[The diagnostic value of whole blood Epstein-Barr virus DNA load in lymphoproliferative diseases after allogeneic hematopoietic stem cell transplantation].

Objectives: To investigate the diagnostic value of whole blood quantitative PCR for DNA load of Epstein-Barr virus (EBV) in post-transplant lymphoproliferative disease (PTLD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: A total of 694 patients with hematologic diseases who underwent allo-HSCT at the Hematology Department of Peking University First Hospital from April 2004 to April 2019 were included, and their data were retrospectively analyzed. Results: ①Among the 694 cases, 29 cases (22 males and 7 females, with a median age of 22 (1-52) years) developed PTLD after allo-HSCT with a cumulative incidence of 4.2% and a median onset time of 2.1 (0.8-20.6) months. ② Univariate analysis showed that age<30 years, diagnosis with aplastic anemia, human leukocyte antigen (HLA) mismatch, use of antithymocyte globulin (ATG) in preconditioning regimens, and EBV reactivation were the risk factors for the occurrence of PTLD. Multivariate analysis showed that EBV reactivation was an independent risk factor for the occurrence of PTLD. ③Further analysis of EBV reactivation cases showed that the peak value of EBV-DNA load was significantly higher in the PTLD group than that in the non-PTLD group (P<0.001) and the incidence of PTLD increased with the increase of EBV-DNA load. Receiver operating characteristic (ROC) curve analysis indicated that PTLD was more likely to be diagnosed when the EBV-DNA load was >1.19×10(6) copies/ml (sensitivity 0.800 and specificity 0.768) . ④All patients with PTLD received rituximab-based treatment, with an overall response rate of 86.2% and an overall survival rate of 54.3%. Conclusion: The PTLD occurrence after allo-HSCT is highly correlated with EBV reactivation, and the higher the EBV-DNA load, the greater the risk of PTLD occurrence. The dynamic monitoring of EBV-DNA load plays an important role in predicting PTLD occurrence.

Let-7 derived from endometrial extracellular vesicles is an important inducer of embryonic diapause in mice.

Embryonic diapause is a maternally controlled phenomenon. The molecule controlling the onset of the phenomenon is unknown. We demonstrated that overexpression of microRNA let-7a or incubation with let-7g-enriched extracellular vesicles from endometrial epithelial cells prolonged the in vitro survival of mouse blastocysts, which developed into live pups after having been transferred to foster mothers. Similar to in vivo dormant blastocysts, let-7-induced dormant blastocysts exhibited low level of proliferation, apoptosis, and nutrient metabolism. Let-7 suppressed c-myc/mTORC1 and mTORC2 signaling to induce embryonic diapause. It also inhibited ODC1 expression reducing biosynthesis of polyamines, which are known to reactivate dormant embryos. Furthermore, the overexpression of let-7 blocked trophoblast differentiation and implantation potential of human embryo surrogates, and prolonged survival of human blastocysts in vitro, supporting the idea that embryonic diapause was an evolutionary conserved phenomenon. In conclusion, let-7 is the main factor inducing embryonic diapause.

[Clinical outcome of allogeneic hematopoietic stem cell transplantation with FLAG sequential busulfan/cyclophosphamide conditioning regimen for refractory/relapsed acute myeloid leukemia].

