with a similar affinity as cetuximab. And antibody from an affinity maturation inhibits the EGF-mediated tyrosine phosphorylation of EGFR in a manner similar to cetuximab.

In vivo hepatitis B virus-neutralizing activity of an anti-HBsAg humanized antibody in chimpanzees

Previously, we constructed a humanized antibody (HuS10) that binds to the common a antigenic determinant on the S protein of HBV. In this study, we evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was intravenously administered with a single dose of HuS10, followed by intravenous challenge with the adr subtype of HBV, while a control chimpanzee was only challenged with the virus. The result showed that the control chimpanzee was infected by the virus, and thus serum HBV surface antigen (HBsAg) became positive from the 14th to 20th week and actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 19th and 23rd week, respectively. However, in the case of the study chimpanzee, serum HBsAg became positive from the 34th to 37th week, while actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 37th and 40th week, respectively, indicating that HuS10 neutralized the virus in vivo and thus delayed the HBV infection. This novel humanized antibody will be useful in the immunoprophylaxis of HBV infection.

Assessment of the anatomic variation of mandibular incisive canal in chin bone harvesting

during bone harvesting from the chin.

Influence of cigarette smoke inhalation on dental implant osseointegration in the rat

Objectives : It is well known that cigarette smoking is harzardous to the osseointegration of dental implant, due to the impaired wound healing accompanied by reduced alveolar bone density. The aim of this study is to evaluate the influence of cigarette smoke on the implant osseointegration by the time factor consideration. Materials and methods : Twenty-four male Spraque-Dawley rats (8 weeks, weighting 200 to 250g) were used in this study. In the experimental group, 13 rats were exposed to cigarette smoke, 8 minutes per day during 6 weeks, and 12 rats in the control group were not exposed at any time. RBM (Resorbed blasting media) surfaced implant (diameter 3.3mm, length 5.0mm, AVANA Co., Korea) was placed in the right femur of each rat. Each implant with surrounding bone was prepared with microtome (cutting band 0.2mm(R), EXAKT Co., Germany) after 1 day, 2, 4, 8 and 12 weeks and stained with toluidine blue (1%). Another clinical investigation of each implant was also done at each evaluation time. Results : Clinical investigation around implant fixture showed that there were no significant differences between the control and experimental group. Microscopic observation around implant fixture showed that there were significant differences between the control and experimental group at the initial stage after implant fixture installation. Experimental group showed a decreased bone to implant contact within 4 weeks compared to control group, but showed similar characteristics after 4 weeks. Conclusion : Smoking inhalation effect on the dental implant showed the impaired wound healing by vasoconstriction and decreased intramedullary blood flow at initial stage of osseointegration. This experimental results can be clinically useful to the implant surgery of smoking patients.

The Effect of Routine and Regular Exercise Treadmill Test to the Civil Airline Pilots

BACKGROUND: As an airplane has been developed, the man could experienced the new environment. In these circumstances, the research of human's physical changes in the air was commended. About 80% of airplane's accidents was occurred by human factors and about 4% was related to the pilot's physical state. According to the many studies, the most common cause of the permanent grounding of the pilot is coronary artery disease(CAD). Therefore the tool of prevention and early detection for CAD was needed. PURPOSE: The purpose of this study is to evaluate the effect of routine and regular Exercise Treadmill Test(ETT) to the civil airline pilots especially 1) in decreasing the correctable risk factors of coronary artery disease 2) in reducing the medical disqualification rate of pilots. METHOD: Routine ETT has been performed to the Asiana Airlines' pilot during his regular physical check up. When the pilot showed abnormal resting ECG or abnormal ETT results, Echocardiography, 24hour Holter monitoring, Cardiac SPECT or Coronary angiography were done to rule out coronary artery disease. Review the medical records from June 1993 through December 1998. Compare the medical disqualification rate between Asiana Airlines pilot & other pilot groups. Compare the individual correctable risk factors of coronary artery disease and the numbers of risk factors between pilot and non-pilot group using SPSS 8.0 t-test. RESULTS: 1. The results showed no evidence of CAD and there was no case of CAD as a pilot's disqualifying conditions. 2. The levels of cholesterol and the numbers of risk factors of pilot had a statistically significantly lower than non-pilot group. CONCLUSIONS: Routine and regular ETT will be effective in educating the pilot to do his best to decrease the correctable risk factors of CAD.

Determinants on Occurrence of Hypertension in the Korean Adult Male Workers in an Industry: a Nested Case-control Study / 대한산업의학회지

OBJECTIVES: In order to find determinants on occurrence of hypertension in the adult male workers in an industry, a nested case-control study was performed in Seoul, Korea at the periodic health examination center. METHODS: A cohort was formed with 1,690 normotensive male workers in 1996 and 1997, who had participated in three consecutive periodic health examinations from 1996 through 1998. Cases were 89 hypertensives in 1998, controls were 356 selected by 1:4 age-matching among 1,601 normotensives in 1998. Baseline health behavior (drinking smoking, exercise), baseline health examination data (height, weight, BMI, blood pressure. hemoglobin, fasting blood sugar, serum total cholesterol), and the changes of data during the study period were compared between case and controls. And multiple logistic regression was performed to find determinants on occurrence of hypertension. RESULTS: There was significant difference in alcohol consumption amount, baseline weight, DM1, blood pressure, total cholesterol, and hemoglobin level. The main determinants on occurrence of hypertension from multiple logistic regression analysis were baseline systolic blood pressure (OR, 1. 13; 95% confidence interval 1.08-1.18%), 15-21 drinks per week (OR, 4.17; 95 % confidence interval 1.44-12.11), and 22 or more drinks per week (OR, 5.29; 95 % confidence interval 1.50-18.58). CONCLUSIONS: This study showed the importance of baseline blood pressure level and adequate drinking habit for the management of hypertension.

Proposals of AME System Improvement: An Aspect of Designated Aeromedical Center

No abstract available.

The carcinogenic effect with the instillation of N-methyl-N-nitro-N-nitrosoaguanidine in Sprague-Dawley rats in the colon and rectum

No abstract available.