Objective:To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population.Methods:Using PCR - SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals, and then using the method of high fidelity and long-fragment PCR - SBT to amplified, sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals.Results:We successfully amplify the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population. 209 KIR2DS4 positive individuals were detected, and the positive rate is 94.1%. By sequence-based typing, we identified 12 genotypes and 7 alleles of KIR2DS4. The 6 known alleles and their detection frequency is as follows: KIR2DS4 * 00101/011 (180, 81.1%), KIR2DS4 * 010 (53, 23.9%), KIR2DS4 * 004 (34, 15.3%), KIR2DS4 * 003 (15 and 6.8%), KIR2DS4 * 006 (2, 0.9%) and KIR2DS4 * 015 (1, 0.5%). In this study, we found a new allele, KIR2DS4 * 016, with the difference in exon 5 comparing its most similar allele KIR2DS4 * 010. In the exon 5 of KIR2DS4 * 010, there is a 22bp-deletion, while the exon 5 of KIR2DS4 * 016 is normal. This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%. The sequence of the new allele sequence has been submitted to GenBank (accession no.: KC414890) and the IPD - KIR database (submission no.: IWS40001804), and was nominated by WHO nomenclature committee for HLA system. Conclusion:In this study, a sequence-based typing method for KIR2DS4 was established, and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.

Objective To investigate the distribution and transmission characteristics of vancomycin -resistant Enterococcus faecium (VREF) carrying both vanA and vanM in the intensive care unit.Methods VREF strains were isolated from patients in the intensive care unit of Jinshan Hospital , Fudan University in Shanghai from 2013 to 2017.Antimicrobial susceptibilities of the VREF strains to nine antibiotics , including vancomycin, teicoplanin, linezolid and chloromycetin , were tested by broth microdilution method.Multiple polymerase chain reaction (PCR) was used for van genotyping and pulsed field gel electrophoresis (PFGE) was used for homology analysis.Results Thirty-five strains were mainly isolated from urine (16 strains), blood (11 strains), feces ( five strains ), bile ( two strains ) and pleural effusion ( one strain ).All the strains (100.00%) were resistant to vancomycin , ampicillin and levofloxacin , but only 40.00% were resistant to teicoplanin.All the strains were sensitive to linezolid.The results of van genotyping showed that 33 (94.3%) strains belonged to vanA and vanM dual genotype VREF, and the other two were vanA type VREF.PFGE results showed that 35 strains could be divided into 14 PFGE patterns, and seven out of 10 strains isolated in 2014 were identical and the other three belonged to three different PFGE patterns.Conclusions A dual genotype VREF carrying both vanA and vanM has been emerging and spreading in the intensive care unit of Jinshan Hospital , Fudan University in Shanghai.

OBJECTIVETo study the distribution of MICA alleles among ethnic Han Chinese blood donors from Shenzhen and their linkage disequilibrium with HLA-B gene.

METHODSFor 143 randomly selected blood donors, the MICA and HLA-B alleles were determined with a PCR-sequence based typing (SBT) method. Allelic frequency, haplotypic diversity and linkage disequilibrium were analyzed with a Pypop software.

RESULTSThirteen MICA and 35 HLA-B alleles were identified among the 143 blood donors, among which MICA*008:01 had the highest frequency (76/286), whilst MICA*008:01-HLA-B*40:01 and MICA*010-HLA-B*46:01 were the most common haplotypes. No novel allele was identified.

CONCLUSIONThe allele frequencies, haplotype diversities and linkage disequilibrium parameters under a high resolution can facilitate further studies and applications of the MICA and HLA-B genes.

OBJECTIVETo study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.

METHODSGenomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.

RESULTSAmong all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.

CONCLUSIONThe present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; Haplotypes ; Humans ; Polymorphism, Genetic ; Receptors, KIR ; genetics ; Sequence Analysis, DNA ; methods

BACKGROUND: Due to the polymorphism of HLA, a large number of ambiguities have been generated by conventional HLA typing techniques, and confirmed stereotypes of ambiguous results based on group-specific haploid full-length typing are rarely reported.OBJECTIVE: To analyze the accuracy of HLA-typing ambigulity based on group-specific haploid full-length sequencing. METHODS: The low-resolution results were used as the starting point for two ambiguous samples. Sanger sequencing (PCR-SBT) based on haploid full-length was performed after group-specific amplification. RESULTS AND CONCLUSION: One case showed a new A*02:03:01 allele, which was found a mutation in NT817 from C to T in comparison with A*11:01:01:01. The other case indicated another new C*07:02:01:01, which was found a mutation in NT879 from A to G in comparison with C*08:01:01. In conclusion, these results indicate that the group-specific haploid full-length sequencing method can be used to accurately classify HLA alleles and to discover new alleles.

