Characterization of Two Novel mAbs Recognizing Different Epitopes on CD43

JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.

Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

Characterization of Monoclonal Antibodies against Human Leukocyte Common Antigen (CD45)

BACKGROUND: The leukocyte common antigen (CD45) is a transmembrane-type protein tyrosine phosphatase that has five isoforms. METHODS: We generated seven murine mAbs against human CD45 by injecting cells from different origins, such as human thymocytes, PBMCs, and leukemic cell lines. By using various immunological methods including flow cytometry, immunohistochemistry, and immunoprecipitation, we evaluated the reactivity of those mAbs to CD45 of thymus as well as tonsil lysates. Furthermore, we transiently transfected COS-7 cells with each of gene constructs that express five human CD45 isoforms respectively, and examined the specificities of the mAbs against the transfected isoforms. RESULTS: In case of thymocytes, lymphocytes, and monocytes, all the seven mAbs demonstrated positive reactivities whereas none was reactive to erythrocytes and platelets. The majority of immune cells in formalin-fixed paraffin-embedded thymus and tonsil tissues displayed strong membranous immunoreactivity, and the main antigen was detected near 220 kDa in all cases. Among the mAbs, four mAbs (AP4, DN11, SHL-1, and P6) recognized a region commonly present in all the five isoforms. One mAb, YG27, recognized four isoforms (ABC, AB, BC, and O). Two mAbs, P1 and P14, recognized the isoforms that contain exon A encoded regions (ABC and AB). CONCLUSION: In this study, we confirmed that AP4, DN11, SHL-1, YG27 and P6, are mAbs reactive with the CD45 antigen whereas P1 and P14 are reactive with the CD45RA antigen.

Characterization of a Novel Monoclonal Antibody (27H2) Recognizing Human CD34 Class III Epitope

BACKGROUND: Monoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia. METHODS: 27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done. RESULTS: Only KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity. CONCLUSION: A novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

Correlation analysis of Each Variable of Fetal Heart rate in Pregnancy induced hypertension and Intrauterine fetal growth restriction / 대한산부인과학회잡지

OBJECTIVE: We aim to analyze each variable of FHR in high risk pregnancies, namely intrauterine growth restriction (IUGR) and pregnancy-induced hypertension (PIH) including chronic hypertensive vascular disease (CHVD), mild and severe preeclampsia to build an objective decision basis using correlation analysis. METHODS: The patients were divided into two groups (500 normal pregnancies and 500 high risk pregnancies related to IUGR, CHVD, mild and severe preeclampsia), and then subdivided into intrauterine pregnancy before 24 weeks, 25-39 weeks, and after 40 weeks. We compared the canonical correlation between each group using variables of FHR after nonstress test (NST). RESULTS: In high risk pregnancies, the linearity was 0.6-0.8 in intrauterine pregnancy before 24 weeks, 0.53-0.68 in 25-29 weeks, 0.50-0.60 in 30-34 weeks, 0.38-0.45 in 35-39 weeks and 0.42-0.55 in after 40 weeks. In normal pregnancies, the linearity was 0.44-0.52 in intrauterine pregnancy before 24 weeks, 0.38-0.45 in 25- 39 weeks which was stable, and there was no specific change in after 40 weeks. Before 32 weeks, canonical variates of FHR_D and FHR_I revealed highest (0.36, 0.47 respectively) in high risk pregnancy and fetal movement and signal loss was the most valuable factors in normal pregnancy. In between 33 to 37 weeks, fetal movement (0.40) and signal loss (0.48) were related most closely in high risk pregnancies and 0.34 and 0.49 respectively in normal pregnancies which show similar pattern. In contrast, FHR_D was most highly related to the duration of pregnancy and FHR_I to fetal movement (0.38) in high risk pregnancy. In normal pregnancies, fetal movement (0.40) and signal loss (0.52) showed the highest linearity. CONCLUSION: The pregnancy with intrauterine growth restriction and pregnancy induced hypertension has more linear relation and less complexity in each variable of FHR than the normal pregnancy group. The formal, functional underdevelopment of fetus may results in the increasement of the linear depedent relation in each variable of FHR in these type of high risk pregnancies.

WEHI-231 cells are defective in the ligand-induced internalization of B cell antigen receptor

No abstract available.

