A Propensity Score Matching Study of Autologous Hematopoietic Stem Cell Transplantation and New Drug Chemotherapy for Newly Diagnosed Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To compare the clinical efficacy, survival, and prognosis of autologous hematopoietic stem cell transplantation (ASCT) with new drug chemotherapy in the treatment of newly diagnosed multiple myeloma (NDMM) in the new drug era.@*METHODS@#The clinical data of 149 patients with NDMM treated with new drug induction regimen in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2012 to December 2019 were retrospectively analyzed. Twenty-four patients who received ASCT were in ASCT group, and 125 patients who did not receive ASCT were in non-ASCT group. The median follow-up time was 43 (1-90) months. The propensity score matching (PSM) method was used to balance confounding factors, then depth of response, overall survival (OS), and progression-free survival (PFS) between the two groups were compared and subgroup analysis was performed.@*RESULTS@#After matching, the covariates were balanced between the two groups. Fifty-one patients (15 cases in ASCT group and 36 cases in non-ASCT group) were included. ASCT patients had a better complete response (CR) rate than non-ASCT patients receiving maintenance therapy (93.3% vs 42.3%, P=0.004), while there were no statistical differences in deep response rate and overall response rate (ORR) between the two groups (93.3% vs 65.4%, P=0.103; 93.3% vs 96.2%, P=1.000). Before matching, the 3 and 5-year PFS rate and median PFS (mPFS) in ASCT group and non-ASCT group were [89.6% vs 66.5%, P=0.024; 69.8% vs 42.7%; non-response (NR) vs 51.0 months], and the 3 and 5-year OS rate and median OS (mOS) were (100% vs 70.6%, P=0.002; 92.3% vs 49.6%; NR vs 54.0 months). After matching, the 3 and 5-year PFS rate and mPFS in ASCT group and non-ASCT group were (83.6% vs 61.7%, P=0.182; 62.7% vs 45.7%; NR vs 51.0 months), the 3 and 5-year OS rate and mOS were (100% vs 65.6%, P=0.018; 88.9% vs 46.9%; NR vs 51.0 months). Subgroup analysis showed that patients with mSMART 3.0 high risk stratification, the 3-year PFS rate and mPFS in ASCT group and non-ASCT group were (83.3% vs 41.5%, P=0.091; NR vs 34.0 months), and the 3-year OS rate and mOS were (100% vs 41.5%, P=0.034; NR vs 34.0 months). Patients with mSMART 3.0 standard risk stratification, the 3-year PFS rate and OS rate in ASCT group and non-ASCT group were (83.3% vs 76.8%, P=0.672; 100% vs 87.2%, P=0.155). The 3-year PFS and OS rate in MM patients who achieved deep response within 3 months after transplantation compared with non-ASCT patients who achieved deep response after receiving maintenance therapy were (83.1% vs 56.7%, P=0.323; 100% vs 60.5%, P=0.042), and the 3-year PFS and OS rate in patients who achieved overall response in both groups were (83.1% vs 62.5%, P=0.433; 100% vs 68.1%, P=0.082). After matching, Cox multivariate regression analysis showed that mSMART 3.0 risk stratification and ASCT were independent prognostic factors for OS.@*CONCLUSION@#In the new drug era, ASCT can increase CR rate and prolong OS of NDMM patients. ASCT patients who are mSMART 3.0 high risk stratification or achieved deep response within 3 months after transplantation have better OS than non-ASCT patients receiving new drug chemotherapy. ASCT and mSMART 3.0 risk stratification are independent prognostic factors for OS in NDMM patients.

Comparison of outcomes after human leukocyte antigen-matched and haploidentical hematopoietic stem-cell transplantation for multiple myeloma / 中华医学杂志(英文版)

