The objective of this study is to establish the genotyping methods of new HLA gene, TAP1 and TAP2, and determine the genetic polymorphisms for database study in Koreans before using in clinical laboratory. Polymerase chain reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) and PCR-Sequence Specific Primers (PCR-SSP) techniques were used for TAP1 and TAP2 genotying, respectively. Restriction enzymes, Bcll and Accl, and 4 oligonucleotide primers were used for the PCR-RFLP analysis of TAP1. Whereas for PCR-SSP assay of TAP2, 12 oligonucleotide primers were synthesized. The results of control cells were correlated well with the types which were analyzed at Xlth histocompatibility international workshop. Arnong three and six different alleles of TAP1 and TAP2 found in 200 unrelated Korean individuals, TAP1A (84%) was the most frequent allele. TAP1B and TAP1C were 15.5% and 0.5% respectively. TAP2A represented more than a half (55.1%). TAP2B and TAP2C were 32.2% and 9.2% respectively. TAP1D, TAP2F and TAP2G were not found in Koreans.
Alleles ; DNA Primers ; Education ; Histocompatibility ; Polymerase Chain Reaction ; Polymorphism, Genetic* ; Polymorphism, Restriction Fragment Length

No abstract available.

BACKGROUND: Rescently, the immunotherapy against infectious disease and cancer is being actively studied, and high yield of pure CD14+ monocytes is a key of concern. In this study, we optimized the percoll gradient method to increase the purity and yield of monocytes from peripheral mononuclear cells. METHODS: We separated mononuclear cells (MNC) from healthy donors, and monocytes from MNC were separated with the various density of percoll gradient. After centrifugation, we determined the most efficient density of the percoll gradient to get the highest yield and purity of monocytes. We also obtained monocytes by the plastic absorption method. Monocytes were differentiated into dendritic cells (DC) and the efficacy of differentiation to DC is confirmed and compared by morphological characteristics and using a flowcytometer. RESULTS: The purity of monocytes was 51.3+/-8.3% by the 35% percoll gradient method and the purity was improved to 82.9+/-4.2% with 100% of yield by repeating the same method. Therefore, the yield of mature dendritic cells was 3.6+/-0.9% of the total input MNC by the 35% percoll repetition method, which was 1.7 times higher than the plastic adherent method (2.1+/-0.5%). CONCLUSION: This study shows a cost-effective method to isolate CD14+ monocytes and these cells demonstrate high differentiation rate to DC. This process will be valuable for obtaining a sufficient number of DC.
Absorption ; Centrifugation ; Communicable Diseases ; Dendritic Cells ; Humans ; Immunotherapy ; Monocytes ; Plastics ; Povidone ; Silicon Dioxide ; Tissue Donors

BACKGROUND: Treatment for chronic hepatitis B aims to suppress virus replication and virus sequestration in hepatocytes. Covalently closed circular (ccc) DNA is the template for transcription of viral genes and is responsible for viral persistence. However, limited data are available for quantification of hepatitis B surface antigen (HBsAg) in Korea. METHODS: We evaluated the Elecsys HBsAg II quant assay (Roche Diagnostics, USA) for within-run, between-run, and between-day precisions, linearity, carryover, and clinical specificity. In total, 156 serum samples were evaluated for correlation between HBsAg and hepatitis B virus (HBV) DNA. Serial samples were obtained from 10 patients at 0, 12, 24, 48, 72, and 96 weeks during follow-up. RESULTS: The assay detected HBsAg in a linear range of 0.5-48,696 IU/mL. Within-run, between-run, and between-day CVs were 2.9-4.1%, 0-1.5%, and 1.5-4.9%, respectively. Cross-reactivity between potentially interfering substances was absent, and the carryover rate was 0.00002%. The correlation of measurements between the Elecsys assay and HBV DNA PCR was weak (r=0.438, P=0.002). For predicting virologic response, cutoff values of 10,275 IU/mL and 3,846 IU/mL at 12 and 24 weeks after treatment initiation showed positive predictive values of 77.1% and 85% and negative predictive values of 84.6% and 50%, respectively. CONCLUSIONS: The Elecsys HBsAg II quant assay showed good performance for precision, linearity, carryover rate, and specificity. HBsAg level at baseline, 12 weeks, and 24 weeks after treatment initiation can predict virologic response, and the assay can be used for HBsAg quantification in clinical practice.