Objective: To investigate the therapeutic effects of allogeneic hematopoietic stem cell transplantation (allo-HSCT) with FLAG sequential busulfan/cyclophosphamide(Bu/Cy) conditioning regimen for refractory/relapsed acute myeloid leukemia. Methods: From February 2012 to June 2017, 21 patients with refractory/relapsed acute myeloid leukemia underwent allo-HSCT with FLAG sequential Bu/Cy conditioning regimen. Transplantation-related complications and clinical outcome were retrospectively analyzed. Results: After conditioning, no hepatic veno-occlusive disease (VOD) and grade Ⅲ hemorrhagic cystitis occurred. 76.2% (16/21) patients had fever with 4 septicemia. One patient died of septic shock before engraftment. Twenty patients achieved neutrophil engraftment with a median time of 13 days (range, 10 to 21 days). Seventeen patients achieved platelet engraftment with a median time of 18 days (range, 9 to 25 days). The cumulative incidence of acute graft-versus-host disease (aGVHD) was 39.5%, and 3 patients developed grade Ⅲ-Ⅳ aGVHD. Of 19 patients who survived more than 100 days after transplantation, 4 had local chronic graft-versus-host disease (cGVHD). Of 21 patients, the median survival time was 15 months (range, 0.5 to 67 months) post-transplantation. Transplantation-related mortality rate was 28.7%. Leukemia relapse occurred in 4 patients with a median time of 4 months (range, 3 to 8 months) after transplantation. The cumulative relapse rate at 1 year was 21.4%. The 1-year and 3-year overall survival (OS) rates were 60.7% and 54.9% respectively. Log-rank analysis revealed that bone marrow blasts ≥ 20% or extramedullary leukemia before transplantation, poor platelet engraftment and grade Ⅲ-Ⅳ aGVHD were significantly related to shortened OS (P<0.05). Conclusions: Allo-HSCT with FLAG sequential Bu/Cy conditioning regimen in patients with refractory/relapsed myeloid leukemia has acceptable transplantation-related risk and relapse rate. The 1-year and 3-year OS rates are comparable with those in remission patients.

Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos.

BACKGROUND: The standard embryo cryopreservation method is still less than optimal for biopsied embryos. The aim of this study was to compare the survival of biopsied embryos cryopreserved with four different methods using non-transferable embryos. METHODS: Abnormal embryos from one or three pronuclei and spare embryos of grade 3 and 4 were used for this study. Non-biopsied embryos were cryopreserved using the standard method as control. Biopsied embryos were cryopreserved using four methods as follows: standard method, modified freezing method, modified thawing method and vitrification. Blastomere survival and blastulation of frozen-thawed embryos were compared between the different methods. RESULTS: The proportion of embryos with > or = 50% blastomere survival and total blastomere survival rate of biopsied embryos were significantly higher with vitrification than the other three methods. Both the modified freezing and modified thawing methods had significantly higher embryo survival and total blastomere survival rates than standard methods. However, there was no significant difference in blastulation of surviving embryos in all the five groups. CONCLUSIONS: Non-transferable embryos derived from clinical IVF/ICSI are useful for evaluation of the optimal freezing procedures for biopsied embryos. Vitrification increases the survival rate of human biopsied embryos above standard and modified cryopreservation methods.

Capillary electrophoresis-based immunoassay.

Capillary electrophoresis-based immunoassay (CEIA) is a developing analytical technique with a number of advantages over conventional immunoassay, such as reduced sample consumption, simpler procedure, easy simultaneous determination of multiple analytes, and short analysis time. However, there are still a number of technical issues that researchers on CEIA have to solve before the assay can be more widely used. These issues include method to improve the concentration sensitivity of the assay, requirement for robust separation strategy for different analytes, and method to increase the throughput of the assay. The approaches to solve these issues are reviewed. Several studies have been devoted to develop general separation strategies for CEIA, and to enhance the sensitivity of detection. The recent development of microchip-based CEIA is encouraging and is likely to address more drawbacks of CEIA, particularly on the throughput issue.

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate-capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay.

A non-competitive immunoassay was performed by sodium dodecyl sulfate-capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 microM under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.

Use of capillary electrophoresis-based competitive immunoassay for a large molecule.

A systematic study on the optimization of capillary electrophoresis-based immunoassay (CEIA) was performed using bovine serum albumin (BSA) and monoclonal anti-BSA. The immunocomplex could not be resolved from free BSA or anti-BSA with UV detection. When fluorescein isothiocyanate-labeled BSA (FITC-BSA) was used as tracer, the free and bound FITC-BSA were well separated giving definite peaks with laser induced fluorescence detection. The factors affecting the separation of the free and bound FITC-BSA, including voltage, pH and ionic strength of the running buffer, were systematically analyzed. Competitive CEIAs were demonstrated in uncoated and coated capillaries with whole or Fab fragment of the antibody. The coefficient of variation for the quantification of BSA in coated capillary was less than that in uncoated capillary. This study demonstrated that competitive CEIA could be applied to quantify high-molecular-mass protein in biological fluids.