No abstract available.

The T cell antigen receptor (TcR) in combination with costimulatory signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC) regulates the activation and growth of T lymphocytes. Calyculin A and Okadaic acid is known to be an inhibitor of serine/threonine phosphatase and RK-682 specifically blocks functions of tyrosine phosphatase. To investigate roles of these inhibitors in TcR-mediated signaling cascade, chimeric molecule CD8-5 which contains the extracellular and transmembrane domains of the human CD8a molecule and the cytoplasmic tail of TcR 5 chain were stably expressed in Jurkat cell line. CD8-5 chimeric protein induced tyrosine phosphorylation of various cytoplasmic substrates and IL-2 gene expression in a NFAT dependent manner by stimulation with anti-CD8 mAb OKT8 as seen in TcR stimulation. When CD8-5 transfectants were preincubated with Okadaic acid, Calyculin or RK682, they differentially affected tyrosine phosphorylation of signaling mediators including CD8-5 molecule. When Jurkat Tag cell line was used where SV40 T antigen is stably expressed and the expression of p-galactosidase is driven by the multiple NFAT binding sites plus minimal IL-2 promoter, these phosphatase inhibitors -RK682, Calyculin A, Okadaic acid- effectively inhibited IL-2 gene expression at the concentration of 1.2832 x 10 ' M, 3.9924 x 10 M, 7.2707 x 10 M respectively. These results suggested that Okadaic acid, Calyculin or RK682 modulate TcR-proximal as well as TcR-distal signaling events during T cell activation.
Antigen-Presenting Cells ; Antigens, Viral, Tumor ; Binding Sites ; Cell Line ; Cytoplasm ; Gene Expression ; Humans ; Interleukin-2 ; Jurkat Cells ; Okadaic Acid* ; Phosphorylation ; Receptors, Antigen, T-Cell ; T-Lymphocytes ; Tyrosine

The systematic study of products from bacteria and fungi has led to the development of two immunosuppressive drugs, cyclosporin A and FK 506 (tacrolimus) which are useful to suppress adaptive immune responses to the grafted tissue. However, they affect all immune responses indiscriminately and are both toxic to kidneys and other organs. To facilitate the development of immunosuppressor to block the T cell receptor (TcR)-mediated signaling cascade specifically, a novel Jurkat T cell transfectants, JK NFAT-SEAP were generated in which the expression of the secreted alkaline phosphatase (SEAP) is driven by the multiple NFAT binding sites plus minimal IL-2 promoter. Upon stimulation with ionomycin or anti-TcR mAb OKT3 in the presence of PMA, these transfectants secreted high level of SEAP into the medium, which was conveniently analyzed by SEAP analysis. The secretion of SEAP was effectively inhibited by cyclosporin A or FK 506 at the concentration of [10 ' ug/ml], [10 ug/ml] respectively. JK NFAT-SEAP transfectants will provide two major advantages for the development of a novel immunosuppressor. First, analysis of SEAP secreted into the culture medium by SEAP analysis enables us to test a large number of samples within a short period of time. Second, Usage of IL-2 promoter for the expression of SEAP makes us identify bioproducts to target specifically on TcR-mediated signaling pathway.
Alkaline Phosphatase ; Bacteria ; Binding Sites ; Cell Line* ; Cyclosporine ; Fungi ; Interleukin-2 ; Ionomycin ; Kidney ; Mass Screening* ; Muromonab-CD3 ; Receptors, Antigen, T-Cell ; T-Lymphocytes* ; Tacrolimus ; Transplants

We report a case of hemolytic disease of the newborn caused by anti-Mia (Miltenberger) antibody. Full term male infant was admitted due to hyperbilirubinemia on second day of life. Total serum bilirubin level was 8.6 mg/dL at 12 hours of age and 12.3 mg/dL at 24 hours of age. The blood group of patient and his mother were both RhD positive B type. Direct antiglobulin test was strongly positive in the patient, and testing of maternal serum and patient's serum against a red cell panel including cells known to carry the antigenic determinants of some Miltenberger phenotypes revealed the presence of anti-Mia . Testing of paternal red cells and patient's red cell against anti-Mia serum revealed positive reaction. This report documents the first case of hemolytic disease of the newborn due to anti-Mi a in Korea.
Bilirubin ; Coombs Test ; Epitopes ; Humans ; Hyperbilirubinemia ; Infant ; Infant, Newborn* ; Korea ; Male ; Mothers ; Phenotype