Background@#Allogeneic stem-cell transplantation (SCT) is a well-established immunotherapeutic strategy for multiple myeloma (MM) with a potent and often sustained graft-vs.-myeloma effect. This multicenter investigation aimed to analyze the complications and survival of haploidentical SCT in patients with MM, and compare the main outcomes with matched-related donors (MRDs).@*Methods@#Haploidentical and MRD SCT was identified from a cohort of 97 patients with MM who received a myeloablative transplantation in 13 hospitals from May 2001 to December 2017. A matched-pair analysis was designed. For each haplo recipient, the recipients were randomly selected from the MRD group and were matched according to the following criteria: year of the hematopoietic SCT (±2 years), disease status at transplantation, and the length of follow-up.@*Results@#Seventy cases received MRD and 27 received haploidentical transplantation. The two groups showed no significant differences regarding age, gender, cytogenetic risk, and diagnostic stage. The cumulative incidences of non-relapse mortality (NRM) at 1 and 3 years based on donor type were 20.5% (95% confidence interval [CI], 10.90–30.10%) and 24.2% (95% CI, 13.81–34.59%) for the MRD group and 16.80% (95% CI, 1.71–31.89%) and 28.70% (95% CI, 8.71–48.69%) for the haplo group, respectively. Cumulative incidence of NRM did not differ significantly between the two groups (χ2 = 0.031, P = 0.861). The cumulative incidences of progression-free survival (PFS) and 1 year and 3 years by type of donors were 59.8% (95% CI, 48.24–71.36%) and 45.4% (95% CI, 33.44–57.36%), and 65.6% (95% CI, 47.18–84.02%) and 26.8% (95% CI, 7.59–46. 01%) for MRD and haploidentical donor, respectively. Cumulative incidence of PFS did not differ significantly between the two groups (χ2 = 0.182, P = 0.670). In multivariate analyses, no statistically significant differences were observed between haploidentical and MRD for relapse, NRM, PFS, and overall survival. There were no statistically differences on main outcomes after haploidentical and MRD.@*Conclusion@#Haploidentical SCT could be performed safely and feasibly for patients with MM in need.

Comparison of outcomes after human leukocyte antigen-matched and haploidentical hematopoietic stem-cell transplantation for multiple myeloma / 中华医学杂志(英文版)

BACKGROUND@#Allogeneic stem-cell transplantation (SCT) is a well-established immunotherapeutic strategy for multiple myeloma (MM) with a potent and often sustained graft-vs.-myeloma effect. This multicenter investigation aimed to analyze the complications and survival of haploidentical SCT in patients with MM, and compare the main outcomes with matched-related donors (MRDs).@*METHODS@#Haploidentical and MRD SCT was identified from a cohort of 97 patients with MM who received a myeloablative transplantation in 13 hospitals from May 2001 to December 2017. A matched-pair analysis was designed. For each haplo recipient, the recipients were randomly selected from the MRD group and were matched according to the following criteria: year of the hematopoietic SCT (±2 years), disease status at transplantation, and the length of follow-up.@*RESULTS@#Seventy cases received MRD and 27 received haploidentical transplantation. The two groups showed no significant differences regarding age, gender, cytogenetic risk, and diagnostic stage. The cumulative incidences of non-relapse mortality (NRM) at 1 and 3 years based on donor type were 20.5% (95% confidence interval [CI], 10.90-30.10%) and 24.2% (95% CI, 13.81-34.59%) for the MRD group and 16.80% (95% CI, 1.71-31.89%) and 28.70% (95% CI, 8.71-48.69%) for the haplo group, respectively. Cumulative incidence of NRM did not differ significantly between the two groups (χ = 0.031, P = 0.861). The cumulative incidences of progression-free survival (PFS) and 1 year and 3 years by type of donors were 59.8% (95% CI, 48.24-71.36%) and 45.4% (95% CI, 33.44-57.36%), and 65.6% (95% CI, 47.18-84.02%) and 26.8% (95% CI, 7.59-46. 01%) for MRD and haploidentical donor, respectively. Cumulative incidence of PFS did not differ significantly between the two groups (χ = 0.182, P = 0.670). In multivariate analyses, no statistically significant differences were observed between haploidentical and MRD for relapse, NRM, PFS, and overall survival. There were no statistically differences on main outcomes after haploidentical and MRD.@*CONCLUSION@#Haploidentical SCT could be performed safely and feasibly for patients with MM in need.

Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations / 华中科技大学学报（医学）（英德文版）

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 华中科技大学学报（医学）（英德文版）

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

No association between cytochrome P450 2D6 gene polymorphism and risk of acute leukemia: evidence based on a meta-analysis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Many studies indicated the human cytochrome P450 2D6 (CYP2D6) gene polymorphism was associated with acute leukemia (AL) susceptibility, however, the results were inconsistent. So we performed this meta-analysis to evaluate the relationship between CYP2D6*3 or CYP2D6*4 polymorphism and AL susceptibility.</p><p><b>METHODS</b>We searched PubMed database up to February 20, 2013, and finally yielded 9 case-control studies including 1343 cases and 1843 controls which tested the association between CYP2D6*3 or *4 polymorphism and AL. After data extraction, we conducted a meta-analysis using the Comprehensive Meta Analysis software.</p><p><b>RESULTS</b>Overall, no significant association between CYP2D6*3 or *4 polymorphism and AL risk was found in this metaanalysis (+ vs. -: OR = 1.13, 95% CI = 0.79-1.63; +/+ vs. -/-: OR = 1.73, 95% CI = 0.99-3.02; -/+ vs. -/-: OR = 1.03, 95% CI = 0.68-1.56; (-/+ and +/+) vs. -/-: OR = 1.08, 95% CI = 0.72-1.63; +/+ vs. (-/+ and -/-): OR = 1.76, 95% CI = 0.98-3.17). Similar results were also been found in stratified subgroup analysis. There was no publication bias.</p><p><b>CONCLUSION</b>CYP2D6*3 or *4 polymorphism might not be associated with AL susceptibility. However, the results need to be further confirmed by well-designed and high quality randomized controlled trials with larger sample sizes.</p>

Arsenic trioxide downregulates the expression of annexin II in bone marrow cells from patients with acute myelogenous leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most patients with acute myelogenous leukemia (AML) suffer from disordered hemostasis. We have previously shown that annexin II (Ann II), a high-affinity co-receptor for plasminogen/tissue plasminogen activator, plays a central role in primary hyperfibrinolysis in patients with acute promyelocytic leukemia (APL). The expression of Ann II in cells from patients with major subtypes of AML and the effect of arsenic trioxide (As2O3) on Ann II expression in AML cells were investigated to determine whether As2O3-mediated downregulation of Ann II could restore hemostatic stability.</p><p><b>METHODS</b>A total of 103 patients (48 females and 55 males; age, 19 - 58 years) were included. Plasma samples were collected before and after treatment as well as after complete remission. Ann II and plasminogen activation were measured in leukemic cells during treatment with 1 micromol/L As2O3.</p><p><b>RESULTS</b>Before As2O3 treatment, Ann II mRNA expression (real-time PCR) was the highest in M3 cells (P < 0.05), higher in M5 cells than that in M1, M2, M4, and M6 cells (P < 0.001), and positively correlated with Ann II protein expression (flow cytometry) (r = 0.752, P < 0.01). Exposure for up to 120 hours to As2O3 (1 micromol/L) had no significant effect on Ann II protein in M1 and M2 leukemic cells, but decreased Ann II protein expression twofold within 48 hours of exposure in M3 cells (P < 0.05) and twofold within 96 hours in M5 cells (P < 0.05). The rate of plasmin generation was higher in APL, M5, and M4 cells than in M1, M2, and M6 cells.</p><p><b>CONCLUSIONS</b>As2O3 may reduce hyperfibrinolysis in AML by downregulation of Ann II. Furthermore, As2O3 affects more than one form of AML (APL, M4 and M5), suggesting its potential role in their management.</p>

