The Role of Endothelial Progenital Cells and Fibrin on Vascularization and Stability in Orbital Implant

PURPOSE: The effects of endothelial progenitor cells (EPCs) and fibrin on fibrovascular growth into porous polyethylene orbital implants (Medpor(R) sheet) were investigated using stem cells. METHODS: EPCs were separated from human adipose fat tissue for culture. Fluorescence-activated cell sorting (FACS) was used to identify the phenotype and to analyze the purity of EPCs cultivated from human adipose tissue. Processed Medpor(R) sheets were inserted in each quadrant of the subcutaneous fat layer under the dorsal surface of 20 anesthetized athymic nude mice, using sterile methods. Medpor(R) sheets processed with endothelial progenitor cells and fibrin were inserted into the two top quadrants, a Medpor(R) sheet processed with fibrin was inserted in the lower right quadrant, and an unprocessed Medpor(R) sheet was inserted in the lower left quadrant of each mouse. The mice were sacrificed on the seventh day. The adhesiveness and blood vessel formation were quantified by weight and the number of blood cells within the Medpor(R) sheets. Hematoxylin and eosin (H&E) and toluidine blue stains were used to analyze fibrovascular and cell growth within the Medpor(R) sheets. RESULTS: The sheets processed with EPCs and fibrin were heavier and contained more white and red blood cells (p<0.001) than the other sheets. The sheets processed with fibrin alone were heavier (p<0.01) and contained more blood cells (p<0.001) than the unprocessed sheets. The degree of vessel formation and tissue adhesiveness was greatest in the group of Medpor(R) sheets processed with EPCs and fibrin. The sheets processed with fibrin only had greater tissue adhesiveness and fibrovascular proliferation than the unprocessed Medpor(R) sheets. CONCLUSIONS: Endothelial progenitor cells and fibrin applied to Medpor(R) sheets improve fibrovascular proliferation and tissue adhesiveness. When both are applied together, a synergistic effect is seen.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein / 대한소화기학회지

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Development of Human Antibody Inhibiting RNase H Activity of Polymerase of Hepatitis B Virus Using Phage Display Technique

BACKGROUND: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. METHODS: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. RESULTS: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of 4.46 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 10(-7) M, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. CONCLUSION: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.

Relationship between the Expression of Platelet-Derived Endothelial Cell Growth Factor(PD-ECGF), Basic Fibroblast Growth Factor(bFGF), Vascular Endothelial Growth Factor(VEGF) and Clinical Stage in Squamous Cell Carcinoma of Larynx / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Normal angiogenesis occurs as a part of the body's repair processes like the healing of wounds and fractures. By contrast, uncontrolled angiogenesis can often be pathological. Vascular remodelling could therefore play an important role in the growth process of solid tumors. The aim of this study was to detect the expression of platelet-derived endothelial cell growth factor (PD-ECGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), and investigate the correlation between these factors. Also, we studied the relationships between the expressions of these factors and the clinical stage, nodal involvement, and histologic grade in the squamous cell carcinoma of larynx. MATERIALS AND METHOD: The authors examined the expression of three angiogenic factors in specimens of laryngeal squamous cell carcinoma (n=17). The mRNA expressions of angiogenic factors were detected by the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Statistics were analysed using the Fisher's exact test, the Wilcoxon signed rank test, and the Spearman correlation coefficiency. RESULTS: PD-ECGF and bFGF were significantly higher in the stages III and IV cancers than in the stages I and II cancers, and thus shows that bFGF was related to severity of the nodal involvement. The expression of more than one factor was significantly related with stages III and IV cancer. PD-ECGF and VEGF were related with each other. CONCLUSION: The authors suggest that angiogenic factors, especially, PD-ECGF and bFGF, may be used as prognostic factors for the squamous cell carcinoma of larynx.