BACKGROUND: The treatment using more potent antiviral agents for the hepatitis B virus (HBV) infection has been performed widely and a highly sensitive quantification method using the polymerase chain reaction (PCR) that measures the HBV viral genome in sera is available. In this study, the HBV DNA level in each disease status including the inactive HBsAg carrier is evaluated. METHODS: Samples were obtained from 227 patients with chronic HBV infection that were grouped into chronic hepatitis (111), liver cirrhosis (71), and inactive HBsAg carrier (45). Quantification of HBV DNA was performed using the automated Cobas Amplicor HBV monitor test(TM). RESULTS: Among the chronic hepatitis B group (9.76 x 10(7) copies/mL), the liver cirrhosis group (4.88x10(5) copies/mL), and the inactive carrier group (3.18 x10(3)copies/mL), the medians of serum HBV DNA levels were significantly different from one group to another (P=0.000). Also, the median of HBV DNA levels in the patients with positive HBeAg (1.77 x10(8) copies/mL) was significantly higher than that of negative HBeAg (2.71 x 10(4) copies/mL) (P=0.000). In the patients with negative HBeAg, HBV DNA level in the inactive carrier group (Median 3.18 x 10(3) copies/mL) was significantly lower than that of the chronic hepatitis group (Median 2.2 x 10(5) copies/mL (P=0.000). CONCLUSIONS: The serum HBV DNA level varied among different disease groups, particularly according to HBeAg positivity. 40% of the chronic hepatitis group with negative HBeAg had HBV DNA levels below 10(5) copies/mL. Therefore, the quantitative analysis of HBV DNA using this sensitive and automated PCR method would be useful in detecting viral proliferation.
Antiviral Agents ; DNA* ; Genome, Viral ; Hepatitis B e Antigens ; Hepatitis B Surface Antigens ; Hepatitis B virus* ; Hepatitis B, Chronic ; Hepatitis, Chronic ; Herpesvirus 1, Cercopithecine* ; Humans ; Liver Cirrhosis ; Polymerase Chain Reaction*

BACKGROUND: Accurate measurement of the concentration of hepatitis B virus (HBV) DNA in clinical samples is important for the appropriate treatment of patients and evaluation of their therapeutic responses. In addition, the concentration of HBV DNA in the serum of patients with chronic HBV infection has a very broad range. Real-time PCR is very sensitive and useful to detect HBV DNA in a wide range of concentrations. We designed new primers and probes for real-time PCR to detect HBV DNA. METHODS: Primers and probes specific for HBV were designed. EUROHEP HBV DNA standards (NIBSC, Hertfordshire, UK) with the HBV DNA concentration of 7.0 x 10(4) copies/mL was used to determine the calibration curve and efficacy for the real-time PCR assay. Sensitivity, dynamic range, and precision were evaluated. The correlation between the real-time PCR and Cobas Amplicor HBV Monitor(TM) assay in the measurement of serum HBV DNA concentrations in 52 patients with chronic HBV infection was evaluated. RESULTS: The sensitivity of the assay was approximately 6.08 x 10(2) copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1 x 0(2) to 6.5 x 10(9) copies/mL without any dilution of specimens. The assay showed low coefficients of variation of repeatability (3.7-24.9%) and reproducibility (7.8-24.7%). The results were found to correlate well with those obtained by Cobas Amplicor HBV Monitor(TM) kit. CONCLUSIONS: We provide a new in-house method for the measurement of serum HBV DNA using real-time PCR, which enables us to detect HBV DNA rapidly, sensitively, and accurately.
Calibration ; DNA* ; Hepatitis B virus ; Humans ; Real-Time Polymerase Chain Reaction*

The aims of this study were to investigate serum hepatitis B virus (HBV) DNA levels at different clinical stages in patients with chronic HBV infection, and to determine the serum HBV DNA level that discriminated HBeAg-negative chronic hepatitis B(CHB) cases from inactive HBsAg carriers. In all, 222 patients, encompassing 68 HBeAg-positive CHB patients (HBeAg-positive, ALT-elevation), 89 HBeAg-negative CHB patients (HBeAg-negative, ALT-elevation), and 65 inactive HBsAg carriers (HBeAg-negative, ALT-normal), were tested. The ALT levels had been tested more than twice during the previous six months, and the serum HBV DNA levels were quantified by a polymerase chain reaction-based assay. The serum HBV DNA levels of the HBeAg-negative patients were significantly lower than those of the HBeAg-positive patients (median 2.7 x 10(4) vs. 1.6 x 10(8) copies/mL; p=0.000). In addition, the HBV DNA levels of the HBeAg-negative CHB patients were significantly higher than those of the inactive HBsAg carriers (median 2.2 x 10(5) vs. 3.2 x 10(3) copies/ mL; p=0.000). The optimal HBV DNA level for discriminating HBeAg-negative CHB cases from inactive HBsAg carriers was 2.0 x 10(4) copies/mL. The serum HBV DNA levels were lower than the cutoff value in 72.3% (47/65) of the inactive HBsAg carriers, and in 31.5% (28/89) of the HBeAg-negative CHB patients. The serum HBV DNA levels differed significantly between these two groups. However, the levels in the two groups overlapped extensively, preventing the definition of a differentiation cut-off value.
Adolescent ; Adult ; Aged ; Child ; DNA/chemistry ; DNA, Viral/*genetics ; False Positive Reactions ; Female ; Hepatitis B/metabolism/*pathology ; Hepatitis B Virus/*metabolism ; Human ; Liver/metabolism ; Male ; Middle Aged ; Polymerase Chain Reaction ; ROC Curve