Fludarabine and cytarabine combined chemotherapy followed by transfusion of donor blood stem cells for treating relapse of acute leukaemia after allogeneic haematopoietic stem cell transplantation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Relapse remains an obstacle to successful allogeneic haematopoietic stem cell transplantation (allo-HSCT) for patients with acute leukaemia and no standard treatment is available. We assessed fludarabine and cytarabine with transfusion of donor haematopoietic stem cell in treating the relapse of acute leukaemia after allo-HSCT.</p><p><b>METHODS</b>Seven patients, median age 34 years, with relapse of acute leukaemia after allo-HSCT received combination chemotherapy of fludarabine with cytarabine for 5 days. Five patients suffered from acute myeloid leukaemia (2 refractory) and 2 refractory acute lymphoblastic leukaemia. After the transplantation, the median relapse time was 110 days (range, 38 - 185 days). Two days after chemotherapy, 5 patients received infusion of donor's peripheral blood stem cells, mobilized by granulocyte colony stimulating factor. No prophylactic agents of graft versus host diseases were administered.</p><p><b>RESULTS</b>Six patients achieved haematopoietic reconstitution. DNA sequence analysis at day 30 after treatment identified all as full donor chimera type. The median observation time was 189 days. After the treatment, the median time for neutrophilic granulocyte value = 0.5 x 10(9)/L and for platelet value = 20 x 10(9)/L were 13 days (range, 10 - 18 days) and 15 days (range, 11 - 24 days), respectively. Graft versus host disease occurred in 2 patients (acute) and 3 (chronic). Five patients suffered from pulmonary fungal infection (2 died), 3 haemorrhagic cystitis and 2 cytomegalovirus viraemia. The other patients died of leukaemia related deaths. Three patients with chronic graft versus host disease who had received donor peripheral blood stem cells reinfusion have survived for 375 days, 232 days and 195 days, respectively.</p><p><b>CONCLUSIONS</b>Fludarabine with cytarabine plus the donor haematopoietic stem cell should be considered as an effective therapeutic regimen for relapse of acute leukaemia after allo-HSCT. The disease free state of patients may increase, though with high risk of secondary fungal infection.</p>

Regulation of tissue factor expression by anti-oncogene PTEN in human neuroblastoma cell line / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of PTEN gene in the regulation of tissue factor (TF) expression in human neuroblastoma cells.</p><p><b>METHODS</b>Expression of PTEN or TF was determined by Western blotting. Transcription of TF was examined by RT-PCR. PTEN gene expressing vector pCMV-PTEN was transfected with Lipofectamine2000. Phosphorylation of AKT was inhibited by LY294002 and then examined by Western blotting.</p><p><b>RESULTS</b>Human neuroblastoma cell line SK-N-SH was PTEN-positive and expressed low level TF, whereas an other neuroblastoma cell line SK-N-MC was PTEN-negative but expressed high level TF. TF level was downregulated in SK-N-MC cells by enforced expression of PTEN in a dose dependent manner. Inhibition of TF was achieved along with inactivation of AKT. Furthermore treatment with PI3K/AKT inhibitor LY294002 also resulted in decrease of TF expression in a dose-dependent manner.</p><p><b>CONCLUSION</b>Expression of TF is inhibited by PTEN gene via inactivating PI3K/AKT pathway, loss of PTEN might be the explanation of aberrant high-level TF in human neuroblastoma. It may be at least one of the mechanisms by which loss of PTEN expression confers to cancer progression.</p>

Inhibition of tissue factor by siRNA enhances doxorubicin-induced apoptosis in human neuroblastoma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the regulation of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma.</p><p><b>METHOD</b>The expression of TF was examined by Western blotting. TF siRNA-pSUPER plasmid was constructed by inserting a specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TF siRNA-pSUPER was performed using lipofectamine 2000. The activation of caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope.</p><p><b>RESULTS</b>(1) Human neuroblastoma cell line SK-N-MC expressed high level of TF. (2) Downregulation of TF expression was achieved by transfection of TF siRNA-pSUPER into SK-N-MC cells in a dose-dependent manner. (3) Cleavage of caspase-3 and PARP was increased in transfected SK-N-MC cell with down-regulation of TF. (4) TF siRNA treatment at 1 microg/ml for 8 h significantly increased apoptotic cell number in transfected SK-N-MC cells compared to that in non-transfected cells (P < 0.05) while exposing to 1 microg/ml doxorubicin for 8 h.</p><p><b>CONCLUSIONS</b>Downregulation of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells.</p>

Effects of arsenic trioxide or retinoic acid on mRNA and protein expression of tissue factor and thrombomodulin and procoagulant activity in NB4 cells / 中国实验血液学杂志

To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.