BACKGROUND: Human leukocyte antigen-E (HLA-E) is one of non-classical HLA class I genes. Up to now, the polymorphism analysis is mainly aimed at the variation in exon 3 of HLA-E, which determines HLA-E*01:01 or HLA-E*01:03. However, the identification of the full-length HLA-E and its novel alleles is rare reported.OBJECTIVE: To establish the method of identification of HLA-E genomic full-length sequence, and to identify its novel alleles in healthy blood donors in Shenzhen, China.METHODS: Peripheral blood DNA samples were extracted from the subjects, and the amplified primers and sequencing primers in conserved regions were designed according to the sequences of HLA-E published in the IMGT/HLA database.A high-fidelity reaction system was used to amplify the genomic full-length of HLA-E, followed by sequencing,assembling, confirming and typing.RESULTS AND CONCLUSION: Herein, we successfully established the method for amplifying genomic full-length sequence and sequence-based typing. Two novel HLA-E alleles were nominated by WHO HLA Nomenclature committee as HLA-E*01:01:01:06 and HLA-E*01:01:01:07. Compared with the most related allele HLA-E*01:01:01:01,HLA-E*01:01:01:06 had one nucleotide change at nt-26(G->T) in 5'-promoter, and HLA-E*01:01:01:07 had one nucleotide change at nt3345(T->C) in 3'-UTR. The polymorphism data of genomic full-length HLA-E in Chinese individuals need to be filled, and the method we developed here supplies the key technique for the further studies.

Objective: To investigate the situations of AIDS related knowledge, sexual behavior with various partners among HIV positive patients in Shenzhen before highly active anti-retroviral therapy, and to provide evidences for health education intervention. Methods: Questionnaire survey and information collection were carried out among HIV positive patients, under the informed consent, during the first physical examination before highly active anti-retroviral therapy. Results: The overall awareness rate of AIDS knowledge was 91%, 94.46% and 80.95% respectively for homosexual and heterosexual populations, with significant difference between the two groups (χ²=21.254, P<0.001). In addition, there were significant differences in the factors related to education, income, route of infection, marital status, residence and other related factors (P<0.05). In HIV positive MSM, primary sexual behavior occurred mainly at the age of less than 30 (96.8%) and in university or earlier stage (58.3%), college students as the first sexual partner accounted for 44.9%, and once used Rush accounted for 42.7%. There were significant differences in the number of temporary sexual partners and condom use frequency between homosexual and heterosexual subjects (P<0.05). Conclusions Individuals infected with HIV via different routes are variant on AIDS cognition. Health education on AIDS related knowledge and sexual security before and after antiviral treatment can not only improve the awareness rate of HIV/AIDS infection, but also can reduce the risk of the spread of HIV/AIDS.

Objective: To investigate the expression levels and clinical significance of serum suppressor of cytokine signaling-3 (SOCS3) and associated cytokines in HIV/TB co-infected patients. Methods: The serum levels of SOCS3, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17 and IL-22 were quantified by using enzyme-linked immunosorbent assays (ELISA) in 50 HIV-infected patients, 48 HIV/TB co-infected patients and 50 healthy donors. Pearson correlation analysis was used to analyze the correlation between SOCS3 and other seven cytokines. Results: Serum levels of SOCS3 expression in HIV/TB co-infection group were significantly higher than those in HIV-infection alone and the control group. There was also significant correlation between SOCS3 and IFN-γ, IL-4, IL-6, IL-10, IL-2 in HIV/TB co-infection group. Conclusions These findings indicated that SOCS3 may play an important role in the immune response of patients with HIV/TB co-infection and it may be helpful in the diagnosis of HIV/TB co-infection.

Objective:To analyze the immunogenicity of the extracellular region of Δ42PD1.Methods: Six fragments ofΔ42PD1 extracellular region-encoding sequence were amplified by PCR, and were cloned into pCTCON2 vector, a yeast surface displaying vector.Yeast cells were transfected with Δ42PD1 fragment-carrying plasmids, then yeast cells were spread on SDCAA plates.Single cell clones were selected and cultured in SGCAA media to induce expression of the target genes.Mouse anti-humanΔ42PD1 anti-serum were generated by immunization of BALB/c mice via intramuscular injection ofΔ42PD1-carrying plasmid plus in-situ electroporation.The binding of anti-serum with yeast cells surface-displaying Δ42PD1 fragments were analyzed using flowcytometry.Results:Nucleotide sequences analysis indicated that the amplified six fragments ofΔ42PD1 sequence length were 110 bp,and the isolated sequence ofΔ42PD1 fragments were 100%homology with PD1 gene previously registered in GenBank.Results from flowcytometry showed that among the six fragments of Δ42PD1 displaying on the surface of yeast cells,F3 and F2 profoundly boundΔ42PD1-specific polyclonal antibodies.Conclusion:F3 and F2 ofΔ42PD1 is an immunogenic dominant region,which pave the way for generation of Δ42PD1-specific monoclonal antibody and epitope mapping.

Objective To evaluate the effects of Reyanbao combined with acupiont application on pain and comfort degree of pregnant women with surgical abortion. Methods One hundred twenty pregnant women suffering from abdominal pain after surgical abortion were randomly divided into control group and treatment group:the control group was treated with conventional nursing care and the treatment group was with Chinese medicine Reyanbao combined with acupiont application from the same treatment as in the control group, pain and comfort of patients were observed after two hours of treatment. Result The pain and comfort of the two groups were statistically significant (P < 0.05). Conclusion Chinese medicine Reyanbao combined with acupiont application can effectively relieve pain after surgical abortion and improve comfort. It is a safe, effective, convenient and practical in use of traditional Chinese medicine nursing analgesia technology.