Matching strategy for patients with platelet transfusion refractoriness caused by compound antibodies against HLA and CD36 / 中国输血杂志

【Objective】 To search compatible and suitable platelets for platelet transfusion refractoriness (PTR) patient caused by compound antibodies against HLA and CD36. 【Methods】 ELISA method was used to detect the antibody against platelet antigens and the specificity of HLA-I antibody in PTR patients. The specificity of HLA-I antibody and corresponding epitopes of patients were analyzed using MATCH IT! and HLA Matchmaker software. The HLA genotype of both donor and patient was obtained by HLA-SSO method. Compatible or suitable donor platelets for PTR patients were searched through cross-reactive group (CREG) of HLA-I and HLA epitope-matched approach (Eplet). The matching degree was identified using monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the platelet suspension immunofluores-cence test (PIFT). Finally, the transfusion effect was evaluated according to the corrected count increment (CCI). 【Results】 Compound antibodies against both CD36 and HLA-I antigens were detected in two PTR patients, and their phenotype of CD36 was conformed to be type I deficient. Through LSA testing, high-frequency of HLA -I antibodies was found in these two patients, and the panel reactive antibody in patients 1 and 2 was 56% (54/96) and 53% (51/96), respectively. According to HLA-CREG and Eplet matching strategies, one donor of grade C-matching with patient 1 and one donor of grade D-matching with patient 2 were screened from the CD36 deficiency donor bank, respectively. And the selected donors avoided the antigen of HLA-I antibody epitope. These results of MAIPA and PIFT also confirmed that no immune response was detected between the patient and the donor. And a CCI of >4.5 within 24 hour of transfusion of compatible platelets was obtained in patient 2. 【Conclusion】 For PTR patients caused by HLA and CD36 compound antibodies, a combination strategy including serological cross-matching, HLA-CREG and Eplet approach should be used to select the CD36 deficient donor platelets which evaded the antigen corresponding to HLA-I antibodies and had the compatible HLA epitopes.

Preliminary study on HUVEC permeability changes in FNAIT induced by CD36 antibody / 中国输血杂志

【Objective】 To explore the pathogenesis of fetal edema caused by CD36 antibody in fetal/neonatal alloimmune thrombocytopenia (FNAIT), and to provide reference for clinical prevention and treatment. 【Methods】 The established CD36 monoclonal antibody was incubated with human peripheral blood mononuclear cells (PBMC), and the concentrations of cytokines (TNF-α and IL-1β) in the supernatant of cell culture were detected by ELISA. The permeability of endothelial cells were investigated by detecting the fluorescence intensity of FITC-albumin by incubating cytokine-rich cell supernatant with human umbilical vein endothelial cells (HUVEC). 【Results】 Flow cytometry showed that CD36 monoclonal antibody could bind to human monocytes. Compared with isotype IgG control, increased cytokine TNF-α (pg/mL) (407.73±20.40 vs 29.38 ±4.72, P<0.05) and IL-1β (pg/mL) (247.14±83.59 vs 53.68±26.96, P<0.05) were detected in the supernatant of cell culture after incubation of CD36 monoclonal antibody with human PBMC. Detection of fluorescence intensity of FITC-albumin in transwell cultured HUVEC showed that cytokine-rich cell supernatant derived from CD36 monoclonal antibody incubated with human PBMC can increase the permeability of endothelial cells significantly (CD36 antibody vs isotype IgG, MFI value: 492±16 vs 320±11, P<0.05). 【Conclusion】 The effect of CD36 monoclonal antibody on PBMC can increase HUVEC permeability, which may be one of the pathogenesis of fetal edema with FNAIT.

Technical guideline for school sanitary and anti epidemic measures after flood disasters / 中国学校卫生

Abstract@#Flood disasters are the common public health emergencies, mainly leading to environmental damage, water pollution, food pollution, vector breeding, infectious disease epidemic and other risk factors of sanitary and anti epidemic work. The guideline has been formulated with reference to the technical documents such as Guideline for Environmental Sanitation Disposal and Preventive Disinfection in Flooded Areas and Technical Proposal for Sanitary and Anti epidemic Measures after Flood Disasters, as well as the latest research progress at home and abroad. In order to guide the sanitary and anti epidemic measures in flooded areas, protect the health and safety of students and teachers and ensure the normal educational and teaching order, the guideline introduces the key measures that should be taken by schools, teachers and students in flood striken areas.