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.
Adolescent ; Adult ; *Amino Acid Motifs ; Chronic Disease ; Female ; Hepatitis B/*drug therapy ; Hepatitis B virus/*genetics ; Humans ; Lamivudine/*therapeutic use ; Male ; Middle Aged ; Mutation ; *Oligonucleotide Array Sequence Analysis ; Polymorphism, Restriction Fragment Length ; Reverse Transcriptase Inhibitors/*therapeutic use ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Variation (Genetics)

Background/Aims: Real-world, clinical practice data are lacking about sofosbuvir/ ribavirin (SOF/RBV) treatment of Korean patients with hepatitis C virus genotype 2 (HCV GT2) infection. This study investigated the efficacy and safety of SOF/RBV in Korean patients with HCV GT2 infection and clinical factors predicting sustained virological response 12 weeks (SVR12) after the end of SOF/RBV treatment. Methods: A total of 181 patients with HCV GT2 with/without cirrhosis were treated with SOF/RBV for 16/12 weeks. Rapid virological response (RVR) was defined as non-detectable HCV RNA at 4 weeks. Results: The RVR rate was 80.7% (146/181), the end of treatment response rate was 97.8% (177/181) and the SVR12 rate was 92.8% (168/181). Of eight patients with relapse, four did not achieve RVR. Three patients had a history of hepatocellular carcinoma (HCC). Multivariable analysis showed that RVR (p = 0.015) and no previous history of HCC (p = 0.007) were associated with SVR12. Factors significantly contributing to RVR included cirrhosis, creatinine concentration, and pre-treatment HCV RNA level. SVR12 rate was significantly higher in RVR (+) than RVR (–) patients (95.2% vs. 82.9%, p = 0.011) and also significantly higher in patients without than with a history of HCC (94.1% vs. 72.7%, p = 0.008). During treatment, 80/181 patients (44.2%) experienced mild to moderate adverse events, with 32 (17.7%) requiring RBV dose reductions due to anemia. Conclusions SOF/RBV treatment was effective and tolerable in HCV GT2 patients. RVR and no previous history of HCC were positive predictors of SVR12.

Background/Aims: Real-world, clinical practice data are lacking about sofosbuvir/ ribavirin (SOF/RBV) treatment of Korean patients with hepatitis C virus genotype 2 (HCV GT2) infection. This study investigated the efficacy and safety of SOF/RBV in Korean patients with HCV GT2 infection and clinical factors predicting sustained virological response 12 weeks (SVR12) after the end of SOF/RBV treatment. Methods: A total of 181 patients with HCV GT2 with/without cirrhosis were treated with SOF/RBV for 16/12 weeks. Rapid virological response (RVR) was defined as non-detectable HCV RNA at 4 weeks. Results: The RVR rate was 80.7% (146/181), the end of treatment response rate was 97.8% (177/181) and the SVR12 rate was 92.8% (168/181). Of eight patients with relapse, four did not achieve RVR. Three patients had a history of hepatocellular carcinoma (HCC). Multivariable analysis showed that RVR (p = 0.015) and no previous history of HCC (p = 0.007) were associated with SVR12. Factors significantly contributing to RVR included cirrhosis, creatinine concentration, and pre-treatment HCV RNA level. SVR12 rate was significantly higher in RVR (+) than RVR (–) patients (95.2% vs. 82.9%, p = 0.011) and also significantly higher in patients without than with a history of HCC (94.1% vs. 72.7%, p = 0.008). During treatment, 80/181 patients (44.2%) experienced mild to moderate adverse events, with 32 (17.7%) requiring RBV dose reductions due to anemia. Conclusions SOF/RBV treatment was effective and tolerable in HCV GT2 patients. RVR and no previous history of HCC were positive predictors of SVR12.