This study was aimed to establish the reliable genotyping method of human platelet antigens of HPA-15 system by PCR-SSP and to use this assay in the further HPA genotyping of volunteer platelet donors. 3 sequence-specific primers recommended by the 11th Platelet Genotyping and Serology Workshop on behalf of International Society of Blood Transfusion (ISBT) were synthesized. The concentration of each primer pair, the concentration of Mg(2+) and the PCR conditions were adjusted to optimize the conditions so that HPA-15 system could be specific amplified. The accuracy and reliability of the developed assay was evaluated and confirmed by typing the coded DNA samples provided by the 11th Platelet Genotyping and Serology Workshop. As a parallel control, a total of 50 volunteer platelet donors in Shenzhen were genotyped by both our assay and the G&T commercial kit at HPA-15 system. 10 coded samples distributed by the 11th Platelet Genotyping and Serology Workshop were genotyped by established PCR-SSP method. The results showed that a concordance rate of 100% was observed between the results obtained by established PCR-SSP method and the results provided by ISBT report. The HPA gene frequencies observed in 50 randomly-selected platelet donors in Shenzhen were 0.5100 and 0.4900 for HPA-15a and HPA-15b respectively. In conclusion, PCR-SSP assay established in our study provides a simple, rapid and accurate method for HPA-15 system genotyping, which assay is suitable for routine clinical HPA genotyping and shows a broad prospect in its further applications.
Antigens, CD ; genetics ; immunology ; Antigens, Human Platelet ; genetics ; GPI-Linked Proteins ; Genotype ; Humans ; Isoantigens ; genetics ; immunology ; Neoplasm Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.
Transplantation, Homologous/immunology ; Signal Transduction/*immunology ; Mice, Knockout ; Mice ; Isoantigens/immunology ; Heart Transplantation/immunology ; Graft Survival/immunology ; Cytotoxicity Tests, Immunologic ; Antigens, CD28/genetics/*immunology/metabolism ; Antibodies/immunology ; Animals ; 4-1BB Ligand/deficiency/genetics/*immunology/metabolism

Neonatal alloimmune neutropenia (NAN) is a disease that can cause severe and prolonged neutropenia in neonates. However, no report is available on the incidence of granulocyte antibody in neonates, the target antigen of this antibody, and the estimated incidence of NAN in Korea. Among a total of 856 neonates admitted to a neonatal intensive care unit (NICU) over a five year period, a total of 105 neonates with neutropenia were enrolled in this study. Positive reactions were observed in the sera of six neonates (5.7%, 6/105) by mixed passive hemagglutination assay (MPHA). To confirm the presence of NAN, MPHA and granulocyte antigen typing (HNA-1a, -1b, -2a, -4a, and -5a) were performed on neonatal and maternal blood. To differentiate granulocyte antibody and HLA antibody, MPHA was also performed using HLA antibody adsorbed serum. We confirmed three cases (2.9%, 3/105) of NAN among neonates with neutropenia in which granulocyte antibody specificities (two anti-HNA-1b and one anti-HNA-1a) and fetomaternal granulocyte antigen mismatches were identified. In this study, the estimated incidence of NAN was 0.35% (3/856) among neonates admitted to NICUs in Korea.
Polymerase Chain Reaction/methods ; Neutropenia/blood/diagnosis/*immunology ; Korea ; Isoantigens/genetics/immunology ; Isoantibodies/*immunology ; Intensive Care Units, Neonatal/statistics & numerical data ; Infant, Newborn ; Humans ; Hemagglutination Tests ; HLA Antigens/immunology ; Granulocytes/*immunology ; Genotype ; Antibody Specificity/immunology

OBJECTIVETo explore the value of HLA-DRB1 gene in predicting the outcome of unexplained recurrent spontaneous abortion (URSA) treated with paternal lymphocyte alloimmunization therapy (PLAT) in Henan Hans.

METHODSThree hundred URSA patients were recruited. Following PLAT treatment, they were divided into two groups according to the outcome of pregnancy. Polymerase chain reaction sequence specific primer (PCR-SSP) were conducted to analyze the HLA-DRB1 gene.

