Epidemiological Survey of Hemoglobinopathies Based on Next-Generation Sequencing Platform in Hunan Province, China / 生物医学与环境科学（英文）

OBJECTIVE@#This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province.@*METHODS@#We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed.@*RESULTS@#The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α 3.7/αα (50.23%) and β IVS-II-654/β N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively.@*CONCLUSION@#Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.

Influence of the Single Live Birth Outcome Following Transfer of Vitrified Blastocyst and Blastocyst Cultured from Thawing Cleavage Embryo / 中山大学学报(医学科学版)

[Objective]To compare the single live birth outcomes of blastocyst transfer between vitrified blastocyst and blastocyst cultured from thawing cleavage embryo,so as to choose the best scheme of blastocyst transfer.[Methods]Retrospective analysis of the single live birth clinical data of 1 037 vitrified blastocyst compared with 690 blastocyst cul-tured from thawing cleavage embryo undergoing frozen embryo transplantation(FET)from January 2014 to October 2016 was performed.Mail outcome were including gestational age,neonatal weight,proportion of male neonate,preterm birth rate,very preterm birth rate,low birthweight rate,very low birthweight rate,congenital anomalies rate.[Results]There were no differences between the two groups for gestational age,neonatal weight,proportion of Live birth,health baby and stillbirth(P>0.05). There were no differences in proportion of male neonate(AOR 1.07,95% CI 0.86~1.34),preterm birth rate(AOR 0.7,95% CI 0.49~1.01),very preterm birth rate(AOR 1.47,95% CI 0.55~3.96),low birthweight rate (AOR 1.38,95% CI 0.86~2.22),very low birthweight rate(AOR 0.76,95% CI 0.20~2.83),congenital anomalies rate (AOR 1.58,95% CI 0.66~3.76,P>0.05).[Conclusion]The blastocyst may be the preferable stage for vitrifying and transfer currently which can obtain good neonatal outcomes.

Prevalence and molecular analysis of β-thalassemia in children of Han ethnicity in Chongqing city / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the incidence and the gene mutation frequencies and patterns of β-thalassemia (β-Thal) in ethnic Han children in Chongqing city.</p><p><b>METHOD</b>A total of 1726 children were screened by using automatic hemocytic analyzer, cellulose acetate electrophoresis and fetal hemoglobin alkali denaturation test. Samples with mean corpuscular volume (MCV) < 80 fl, cell hemoglobin content (MCH) < 27 pg and hemoglobin A2 (HbA2) levels >3.3%, fetal hemoglobin (HbF) >2% for β-Thal screening indicators. The positive samples of screening indicators were detected and identified by PCR-reverse dot blot method for 18 common β-Thal mutations in Chinese populations, unknown mutations samples were subjected to DNA sequencing analysis of the β-globin gene.</p><p><b>RESULT</b>Twenty-five cases of β-Thal carriers were observed from the 1726 samples, with 24 cases of β-Thal heterozygote and one case of double heterozygote. Therefore, the β-Thal carrier rate was 1.51%. After 1726 peripheral venous blood samples analyzed by hematological parameters, 164 positive cases of β-Thal screening indicators were found, with the positive rate being 9.50% (164/1726). A total of 6 different gene mutations were detected, the four most common mutations were as the following: CD41-42, IVS-II-654, CD17 and beta E. These four mutations as the major types in this area accounted for 88.00% of all the mutations. In addition, one rare mutation of 5 'UTR; + (43 -40) was found, and one case of the hemoglobin variant of Hb Zurich was reported in Chinese people for the first time.</p><p><b>CONCLUSION</b>Chongqing is a high risk region of the β-Thal. Epidemiological Data from the research was useul for the genetic counseling and the prevention of β-Thal major.</p>

Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH.</p><p><b>METHODS</b>CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted.</p><p><b>RESULTS</b>The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01).</p><p><b>CONCLUSIONS</b>The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.</p>

Comparative study on anti-tumor immune response of autologous cytokine-induced killer (CIK) cells, dendritic cells-CIK (DC-CIK), and semi-allogeneic DC-CIK / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Cytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.</p><p><b>METHODS</b>Peripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively.</p><p><b>RESULTS</b>Compared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis.</p><p><b>CONCLUSIONS</b>Semi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.</p>