Treatment of platelet transfusion refractory caused by anti-HLA in pregnancy complicated with aplastic anemia / 中国输血杂志

【Objective】 To find the HLA-matched platelets from platelet donor registry and track the transfusion effect for aplastic anemia patients in pregnancy with platelet transfusion refractory (PTR) caused by anti-HLA, so as to support the childbirth and follow-up treatment of the patients. 【Methods】 Antibodies to HPA and HLA were detected by ELISA kit and Luminex, respectively. DNA of the patient and 523 platelet donors from the donor registry were extracted for high-resolution HLA genotyping. The Risk Factors Evaluation Software of PTR was used to select the ABO-compatible and HLA-matched donors, without HLA Eplets specific to the patient. After MASPAT cross matching, the patient was transfused with 1 U of platelets, and the 24h post-transfusion effect was recorded. 【Results】 Only anti-HLAⅠantibody was found in the patient serum, and the specificity Eplet was 65QIA, including HLA-B*27∶08, B*27∶05, B*07∶02, B*55∶01, B*67∶01 and B*54∶01; anti-HLA Ⅱ antibody was negative. The HLA genotypes of both the patient and donor were HLA-A*02∶07, A*11∶01, B*46∶01, B*46∶01, C*01∶02, 01∶03, DRB1*04∶05, DRB1*0901, DQB1*03∶03 and DQB1*04∶01. The results of MASPAT matching were negative. HLA-matched platelets transfusion provided a satisfactory posttransfusion platelet responses in patients(1 before vs 33 ×109/L after). A baby boy was delivered by cesarean section 4 weeks later, and the same donor was recruited due to the mother′s low Plt and bleeding trend. The 24h posttransfusion Plt (×109 / L) rose from 5 to 37 after the secondary transfusion of 1U platelet. The vital signs of the mother and her baby were normal during the two-day follow up. 【Conclusion】 The establishment of blood donor registry and screening of HLA-matched donors is an effective approach to treat PTR caused by HLA antibodies in pregnancy complicated with aplastic anemia.

Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells / 中华医学遗传学杂志

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.

Smoking epidemiology among primary and middle school students in Beijing during 2005-2015 / 中国学校卫生

Objective@#To understand the changes of smoking behavior among primary and middle school students in Beijing during 2005-2015.@*Methods@#The primary and middle schools in Beijing were classified and then taken as a sampling frame. Twophase stratified random cluster sampling was conducted with school as primary sampling unit (PSU) and class as the minimum sampling unit, respectively. Beijing Schoolbased Smoking Monitoring Questionnaire was surveyed in 2005 and 2005, 2010, 2011, 2013 and 2015 anonymously.@*Results@#In 2015, the smoking rate among primary and middle school students in Beijing was 9.41%. Among them, for primary, junior, high school and vocational high school students, the rates were 7.05%, 7.06%, 12.41% and 34.11%, respectively. The smoking rates were now 3.26%, with 1.99%, 1.80%, 3.48% and 20.22%, respectively, among primary, middle and high school and vocational high school students. male, vocational school students were more likely to report smoking across six waves of surveillance. Results from the surveillance in 2015 showed a decreasing trend in ever smoking rate and current smoking rate compared with previous survey. About half of the current student smokers were reported to take 1 cigarette/day, and about 10% smokers were reported to take 10 cigarettes/day. Students were most likely to smoke at home(24.02%), followed by smoking at schools(12.74%). The percentage of buying cigarettes by themselves was increased from 17.10% in 2008 to 66.09% in 2015.@*Conclusion@#From 2005 to 2015, both ever smoking and current smoking rate among middle school students in Beijing decreased in general, however smoking among vocational high school students warrants further attention.

Establishment of a cell line expressing the CD36 on human platelets and its application to the detec-tion of anti-CD36 antibodies / 中华微生物学和免疫学杂志

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.