RESULTSFor those who have received PLAT treatment, the frequency of HLA-DRB1*11 was significantly lower in successfully treated cases than those with abortion (0.052 vs. 0.110, P < 0.05, OR=0448), whilst the frequency of HLA-DRB1*15 was significantly greater in the former (0.207 vs. 0.100, P < 0.05, OR=2.352).

CONCLUSIONFor patients who have received PLAT treatment, those with HLA-DRB1*15 are more likely to conceive that those with HLA-DRB1*11.

Abortion, Spontaneous ; ethnology ; genetics ; immunology ; therapy ; Asian Continental Ancestry Group ; ethnology ; genetics ; China ; Female ; Genetic Predisposition to Disease ; ethnology ; HLA-DRB1 Chains ; genetics ; Humans ; Immunotherapy ; Isoantigens ; immunology ; Lymphocytes ; immunology ; Male ; Pregnancy ; Treatment Outcome

Neonatal alloimmune neutropenia (NAN) is an uncommon disease of the newborn provoked by the maternal production of neutrophil-specific alloantibodies, whereby neutrophil IgG antibodies cross the placenta and induce the destruction of fetal neutrophils. Affected newborns are usually identified by the occurrence of bacterial infections. The most frequent antigens involved in NAN are the human neutrophil antigen-1a (HNA-1a), HNA-1b, and HNA-2a. We report a neonate who was delivered at 36 weeks and had a severe neutropenia but who responded well to recombinant human granulocyte colony-stimulating factor (rhG-CSF). Anti-HNA-1a antibody was identified by mixed passive hemagglutination assay in both the sera of the baby and the mother. The baby had HNA-1a and HNA-1b but the mother had only HNA-1b on granulocytes. This is the first Korean report of NAN in which the specificity of the causative antibody was identified.
Pregnancy ; Neutrophils/immunology ; Neutropenia/drug therapy/etiology/genetics/*immunology ; Maternal-Fetal Exchange/immunology ; *Isoantigens/genetics ; Isoantibodies/*blood ; Infant, Newborn ; Immunoglobulin G/blood ; Humans ; Granulocyte Colony Stimulating Factor, Recombinant/therapeutic use ; Genotype ; Female ; DNA/genetics ; Base Sequence ; Antibody Specificity ; Adult

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.
ABO Blood-Group System ; classification ; immunology ; Blood Group Incompatibility ; prevention & control ; Blood Transfusion ; methods ; Coffee ; enzymology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; immunology ; metabolism ; Humans ; Isoantigens ; drug effects ; metabolism ; Recombinant Proteins ; metabolism ; pharmacology ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology

BACKGROUNDPrevious studies showed that anti MHC-II monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope (188)Re marked MHC-II antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-II antibody.

METHODS188Re was incorporated to 2E9/13F (ab')(2) which is against swine MHC class II antigen (MAb-(188)Re). Porcine peripheral blood mononuclear (PBMC) cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-II MAb or MAb-(188)Re.

RESULTSThe proliferative response of recipient PBMCs in mixed lymphocyte reaction (MLR) to donor alloantigen showed that the stimulation index of MAb-(188)Re group was significantly lower than the MHC-II MAb group and control (P < 0.05). mRNA expression of interleukin 2, interferon Υ and tumor necrosis factor α (type 1 cytokines) was lower in MAb-(188)Re group than the MHC-II MAb group, while interleukin 10 (type 2 cytokines) was higher in MAb-(188)Re group in the first 24 hours.

CONCLUSIONMAb-(188)Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.

Animals ; Antibodies, Monoclonal ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Interleukin-10 ; genetics ; Interleukin-2 ; genetics ; Isoantigens ; immunology ; Leukocytes, Mononuclear ; drug effects ; radiation effects ; Lymphocyte Culture Test, Mixed ; Mitomycin ; pharmacology ; Radioisotopes ; Reverse Transcriptase Polymerase Chain Reaction ; Rhenium ; Swine ; Tumor Necrosis Factor-alpha ; genetics