The Analysis of FHR Parameters and Canonical Correlation of Fetuses with Breech Presentation / 대한주산의학회잡지

No abstract available.

Development of Algorithm for Estimation of Gestational Age using each Variable of Fetal Heart Rate / 대한산부인과학회잡지

OBJECTIVE: The objective of this study is to estimate gestational age by using FHR parameters after linear and nonlinear analysis of FHR data. Linear spectral decomposition has a limit to describe and analyze the nonlinear and complex physiological nature. Random processing and chaotic analysis assist to quantify some patterned energies, interaction of the physiological system in body system, which the constancy is preserved, and complexity of physical cardiovascular system. But there are no great development of new device which notify fetal age. METHODS: From March 1995 to December 2000, 2,548 cases who received NST during antenatal visit were included in this study. Among these cases, 1000 cases were sampled randomly. We divided this cases into a study and a control group, 500 cases for model building set and the other 500 cases for validation set. Each FHR parameters such as baseline FHR, variability (AMP, MMR), acceleration & deceleration (15 bpm-15 seconds), loss of signal, the number of fetal movement were analysed by using our own computerized HYFM-I, II software system. The linear and non-linear analysis of FHR were done after extracting approximate entropy (ApEn) value. Finally, we performed regression analysis and extract an equation for estimation of gestational weeks using bootstrap method. RESULTS: We extract a following equation using above study method: Log(GA)=5.870-0.051(Sloss*)-0.065(Mean FHR*)+0.049(Mean FHR*)2-0.058(FM*)+0.048(AMP*)2+0.121(A1515*)-0.031(A1515*)2+0.036(ApEn*)2. CONCLUSIONS: Accurate dating of pregnancy, namely gestational week is very important for antenatal diagnosis in the field of perinatal medicine. This study will propose scientific research results which are useful for studying normal and morbid generating physiological fetal condition. We extract an appropriate equation for estimation of gestational weeks only using FHR parameters. We hope to compare our result with other authors' results in the near future.

Development of Algorithm for Estimation of Gestational Age using each Variable of Fetal Heart Rate / 대한산부인과학회잡지

OBJECTIVE: The objective of this study is to estimate gestational age by using FHR parameters after linear and nonlinear analysis of FHR data. Linear spectral decomposition has a limit to describe and analyze the nonlinear and complex physiological nature. Random processing and chaotic analysis assist to quantify some patterned energies, interaction of the physiological system in body system, which the constancy is preserved, and complexity of physical cardiovascular system. But there are no great development of new device which notify fetal age. METHODS: From March 1995 to December 2000, 2,548 cases who received NST during antenatal visit were included in this study. Among these cases, 1000 cases were sampled randomly. We divided this cases into a study and a control group, 500 cases for model building set and the other 500 cases for validation set. Each FHR parameters such as baseline FHR, variability (AMP, MMR), acceleration & deceleration (15 bpm-15 seconds), loss of signal, the number of fetal movement were analysed by using our own computerized HYFM-I, II software system. The linear and non-linear analysis of FHR were done after extracting approximate entropy (ApEn) value. Finally, we performed regression analysis and extract an equation for estimation of gestational weeks using bootstrap method. RESULTS: We extract a following equation using above study method: Log(GA)=5.870-0.051(Sloss*)-0.065(Mean FHR*)+0.049(Mean FHR*)2-0.058(FM*)+0.048(AMP*)2+0.121(A1515*)-0.031(A1515*)2+0.036(ApEn*)2. CONCLUSIONS: Accurate dating of pregnancy, namely gestational week is very important for antenatal diagnosis in the field of perinatal medicine. This study will propose scientific research results which are useful for studying normal and morbid generating physiological fetal condition. We extract an appropriate equation for estimation of gestational weeks only using FHR parameters. We hope to compare our result with other authors' results in the near future.