A novel human leukocyte antigen-A*33:44 allele revealed by sequence analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the sequence of a novel human leukocyte antigen (HLA)-A*33:44 allele.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>Genomic 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.</p><p><b>CONCLUSION</b>A mutation has been found in exon 4 of the novel HLA-A*33:44 allele, which may provide more information for HLA gene study.</p>

Difference between the bone marrow and peripheral blood engraftment evidence after allogeneic hematopoietic stem cell transplantation by PCR-STR / 中国输血杂志

Objective To observe the difference between the bone marrow and peripheral blood engraftment evidence after allogeneic haemopoietic stem cell transplantation(Allo-HSCT) using PCR-STR. Methods DNA from peripheral blood or bone marrow of donors and recipients in different phases were extracted,and 16 STR loci with high polymorphism were amplified by PCR.Separation of the PCR products and fluorescence detection were performed by ABIprism 3100 Genetic Analyzer with capillary electrophoresis.Results The 16 patients included in the study had different levels of engraftment.Twelve patients displayed complete chimerism,while 5 patients showed mixed chimerism.One patient was keeping continuance of remission.The decrease of donor DNA amounts in mixed chimerism was earlier in bone marrow than that in peripheral blood(P

Human mesenchymal stem cells differentiate into neuron-like cells with bFGF / 中华神经科杂志

Objective To investigate the differentiation from human mesenchymal stem cells (hMSC) into neuron-like cells. Methods hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with DMEM/BHA/DMSO or DMEM/monothioglycerol, respectively. Neuron-specific enolase (NSE), neurofilament (NF), nestin, glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. Results hMSC were expanded to be undifferentiated cells in culture for more than 10 passages. The isolated and cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. Simple method induced hMSC exhibiting a neuronal phenotype, with a positive expression of NSE, NF-M and nestin at 5 hours. But the neuron-like cells did not express the glial astrocyte marker GFAP. Conclusion It suggests that hMSC can be differentiated into neurons in vitro .

Human mesenchymal stem cells differentiate into neuron-like cells / 中国病理生理杂志

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate with DMEM/monothioglycerol or DMEM/β-mercaptoethanol, respectively. Neuron-specific enolase(NSE), neurofilament(NF), and glial fibrillary acidic protein(GFAP) were detected by immunohistochemistry. RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 5 passages. When treated with monothioglycerol or β-mercaptoethanol for 5 hours, hMSC exhibited neuronal phenotype. The expression of NSE and NF in the neuron-like cells was positive, but the glial astrocyte marker GFAP didn't express. CONCLUSION:hMSC can be induced to differentiate into neurous.

Genetic polymorphism of HPA-1 to-17 alloantigen system in Guangzhou population / 中国免疫学杂志

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.

Adult human mesenchymal stem cell differentiates into adipocytes / 中国病理生理杂志

AIM: To investigate the differentiation from human mesenchymal stem cells (hMSC) to adipocytes.METHODS: hMSC were separated from rib marrow and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced with dexamethasone, insulin, 1-methy1-3-isobutylxanthine and indomethacin which acted as adipocyte differentiation inducer. The cells were stained with Oil Red O. The number of adipocytes were counted on a phase-contrast microscope.RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 5 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded, attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD14, CD34, CD45, CD11a. After induced with induction medium, lipid vacuoles were first detectable within the cells at 48 hours. Two weeks later, more than 85% MSC differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. CONCLUSION: hMSC can be induced to differentiate into adipocytes. [

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP / 中国病理生理杂志

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than 10 passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.

Effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts / 中国病理生理杂志

AIM: To investigate the effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts.METHODS: hMSC were separated from human marrow and expanded in cuture medium. hMSC were induced with dexamethasone, ?-glycerophosphate, vitamin C which acted as osteoblast differentiation inducer. PD98059 was added into the osteoblasts induction medium. The cells were assayed with cell morphology, alkaline phosphatase (AP) activity and calcium deposition. RESULTS: The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. After induced with osteoblasts induction medium, the cells showed changes in cell morphology from spindle-shape to cuboidal and polygonal. The AP activity increased gradually and reached the peak in 12 days, then decreased. Many scattered tangerice calcium nodes were observed. PD 98059 significantly inhibited AP activity and calcium deposition in a dose-dependent manner. A striking observation of the present study was that a few adipocytes appeared in cultures that were treated with PD 98059 and osteogenic differentiation medium. CONCLUSION: These results indicated that osteogenic diferentiation from the hMSCs was related to the activation of the ERK.inCwhichactedasost