Antitumor response induced by vaccine of autologous dendritic/tumor fusion cells against renal cell / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether dendritic cells fused with tumor cells could elicit in vitro antitumor responses against renal cell carcinoma (RCC) cells.</p><p><b>METHODS</b>Renal carcinoma cells were purified from tumor tissue excised from patients with metastatic RCC through tumor cell purifying technique and cultured in RPMI-1640 medium containing 10% FCS. Monocyte-derived DCs generated from peripheral blood mononuclear cell of RCC patients were cultured in the presence of human recombinant granulocyte-macrophage colony stimulating factor and interleukin-4. Tumor cells and DCs were cocultured in the presence of polyethylene glycol (PEG) to generate cell fusion. The phenotype of tumor cells, DCs and fusion cells were detected by flow cytometry. MTT was used to measure the ability of fusion cells to stimulate T cell proliferation. T cell-mediated antitumor responses were measured by lactate dehydrogenase release (LDH) assay for lysis of autologous tumor cells.</p><p><b>RESULTS</b>The DCs expressed MHC class I, MHC class II and costimulatary molecules (CD80 and CD86), while the renal carcinoma cells expressed a high molecular glycoprotein MUC-1. The DC/tumor fusion cells coexpressed MUC-1 and the phenotype of DCs, and could stimulate T cell proliferation effectively. CTLs stimulated by the fusion vaccine showed distinct lytie activity in vitro to autologous tumor cells.</p><p><b>CONCLUSION</b>Dendritic cells fused with tumor cells can elicit distinct antitumor responses in vitro against tumor cells from patients with metastatic RCC, providing a basis for further research on the clinical application of fusion vaccine in treatment for renal cancers.</p>

Detection of AMACR (P504S), P63 and 34betaE12 cocktail in the early diagnosis of prostate cancer / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the value of detection of AMACR (P504S), P63 and 34betaE12 cocktail in the early diagnosis of prostate cancer (PCa).</p><p><b>METHODS</b>The expressions of AMACR, P63 and 34betaE12 were examined in the biopsy specimens of 42 cases of prostate cancer, 12 cases of high-grade prostatic intraepithelial neoplasia (HGPIN) and 30 cases of benign prostatic hyperplasia (BPH) using the Maxvision single-step immunohistochemical method with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens in single paraffin sections .</p><p><b>RESULTS</b>The expressions of AMACR, P63 and 34betaE12 were significantly different between PCa and BPH (P < 0.01). The staining of PCa was positive for AMACR and negative for P63 and 34betaE12, and the positivity rate of AMACR was 100%. BPH was strongly expressed for P63 and 34betaE12, but negatively for AMACR. The expression of AMACR was significantly different between HGPIN and BPH (P < 0.01), but not between HGPIN and PCa (P > 0.05), and the positivity rate of AMACR in HGPIN was 91.67%. However, the expressions of P63 and 34betaE12 were significantly different between HGPIN and PCa (P < 0.01), but not between HGPIN and BPH (P > 0.05), and the positivity rate of AMACR in HGPIN was 100%. The level of AMACR expression was not correlated with PCa Gleason score (P > 0.05).</p><p><b>CONCLUSION</b>AMACR is a sensitive and specific marker for PCa. P63 and 34betaE12 cocktail staining can increase the sensitivity and specificity of the basal cell detection. The immunohistochemical analysis with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens can improve diagnostic accuracy and has an important applied value for the early diagnosis of prostate cancer.</p>

Clinical application of alpha1 adrenoceptor antagonist Naftopidil to the treatment of chronic non-bacterial prostatitis / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the efficacy and safety of alpha1 adrenoceptor antagonist Naftopidil in the treatment of chronic non-bacterial prostatitis.</p><p><b>METHODS</b>An opened, self-controlled, multicentral clinical trial was conducted. One hundred and six cases of patients who had been diagnosed as chronic non-bacterial prostatitis (NBP) were treated with Naftopidil (25 mg once a day) for 4 weeks. The efficacy was evaluated by the NIH Chronic Prostatitis Symptom Index (NIH-CPSI) and the WBC in the examination of prostatic secretion (EPS) after the treatment.</p><p><b>RESULTS</b>After 4 weeks therapy, 105 cases were evaluable. After treatment, NIH-CPSI total score were averagely decreased 12.0 points (P <0.001), symptom score 7.9 points (P <0.001) and QOL score 4.1 points (P <0.001). There was a statistically significant difference in WBC count ([(15.2 +/- 15.1)/HP vs (9.5 +/- 12.0)/HP, P < 0.01] and max flow rate(MFR) [(19.2 +/- 4.8) ml/s vs (22.7 +/- 4.9) ml/s, P < 0.01]. The total effective rate were 84.8% in the whole group. The clinical adverse rate was 3.81%, including 3 cases of mild dizziness and 1 case of mild inappetence.</p><p><b>CONCLUSION</b>alpha1 adrenoceptor antagonist Naftopidil is effective and safe for the treatment of chronic non-bacterial prostatitis.</p>

Preliminary result on the nosocomial infection of severe acute respiratory syndrome in one hospital of Beijing / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the transmission route of severe acute respiratory syndrome (SARS) nosocomial infection.</p><p><b>METHODS</b>Ten identified SARS patients were selected from a general hospital in March. Survey was carried out through a standardized questionnaire provided by Chinese Center for Disease Control and Prevention. Contents of the questionnaire would include: history of contact with SARS patient, route of infection, methods used for protection and so on.</p><p><b>RESULTS</b>(1) Distribution os SARS patients were confined to 3 wards: 4, 5, and 6 on the 7, 8, 12, 13 and 14 floors in the west unit of the inpatient building. Most of the inpatients were elderly and having severe original diseases. (2) Index patients were the first generation source of transmission and they infected inpatients and medical staff, making them the second generation. People with latent infection who had close contact with SARS patients might also serve as the possible source of transmission. (3) The major transmission routes were: near distant droplet infection and close contact infection. There was also a clue to the probability of aerosol or droplet nuclei infection through air-conditioning and ventilation system.</p><p><b>CONCLUSION</b>Nosocomial infection appeared to be the main characteristic of the SARS epidemic in the early stage of this hospital. Other than close contact and near space airborne transmission of SARS virus, the possibility of long-distance aerosol transmission called for further epidemiological and experimental studies in the future.</p>