OBJECTIVE: The purpose of this study was to investigate the incidence of apoptosis and expression of bcl-2 in the placenta of normal pregnancy, Pregnancy Induced Hypertension, and Intrauterine Growth Restriction. METHODS: Placenta samples were collected from 15 cases of normal full-term pregnancies, 15 cases of second trimester pregnancies, 17 cases of Pregnancy Induced Hypertension, and 13 cases of Intrauterine Growth Restriction. Hematoxylin and eosin staining and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate marker nick end-labeling) staining were used to quantify the incidence of apoptosis and the electron microscopy was used to confirm it. Expression of bcl-2 was confirmed by using immunohistochemical stain in relation to apoptosis. RESULTS: 1. In TUNEL staining, quantification of apoptosis was 1.05 in 2nd trimester (n=15), 3.65 in pregnancy induced hypertensive pregnancy (n=17), 2.92 in intrauterine growth restrictive pregnancy (n=13) and 1.93 in normal full-term pregnancy (n=15). The incidence of apoptosis was significantly higher in placental tissues from full-term pregnancies than second trimester pregnancies (p<0.05, t test), and higher in pregnancy induced hypertensive pregnancy and in intrauterine growth restrictive pregnancy than in normal full-term pregnancy (p<0.05, t test). 2. Bcl-2 expression was significantly higher in placental tissues from second trimester pregnancies than full-term pregnancies (p<0.05 t test). But, there was no statistically significant difference between pregnancy induced hypertension and normal full-term pregnancy (p>0.05, t test), and between intrauterine growth restriction and normal full-term pregnancy (p>0.05, Mann-Whitney U test). CONCLUSION: These data suggest that apoptosis increases with gestational age, and in pathophysiologic states such as pregnancy induced hypertension and intrauterine growth restriction, and that bcl-2 expression is lower with gestational age.
Apoptosis* ; Deoxyuridine ; Eosine Yellowish-(YS) ; Female ; Gestational Age ; Hematoxylin ; Humans ; Hypertension, Pregnancy-Induced* ; In Situ Nick-End Labeling ; Incidence ; Microscopy, Electron ; Placenta ; Pregnancy Trimester, Second ; Pregnancy*

OBJECTIVE: The purpose of this study was to investigate the incidence of apoptosis and expression of bcl-2 in the placenta of normal pregnancy, Pregnancy Induced Hypertension, and Intrauterine Growth Restriction. METHODS: Placenta samples were collected from 15 cases of normal full-term pregnancies, 15 cases of second trimester pregnancies, 17 cases of Pregnancy Induced Hypertension, and 13 cases of Intrauterine Growth Restriction. Hematoxylin and eosin staining and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate marker nick end-labeling) staining were used to quantify the incidence of apoptosis and the electron microscopy was used to confirm it. Expression of bcl-2 was confirmed by using immunohistochemical stain in relation to apoptosis. RESULTS: 1. In TUNEL staining, quantification of apoptosis was 1.05 in 2nd trimester (n=15), 3.65 in pregnancy induced hypertensive pregnancy (n=17), 2.92 in intrauterine growth restrictive pregnancy (n=13) and 1.93 in normal full-term pregnancy (n=15). The incidence of apoptosis was significantly higher in placental tissues from full-term pregnancies than second trimester pregnancies (p<0.05, t test), and higher in pregnancy induced hypertensive pregnancy and in intrauterine growth restrictive pregnancy than in normal full-term pregnancy (p<0.05, t test). 2. Bcl-2 expression was significantly higher in placental tissues from second trimester pregnancies than full-term pregnancies (p<0.05 t test). But, there was no statistically significant difference between pregnancy induced hypertension and normal full-term pregnancy (p>0.05, t test), and between intrauterine growth restriction and normal full-term pregnancy (p>0.05, Mann-Whitney U test). CONCLUSION: These data suggest that apoptosis increases with gestational age, and in pathophysiologic states such as pregnancy induced hypertension and intrauterine growth restriction, and that bcl-2 expression is lower with gestational age.
Apoptosis* ; Deoxyuridine ; Eosine Yellowish-(YS) ; Female ; Gestational Age ; Hematoxylin ; Humans ; Hypertension, Pregnancy-Induced* ; In Situ Nick-End Labeling ; Incidence ; Microscopy, Electron ; Placenta ; Pregnancy Trimester, Second ; Pregnancy*

Ael is a rare blood type which has the least amount of A antigen among A subgroups. It can be detected by special tests performed to resolve the discrepancy between red cell and serum typing in routine serological typing. The presence of A antigen on Ael red cell is demonstrable only by adsorption and elution tests. An Ael individual does not secret A substance in the saliva and may have anti-A antibody in the serum which is usually less reactive with the reagent red cells than anti-B antibody. In Korea, Ael02 has been reported more frequently than other Ael alleles. We report a case of Ael02/O04 who presented as typical phenotype O with strong anti-A and anti-B antibodies and no A antigen detected even by adsorption and elution tests. The case has been proved to be Ael02/O04 by direct sequencing analysis. In individuals with history of discrepancies in the results of ABO phenotyping, ABO genotyping is needed for an accurate evaluation of their blood type.
ABO Blood-Group System/classification/*genetics ; Alleles ; Child ; Genotype ; Heterozygote ; Humans ; Male ; Pedigree ; Phenotype ; Sequence Analysis, DNA

