Objective:To analyze the polymorphism and haplotypes of HLA-A,B,DRB1 loci in Guangzhou blood donors.Methods:PCR-SSP methods were applied to determine HLA-A,B,DRB1 genotypes of 1 691 unrelated healthy individuals from Guangzhou,and the frenquencies of alleles and haplotypes were calculated with software of Pypop.Results:37 HLA-A alleles,76 HLA-B alleles and 43 HLA-DRB1 alleles were detected,and A02:07-B46:01-DRB109:01 and A33:03-B58:01-DRB103:01 were the most common haplotypes in Guangzhou blood donors.Conclusion:The distribution of alleles and haplotypes of Guangzhou blood donors are close to those of southern Chinese population and provide more genetic information in anthropology,transplantation and studies involoing disease association.

Objective:To characterize the allele frequencies and its polymorphisms of human platelet antigen (HPA) in Guangzhou population.Methods:A total of 500 samples from healthy voluntary platelet donors in Guangzhou were genotyped for HPA-1 to-17 by PCR-SSP.Results:HPA-1a to-17a alleles were found in each of the samples;The gene frequencies of HPA-1a to-17a were 99.8%,99.85%,56.3%,99.9%,98.8%,98.6%,100%,100%,100%,100%,100%,100%,100%,100%,55.1%,and 100%,100% respectively.The gene frequencies of HPA-1b to-6b and-15b alleles were 0.2%,0.15%,43.7%,0.1%,1.2%,1.4% and 44.9% respectively;HPA-7b to-14b and-16b-17b were not detected.In summary Guangzhou population displayed higher frequency of HPA-1a to-17a and HPA-3b,-15b.Compared with those of other Han populations in China,HPA frequency of Guangzhou people was significantly different from that of Beijing;Compared to that of the European,American,English and Egyptians,HPA frequency was different significantly.While HPA frequency was different from those of Japanese and Thais.This study for the first time investigated the assortment of HPA genes and its frequency,there were 40 assortments in Guangzhou population,only 5 assortment of HPA gene frequencies more than 10%,35 assortment of HPA gene frequencies less than 9%.Conclusion:HPA distribution in Guangzhou population appears to have local characteristics.This study confirms the ethnic and original difference of HPA.The allele frequencies and its polymorphisms of HPA in Han population are shown North-South differences.Races and countries outside Asia are also shown differences.The basic information provided by this study of the HPA system polymorphisms is useful to guide the design of the local HPA genotype database of volunteer platelet donors.It's also useful to avoid the PTR,and the HPA related clinical research.

Objective To evaluate the effect of combination therapy of tolterodine and tamsulosin in improving symptoms in patients with indwelling double-J ureteral stents.Methods A total of 96 patients underwent placement of a double-J ureteral stent after retrograde ureteroscopy for urinary stone disease,which were prospectively randomized ( random numbers table) into two groups.The tamsulosin group ( n =48) was administered tamsulosin 0.2 mg once daily,the combination group (n =48) was administcred tamsulosin 0.2 mg once daily and tolterodine 2 mg twice daily.All the patients completed a validated Ureteral Stent Symptom Questionnaire (USSQ),the Overactive Bladder Symptom Score (OABSS),the International Prostate Symptom Score (IPSS) one day before he placement and 4 weeks after stent placement of stent.Results The mean urinary symptom index score (25.4 ± 4.0 vs 15.3 ± 2.9,P ＜ 0.0001 ),the mean pain index score (15.0 ±2.6 vs10.3 ±2.1,P ＜0.0001),the mean general health index score (15.4 ±2.2 vs 11.0±1.5,P＜0.0001),the work performance index score (13.0±2.2vs9.7±1.9,P＜0.0001),the sex index score (3.6 ± 1.1 vs 2.2 ± 0.9,P ＜0.0001 ) improved significantly in combination group than those in tamsulosin group.There was a statistically significant difference between tamsulosin group and combination group in the IPSS score ( 12.8 ± 2.0 vs 9.2 ± 1.7,P ＜ 0.0001 ) and OABSS score ( 6.7 ± 1.4 vs 4.2 ± 1.4,P ＜ 0.0001 ) at the 4 weekfollow-up.Conclusions Indwelling ureteral stents have a significant impact on health related quality of life.It is effective for improving symptoms in patients with indwelling double-J ureteral stents by combination therapy with tolterodine and tamsulosin.

OBJECTIVETo analyze the sequence of a novel human leukocyte antigen (HLA)-A*33:44 allele.

METHODSA novel HLA-A allele was found by double-stranded sequencing combined with single-stranded sequencing. The frequency of the novel allele was determined by population survey.

RESULTSGenomic sequence of this novel HLA-A*33:44 allele (accession No. HQ873871) has differed from HLA-B*33:03:01 by one nucleotide in exon 4, which resulted in nt 866 G→ A substitution, which results in an amino acid substitution from Gly(GGT) to Asp(GAT) at codon 265. This alternation is a new single nucleotide polymorphism compared with other HLA-A alleles. The frequency of this new allele is less than 0.0003 in Chinese Han population.

CONCLUSIONA mutation has been found in exon 4 of the novel HLA-A*33:44 allele, which may provide more information for HLA gene study.