Application of double immunomagnetic positive sorting to ex vivo expansion of marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria / 中国实验血液学杂志

Since flow cytometry was not feasible for sorting a huge amount of cells for clinical use, the method of double immunomagnetic positive sorting was used for selection of CD34(+)CD59(+) cells from bone marrow mononuclear cells in patients with paroxysmal nocturnal hemoglobinuria (PNH), which laid the groundwork for clinical ABMT/APBSCT of patients with PNH. Immunomagnetic positive selection was used for two times, the microbeads were removed from the CD34(+) cells selected firstly by means of overnight culture, then the sufficient CD34(+)CD59(+) cells were used for ex vivo expansion. The results showed that the survival, proliferation and colony-forming units of the selected CD34(+)CD59(+) cells by double immunomagnetic positive sorting had no significant difference as compared with that of CD34(+)CD59(+) cells selected by flow cytometry technique. It is suggested that the double immunomagnetic positive sorting promotes the use for separation and purification hematopoietic stem/progenitor cells and other cells with double or multiple markers cells for autologous hematopoietic stem cell transplantation in PNH patients.

Study of biological characteristics of the CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients' bone marrow / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the characteristics of CD(34)(+) CD(59)(+) cells from paroxysmal nocturnal hemoglobinuria(PNH) patients' bone marrow and the possible reasons of hematopoietic clonal dominance of PNH clones.</p><p><b>METHODS</b>CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) cells from normal control were selected from the bone marrow mononuclear cells by means of immunomagnetic microbead-flow cytometry two step sorting method undergone ex vivo expansion in liquid culture for two weeks and performed semisolid cultures before and after expansion.</p><p><b>RESULTS</b>(1) Cultivation for seven days was the optimum for ex vivo expansion of PNH CD(34)(+) CD(59)(+) cells and normal CD(34)(+) cells, both cell populations remained CD(59) positive after expansion. (2) Normal CD(34)(+) cells had higher capacities of proliferation and expansion, and stronger potential to survival than that of both PNH CD(34)(+) CD(59)(+) and PHN CD(34)(+) CD(59)(-) cells. (3) In terms of semisolid culture, there was no significant difference in the yields of CFU formation between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. (4) In liquid culture with combinations of hematopoietic factors SCF + IL-3 + IL-6 + FL + Tpo or SCF + IL-3 + IL-6 + FL + Tpo + Epo, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells; but with combination of SCF + IL-3 + IL-6 + FL + Tpo + Epo + GM-CSF, CD(34)(+) CD(59)(-) cells had better proliferation and expansion capacities and stronger potential to survival than that of CD(34)(+) CD(59)(+) cells.</p><p><b>CONCLUSIONS</b>(1) Normal CD(34)(+) cells had better proliferation, expansion capacities and stronger potential to survival than that of PNH CD(34)(+) CD(59)(+)cells. (2) In semisolid and liquid culture with hematopoietic factor combinations, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. It was suggested that CD(34)(+) CD(59)(-) cells had no clonal hemotopoiesis dominance. GM-CSF might be one of the reasons for PHN clones to possess clonal hematopoiesis dominance.</p>

Comparison of capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore in vitro expansion of CD34+CD59+ cells from patients with PNH, and compare the capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control.</p><p><b>METHODS</b>CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control were selected from the bone marrow mononuclear cells by means of two-step sorting method with immunomagnetic microbead-flow cytometry, then underwent in vitro expansion for two weeks and semi-solid culture in vitro before and after expansion.</p><p><b>RESULTS</b>(1) CD34+CD59+ cells from patients with PNH can be expanded effectively in vitro, and the biggest expansion of CD34+CD59+ cells was about 23.49 fold on the 7th day. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, such as: the best combination of hematopoietic factors for in vitro expansion was SCF+ IL-3 + IL-6 + FL + Tpo + Epo, and the seventh day was the most suitable in course of 4-14 days for in vitro expansion, and after in vitro expansion, the cells remained CD59 positive and strong capability of performing colony-forming. (3) CD34+ cells from normal control had better proliferation, expansion and stronger potential to survive than CD34+CD59+ cells from patients with PNH.</p><p><b>CONCLUSIONS</b>(1) In vitro expansion of CD34+CD59+ cells from patients with PNH can be performed. The present study showed the possibility of performing ABMT or APBSCT clinically for patients with PNH. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, but the latter had better proliferation, expansion and stronger potential to survive than the former. CD34+CD59+ cells from patients with PNH were not completely normal cells.</p>