BACKGROUND: ABO genotyping is a useful tool in case of ABO discrepancies and in legal medicine. Recent knowledge of various alleles in the ABO gene has led to the need of a different method that can cover numerous polymorphisms. We performed a polymerase chain reaction using sequencespecific priming (PCR-SSP) with 12 primer sets and evaluated its value in the detection of 8 ABO alleles. METHODS: Genomic DNA was isolated from peripheral blood of 222 unrelated Koreans. Sequencespecific primer sets for the nucleotides 261, 297, 467, 802, 803, and 1059 were selected, and 12 PCR reactions were performed for each sample. Direct sequencing was performed to evaluate the accuracy of discrimination between A1(Pro) and A1(Leu) and between O1 and O1v. RESULTS: All the ABO genotype patterns were in an exact match with the ABO phenotypes. The results from sequencing and PCR-SSP were equivalent. The allele frequencies of A1, B, O1, and O1v were 27.25%, 19.82%, 27.25%, and 25.68% respectively. Out of total 121 A1 alleles, 6 (4.96%) were A1(Pro) alleles and 115 (95.04%) were A1(Leu) alleles. No A2, O2, or CisAB alleles were found in this study. CONCLUSIONS: The proportion of O1v allele was similar to that of O1 allele. This was an unexpected result. We developed a method for detecting 8 ABO alleles by PCR-SSP; the method was accurate and was able to discriminate between A1(Pro) and A1(Leu) and between O1 and O1v.
Alleles* ; Discrimination (Psychology) ; DNA ; Forensic Medicine ; Gene Frequency ; Genotype ; Nucleotides ; Phenotype ; Polymerase Chain Reaction

BACKGROUND: The number of reagent red cells has an effect on the results of an unexpected antibody screening test. We evaluated the effect of the number of reagent red cells on antibody screening and identification using test panels of a DG Gel (Diagnostic Grifols, Barcelona, Spain). METHODS: A total of 310 samples were tested in parallel using SeraScan Diana 2 and SeraScan Diana 4 (Diagnostic Grifols, Barcelona, Spain) and ID-DiaCell (DiaMed, Cressier, Morat, Switzerland). Positive samples as determined by the use of SeraScan and ID-DiaCell were identified on an ID-Dia panel (DiaMed), Identisera Diana and Identisera Extend (Diagnostic Grifolsn). RESULTS: Among the 310 samples, 54, 59 and 59 samples were determined as antibody positive by the use of SeraScan Diana 2, SeraScan Diana 4 and ID-DiaCell, respectively. Unexpected antibodies were identified in 10/59 samples (17%) by the use of SeraScan Diana 4, and were identified in 34/59 samples (57.6%) by the combined use of SeraScan Diana 2 and SeraScan Diana 4. Identification of unexpected antibodies by the use of Identisera Diana or Identisera Extend was not different. CONCLUSION: When the results of SeraScan Diana 2 and SeraScan Diana 4 are integrated, unexpected antibodies could be identified in 57.6% of the screening-positive samples. Therefore, if the number of reagent red cells is increased, some antibodies can be identified by antibody screening tests, and the results can be used to validate those of antibody identification tests.
Antibodies ; Mass Screening*

BACKGROUND: The risk of transfusion-transmitted bacterial infection has been reported in addition to the risk of transmission of viral disease. Especially for platelets that are stored at 20degrees C~24degrees C with agitation to sustain platelet function. This storage method facilitates bacterial proliferation. Therefore, sensitive and rapid detection of bacteria must be considered for stored platelets. METHODS: Six concentrations of platelets were spiked (1.5 mL) with five different bacteria and were analyzed by the 16S rDNA real-time polymerase chain reaction (PCR) using the ABI PRISM 7500 (Applied Biosystems, Foster, CA, USA) and the LightCycler 2.0 (Roche, Penzberg, Germany). The 16S rDNA gene was analyzed in three lots by real-time PCR reagents with sterile water. Three concentrations of spiked platelets (0.5 mL) with three different bacteria were preincubated in thioglycollate medium at 37degrees C for 8, 12, 16, 20, and 24 hours and then were analyzed by the 16S rDNA real-time PCR. The spiked platelets (0.5 mL) with fast-growing (8 hours of preincubation) and slow-growing (24 hours preincubation) bacteria were analyzed for the minimum incubation time. RESULTS: The average crossing points (Cps) of the five bacteria were 21.2 with the ABI PRISM 7500 and 22.2 in the LightCycler 2.0. All five bacteria (10(1) bacteria/mL) were detected by both instruments. The average Cps of the three lots by real-time PCR was 29.3, 31.5 and 35.6. The contamination levels of the 16S rDNA were different. Fast-growing and slow-growing bacteria, preincubated at 8 and 24 hours, respectively, were detected at levels of 10(1) bacteria/mL. CONCLUSION: The results of this study suggest that slow-growing bacteria can be detected at concentrations of 10(1) bacteria/mL in platelets preincubated with thioglycollate medium, at 37degrees C for 24 hours using platelets segment.
Bacteria ; Bacterial Infections ; Blood Platelets ; Dihydroergotamine ; DNA, Ribosomal ; Indicators and Reagents ; Real-Time Polymerase Chain Reaction ; Virus Diseases ; Water