Human mesenchymal stem cells differentiate into osteoblasts / 中国病理生理杂志

AIM: To investigate the differentiation from human mesenchymal stem cells (hMSC) into osteoblasts. METHODS: MSC were separated from human marrow with Ficoll-Paque reagent and expanded in cuture medium. To detect the surface antigens, The labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate from mesenchymal stem cells into osteoblasts with dexamethasone, vitamin C, ?-GP. Cell morphology?AP activity?calcium deposition and osteopontin were detected. P10 MSC were compared to P3 MSC in the tendency of osteoblastic differentiation. RESULTS: The cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. hMSC showed a strong self-renewal capacity. After primary culture, approximately (5-6)?10 5 cells were obtained. These expanded attached MSC were uniformaly positive for CD29,CD44,CD59,CD105,CD166 and didn't express CD11a, CD14, CD33, CD34, CD45, CD38, CD80, CD86, CD117. After osteoblasts induction, the cells changed from spindle-shape to cuboidal and polygonal in cell morphology. The AP activity increased gradually and many scattered calcium nodes were observed. The expression of osteopontin was positive. CONCLUSION: hMSC can be induced to differentiate into osteoblasts.

Establishment of eight human platelet antigen systems and HLA-Ⅰ antigen donor bank in Guangzhou area / 中国输血杂志

Objective To establish eight human platelet antigen systems and HLA-Ⅰ antigen donor bank,and to determine the gene frequencies of human platelet antigen(HPA) and HLA-Ⅰin Guangzhou area.Methods A total of 805 blood samples from Chinese Han voluntary platelet donors were included in this study.PCR-SSP was used to detect single-nucleotide polymorphism in HPA systems.Luminex-SSO was used to detect the HLA-Ⅰantigens.Results The distribution of HPA 1,2,3,4,5,6,9,15 was in Hardy-Weiberg equilibrium among study subjects.Allele frequencies of 0.998 and 0.002 were observed for HPA 1a and 1b,0.952 and 0.048 for HPA 2a and 2b,0.553 and 0.447 for HPA 3a and 3b,0.999 and 0.001 for HPA 4a and 4b,0.976 and 0.024 for HPA 5a and 5b,0.982 and 0.018 for HPA 6a and 6b,1 and 0 for HPA 9a and 9b,0.518 and 0.481 for HPA 15a and 15b.The high frequency HLA-Ⅰ alleles were A*02,0.286;A*24,0.162;A*11,0.323;B*46,0.147;B*75,0.100;C*01,0.177;C*03,0.289;C*07,0.179.Conclusions This study confirmed the ethnic and territorial difference of HPA and HLA-Ⅰ.The establishment of HPA and HLA-Ⅰ matched plateletpheresis donor registry is helpful in the improvement in platelet transfusion.

Allelic polymorphism of human leukocyte antigen-DRB1~* 04 in Chinese Han populations / 中国输血杂志

Objective To investigate the allelic polymorphism of Human Leukocyte Antigen(HLA)-DRB1*04 in Southern and Northern Chinese Han populations.Method A total of 104 and 168 samples of HLA-DRB1*04 alleles from Southern and Northern Chinese Han populations respectively were genotyped at high-resolution by PCR-SBT,and then the DRB1*0406/0449 ambiguous allele pairs were identified via high-resolution PCR-SSP.The allelic distribution of DRB1*04 in Southern and Northern Chinese Han populations were compared with other different populations by chi-square test.Results All 56 ambiguous allelic pairs of DRB1*0406/0449 were identified as DRB1*0406.A total of 5 alleles were observed in Southern Chinese Han population and the most frequent alleles were DRB1*0405(47.32%),DRB1*0406(24.24%)and DRB1*0403(16.35%).There were 10 alleles tested in Northern Chinese Han population,and the major alleles were DRB1*0405(40.48%),DRB1*0406(18.45%) and DRB1*0403(17.26%).The overall distribution of DRB1*04 alleles in Southern and Northern Chinese Han differed significantly from that of the Caucasian,African American,Hispanics,and Asian and Pacific Islanders,but not from the Korean.Conclusions The overall distribution of HLA-DRB1*04 alleles differed significantly between Southern and Northern Chinese Han populations,but the major alleles DRB1*0405,DRB1*0406 and DRB1*0403 showed the same distribute characteristic.The distribution of DRB1*04 alleles in different populations has their own characteristics.