Adult ; Alleles ; Amino Acid Substitution ; Asian Continental Ancestry Group ; genetics ; Female ; HLA-A Antigens ; genetics ; Humans ; Male ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; methods

Objective To establish a cell line stably expressing the human CD36 by using TA clo-ning and cell transfection technology and to analyze its application to the detection of anti-CD36 antibodies. Methods Total RNA was isolated from human platelets and then used to synthesize complementary DNA ( cDNA) . Sequence of the gene encoding CD36 on human platelets was obtained by PCR amplification. The recombinant vector was transformed into TOP10 E. coli after TA cloning. The positive recombinant pcDNA3. 1/V5-CD36 plasmid was screened out by blue-white selection and then sequenced. The correctly constructed plasmid coated with Effectene? Transfection Reagent was transferred into HEK293T cells. Fluo-rescence-activated cell sorting was performed to screen out the cell line that could stably express the CD36 on human platelets. The transfected cell line-based flow cytometry analysis and antibody capture assay ( ACA) were established and used for antibody detection in nine serum samples positive for anti-CD36 antibodies. Results The HEK293T cell line stably expressing the recombinant CD36 was successfully established. Compare with the monoclonal antibody immobilization of platelet antigens assay ( MAIPA) , anti-CD36 anti-bodies could be easily identified in nine serum samples by using the transfected cell line-based flow cytome-try analysis and ACA. Conclusion This study suggests that the HEK293T cells stably expressing the re-combinant CD36 could be used in flow cytometry analysis and ACA for the detection of anti-CD36 antibod-ies. It also paves the way for further researches on the mechanism of CD36 in other diseases.

OBJECTIVE: To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells. METHODS: RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting. RESULTS: An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting. CONCLUSION The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.
Blood Platelets ; CD36 Antigens ; Cloning, Molecular ; Escherichia coli ; Eukaryota ; Genetic Vectors ; HEK293 Cells ; Humans ; Plasmids ; Transfection

Antibodies against human CD36 are responsible for several immune-mediated disorders. The detection of anti-CD36 antibodies using the standard monoclonal antibody (mAb) immobilization of platelet antigens (MAIPA) assay is hampered by a high frequency of false-negative results, most likely due to competitive inhibition of the mAb used as the capture antibody. We generated a panel of mouse mAbs against CD36 and seven hybridomas (GZ-3, GZ-13, GZ-70, GZ-143, GZ-413, GZ-507, and GZ-608), which were selected for MAIPA assays, as they reacted with mouse and human CD36. Fourteen anti-CD36 sera were assayed; all of which showed a positive reaction in a PakPlus (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA) ELISA-based screening (optical density: 0.257–2.292). When the reference anti-CD36 mAb FA6-152 was used in the MAIPA assay, only 6/14 (42.9%) sera displayed a positive reaction. In contrast, anti-CD36 antibodies were detected in 13/14 (92.9%) sera when GZ-70 and GZ-608 mAbs were used. This significant improvement resulted in the identification of anti-CD36 antibodies by an antigen capture assay. Since patient’s platelets possibly carrying rare native antigens are used, this method will facilitate the identification of new platelet antibodies against CD36 that are involved in immune-mediated thrombocytopenia and other diseases, such as transfusion-related acute lung injury.

Objective To analyze the structure of CD36,search the possible B cell epitopes and prepare multi-antigen peptides(MAP)with B cell epitopes,so as to provide a preliminary experimental basis for the preparation of CD36 antibod-ies using MAP with B cell epitopes.Methods The potential B cell epitopes of CD36 were analyzed by bioinformatics meth-ods,including physical and chemical properties,secondary structure,potential phosphorylation and glycosylation sites.Eight-branch MAP with CD36 B cell epitopes were synthesized by FMOC using polylysine as the core matrix.The purity of MAPs was analyzed by reverse high-performance liquid chromatography chromatography(RP-HPLC),and the molecular weight of MAPs was determined by mass spectrometry.Results CD36 is a stable and hydrophilic alkaline protein,with multiple phosphorylation and glycosylation sites and strong antigenicity,and its secondary structure is mainly characterized by irregular curls.Four potential B cell epitopes were obtained and 4 MAPs containing potential B cell epitopes were pre-pared.RP-HPLC analysis showed that the purity of the MAPs were above 85%,and the molecular weight of 3 MAPs was consistent with the expected theoretical molecular weight.Conclusion CD36 on platelet has strong antigenicity.MAPs con-taining CD36 B cell epitopes can provide the experimental basis for the preparation and related research of CD36 antibodies.

Objective: To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia (FNAIT) through its expression in vitro. Methods: The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay (ELISA), solid-phase agglutination, flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The common human platelet antigen (HPA) genotypes of the neonate and her parents were obtained using the HPA-SSP method. The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes. The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA. The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology. The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation, and the plasmid DNA was co-transfected with that of ITGA2B (αⅡb) into HEK293 cells. The transfected cells stably expressing GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA. Results: Antibodies against HLA-class Ⅰ and GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against HLA-B 57∶01 and A 02∶05. ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation, which results in the change of glutamate to lysine at position 200. Antibodies against GP Ⅱb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum. Conclusion: The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT. The establishment of this method facilitates the discovery of new platelet low-frequency antigens, and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.

Objective: To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia (FNAIT) through its expression in vitro. Methods: The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay (ELISA), solid-phase agglutination, flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The common human platelet antigen (HPA) genotypes of the neonate and her parents were obtained using the HPA-SSP method. The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes. The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA. The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology. The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation, and the plasmid DNA was co-transfected with that of ITGA2B (αⅡb) into HEK293 cells. The transfected cells stably expressing GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA. Results: Antibodies against HLA-class Ⅰ and GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against HLA-B 57∶01 and A 02∶05. ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation, which results in the change of glutamate to lysine at position 200. Antibodies against GP Ⅱb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum. Conclusion: The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT. The establishment of this method facilitates the discovery of new platelet low-frequency antigens, and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.