The Effect of Oral Steroid for the Expression of Tumor Necrosis Factor-alpha (TNF-alpha), Transforming Growth Factor-beta1 (TGF-beta1), Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF) in Nasal Polyp / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Glucocorticoids are currently the most potent medication available for the treatment of nasal polyposis, but exact mechanisms are uncertain. The purpose of this study was to evaluate the response of transforming growth factor-beta1 (TGF-beta1), tumor necrosis factor-alpha (TNF-alpha), and platelet-derived endothelial cell growth factor (PD-ECGF) to oral corticosteroid in nasal polyps. MATERIALS AND METHOD: Oral steroid-untreated nasal polyps (n=10) and oral steroid-treated nasal polyps (n=13) (prednisolone 30 mg per day for 7 days) underwent nasal endoscopy and biopsy of the polyps. The mRNA expression of these factors were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of these factors in nasal polyp tissues were evaluated by immunohistochemistry. RESULTS: The TNF-alpha mRNA and PD-ECGF mRNA expressions were significantly decreased in the oral steroid-treated nasal polyps when compared with the oral steroid-untreated nasal polyps, but TGF-beta1 mRNA was not significantly decreased. The immunohistochemical studies revealed that levels of expression for TNF-alpha protein and PD-ECGF protein in the oral steroid-treated nasal polyps were significantly decreased, but TGF-beta1 protein was not decreased when compared with oral steroid-untreated nasal polyps. CONCLUSION: We suggest that oral corticosteroids may exert a beneficial effect by significantly reducing the levels of TNF-alpha and PD-ECGF in nasal polyps.

Definition of the peptide mimotope of cellular receptor for hepatitis C virus E2 protein using random peptide library

BACKGROUND: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell . METHODS: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. RESULTS: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patient s captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. CONCLUSION: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

Quantification of Hepatitis C Virus RNA in Patients Sera by Competitive RT-nested PCR

No Abstract Available.

Quantification of Hepatitis C Virus RNA in Patients Sera by Competitive RT-nested PCR

No Abstract Available.

Two Purification of Maltose-binding Protein(MBP) PNase H Fusion Protein with a c-myc Tag and Histidine Tail

No Abstract Available.

Two Purification of Maltose-binding Protein(MBP) PNase H Fusion Protein with a c-myc Tag and Histidine Tail

No Abstract Available.

Seroepidemiological Survey of Hemorrhagic Fever with Renal Syndrome in Korea , 1994 - 1996

To understand the seroepidemiological patterns of haemorrhagic fever with renal syndrome in Korea, a nation-wide survey collaborated with fourteen clinics was carried out from 1994 to 1996. Sera of 4,547 patients with acute febrile episodes were tested by indirect immunofluorescent antibody test and the seroepidemiological analysis including sex, age, seasonal and regional distributions were performed. According to the results obtained in this study, the epidemiological characteristics of haemorrhagic fever with renal syndrome in Korea were summarized as follows: 1. Seropositive rate of hemorrhagic fever with renal syndrome among the patients with acute febrile episodes was 6.4% by the cut-off point of 1:40. 2. Among the seropositives, male outnumbered female and the ratio of males to females was 2.0:1.0. 3. Seventy six % of the seropositive patients were 21-60 years old. 4. The number of seropositive cases increased from October and reached maximum in December and began to decrease gradually from January. 5. The geographical distribution of the seropositives cover most areas including Cheju province in Korea.

Hyperbaric Oxygen Therapy in Decompression Sickness

BACKGROUND: Scuba diving has become increasingly popular in Korea. Medical problems are common with dives, especially decompression sickness(DCS). This study was performed to obtain an useful information of hyperbaric oxygen therapy in DCS in Korea. METHOD: We reviewed the 62 cases of Korean divers, who were diagnosed as DCS and received recompression therapy according to U.S. Navy Standard Recompression Treatment Table at Ocean and Underwater Medical Research and Training Center of ROK Navy, for 6 years from Jan. 1993 to Nov. 1998. RESULT: 1) the mean no-decompression limit excess time between type I DCS group(72.7 min.) and type II DCS group(92.8min.) showed significant difference. 2) The rate of symptoms appeared on surfacing and within 10min. after surfacing of type I and type II DCS were 41.4%and 72.7% respectively. 3) The cure late of type I and type II were 75.9%and 42.4% respectively. In type II DCS group, the cure rate of the group within 12 hour-delayed recompression treatment and the group above 12 hour-delayed treatment were 64.3%and time 26.3% respectively, and in type I DCS group, 100% and 66.7% respectively. CONCLUSION: These findings suggest that the education of safety, the strict observance of the standard decompression table, and the avoidance of excessive repeated diving are important for reducing the risk of diving related disease. And to offer proper management of DCS, there should be more multiplace hyperbaric oxygen chambers, the suitable transport system, and the specialist of diving medicine or hyperbaric medicine in Korea.