The analysis of FHR parameters, approximate entropy and canonical correlation of fetuses with mild baseline tachycardia / 대한산부인과학회잡지

OBJECTIVE: This study was to determine differences of fetal heart rate (FHR) parameters, approximate entropy, canonical correlation coefficient and perinatal outcome between fetuses with mild baseline tachycardia (FHR range : 161-180 bpm) and fetuses with normal baseline FHR. METHODS: Using our own software developed by Hanyang University Hospital in Korea, non-stress tests were performed for 20 minutes and cases were divided into two groups; mild tachycardia group (FHR range ; 161-180 bpm, 100 cases) and normal group (FHR ranges ; 120-160 bpm, 197 cases). And then, we analyzed various FHR parameters, approximate entropy, adjusted canonical correlation coefficient and perinatal outcome in each group. RESULTS: Perinatal outcomes between two groups were not significant. However, mean minute range (MMR) was lower and the number of deceleration (15 bpm for 15 sec, more than one) was significantly higher in fetuses with mild tachycardia than normal group (p<0.05). The apprximate entropy and canonical correlation coefficient showed respectively 0.77+/-0.02, 0.45+/-0.06 in mild tachycardia group and 0.80+/-0.01, 0.43+/-0.07 in normal group, there were no significant differences. CONCLUSIONS: The results showed that mild baseline tachycardia in this range were not associated with an increase in the incidence of adverse perinatal outcome, although decreased MMR and increased number of deceleration in mild tachycardia group may suggest hidden subtle change of FHR parameters. Fetuses with mild tachycardia were not different irregularity of FHR quantified by approximate entropy from normal fetuses and were also similar tendency to correlation with perinatal outcome by adjusted canonical correlation coefficient, which is a multivariate extension of the univariate correlation analysis.

The analysis of FHR parameters, approximate entropy and canonical correlation of fetuses with mild baseline tachycardia / 대한산부인과학회잡지

OBJECTIVE: This study was to determine differences of fetal heart rate (FHR) parameters, approximate entropy, canonical correlation coefficient and perinatal outcome between fetuses with mild baseline tachycardia (FHR range : 161-180 bpm) and fetuses with normal baseline FHR. METHODS: Using our own software developed by Hanyang University Hospital in Korea, non-stress tests were performed for 20 minutes and cases were divided into two groups; mild tachycardia group (FHR range ; 161-180 bpm, 100 cases) and normal group (FHR ranges ; 120-160 bpm, 197 cases). And then, we analyzed various FHR parameters, approximate entropy, adjusted canonical correlation coefficient and perinatal outcome in each group. RESULTS: Perinatal outcomes between two groups were not significant. However, mean minute range (MMR) was lower and the number of deceleration (15 bpm for 15 sec, more than one) was significantly higher in fetuses with mild tachycardia than normal group (p<0.05). The apprximate entropy and canonical correlation coefficient showed respectively 0.77+/-0.02, 0.45+/-0.06 in mild tachycardia group and 0.80+/-0.01, 0.43+/-0.07 in normal group, there were no significant differences. CONCLUSIONS: The results showed that mild baseline tachycardia in this range were not associated with an increase in the incidence of adverse perinatal outcome, although decreased MMR and increased number of deceleration in mild tachycardia group may suggest hidden subtle change of FHR parameters. Fetuses with mild tachycardia were not different irregularity of FHR quantified by approximate entropy from normal fetuses and were also similar tendency to correlation with perinatal outcome by adjusted canonical correlation coefficient, which is a multivariate extension of the univariate correlation analysis.

A Case of T3 Thyrotoxicosis developed after Cesarean Section / 대한산부인과학회잡지

Thyroid storm (thyrotoxicosis) is rarely encountered during pregnancy or the puerperium, even in untreated women and characterized by hypertension, hyperthermia, and multiple systems involvement. Much more common is heart failure, apparently caused by the long-term myocardial effects of thyroid hormone and intensified by other pregnancy complications that include severe preeclampsia, infection, anemia, or combinations of these. Prompt diagnosis and treatment of thyroid storm are essential in reducing morbidity and mortality of a patient from this disorder. We experienced a case of triiodothyroxine induced thyroid storm (T3 thyrotoxicosis) developed after Cesarean section. This case highlights both physicians should be aware of the symptoms and risk factors and reversing of heart failure successfully by reducing cardiac workload is important through immediate medical treatment - adequate hydration, thermoregulation, and control of hypertension, infection and anemia et al.

Correlation Analysis of Each Variable of Fetal Heart Rate in Normal and Abnormal Pregnancies / 대한주산의학회잡지

No abstract available.