The clinical significance of dynamic changes of CD4~+ CD25~+ regulatory T cells in patients subject to allogeneic hematopoietic stem cell transplantation / 中华器官移植杂志

Objective To investigate the clinical significance of dynamic changes of CD4~+ CD25~+ regulatory T cells(Treg)in patients subject to allogeneic hematopoietic stem cell transplanta- tion(alIo-HSCT).Methods Forty-five patients received allo-HSCT.The graft-vesus-host disease (GVHD)was prevented by cyclosporine A and short-term MTX regimen in 31 patients.Fourteen of all the patients received Zenapox(CD25MAb)at the day of transplantation and day 4 after transplan- tation.The levels of Treg in peripheral blood were detected by flow cytometry from 45 patients at 2nd,4th,8th and 12th week after allo-HSCT and the time of aGVHD development,respectively.Re- suits Anti-CD25 could suppress the peripheral blood levels of Treg significantly.The Treg levels were significantly higher in patients with grade 0-1 aGVHD than those with 2-4 aGVHD at 8th and 12th week after transplantation.Among patients with 2-4 aGVHD,Treg levels were significantly low- er after development of aGVHD than before.Conclusions Treg are important for the aGVHD preven- tion and can be a useful clinical surveillant index for the development of aGVHD.It can significantly decrease the levels of Treg in the peripheral blood with anti-CD25.

CML cell line K562 cell apoptosis induced by mangiferin / 中国实验血液学杂志

This study was aimed to investigate the effects and the mechanism of mangiferin on chronic myeloid leukemia cell lines K562 cells in vitro. The antiproliferation effects of mangiferin on K562 leukemia cells were tested by tetrazolium salt (MTT) method; the apoptosis induced by mangiferin on K562 cell line was explored by means of cell morphology, DNA gel electrophoresis and flow cytometry. The changes in bcr/abl gene expression was detected by using reverse transcriptase (RT)-PCR. The results showed that five different concentrations of mangiferin (25 - 200 micromol/L) dose-dependently and time-dependently inhibited the proliferation of K562 cells, and induced apoptosis in K562 cell line. RT-PCR revealed that bcr/abl gene expression was down-regulated when K562 cells had been treated with different concentrations of mangiferin. In conclusion, mangiferin remarkably inhibits the proliferation of K562 leukemia cells in vitro, and induces apoptosis in K562 cell line probably through down-regulation of bcr/abl gene expression.

Coagulation factor VII levels in uremic patients and theirs influence factors / 中国实验血液学杂志

This study was aimed to investigate coagulation factor VII level in uremic patients with chronic renal failure and to explore theirs influence factors. The plasma levels of coagulation factor VII were detected in 30 uremic patients with chronic renal failure before and after hemodialysis for 1 month, the factor VII activity (FVII:C) was determined by one-stage coagulation method, while activated factor VII (FVIIa) was measured by one-stage coagulation method using recombinant soluble tissue factor, and factor VII antigen was detected by ELISA. The results showed that: (1) The FVIIa, FVII:C and FVIIAg levels in chronic uremic patients before hemodialysis were 4.00 +/- 0.86 microg/L, (148.5 +/- 40.4)% and (99.8 +/- 21.1)% respectively, which were significantly increased, as compared with healthy controls [2.77 +/- 1.02 microg/L, (113.1 +/- 33.0)% and (73.7 +/- 18.3)% respectively, P < 0.05]. (2) After hemodialysis the FVIIa, FVII:C and FVIIAg levels in uremic patients significantly enhanced to 5.56 +/- 1.45 microg/L, (200.8 +/- 68.7)% and (124.1 +/- 19.3)% respectively (P < 0.05). (3) The abnormal increase of coagulation factor VII was positively correlated with levels of blood uria nitrogen and serum creatinine before hemodialysis but not after hemodialysis. It is concluded that the enhanced levels of coagulation factor VII in chronic uremic patients suggested abnormal activated state, herperactivity and elevated production of factor VII which correlated with renal functional injury. The abnormality of factor VII in uremia may be aggravated by hemodialysis. Coagulation factor (FVII) may be a risk factor for cardiovascular events in uremic patients who especially had been accepted long-term hemodialysis.

Study of the effect of TF/FVIIa complex on the expression of u-PAR mRNA in human ovarian cancer / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct the expression vector of human tissue factor (TF), and investigate the influence of TF/coagulant factor VIIa (FVIIa) complex on the transcriptional expression of urokinase plasminogen activator (u-PA) and u-PA receptor (u-PAR) in human ovarian cancer.</p><p><b>METHODS</b>The human TF cDNA was obtained from placenta by RT-PCR and then inserted into eukaryotic expression vector pcDNA3 to obtain the TF-pcDNA3 combinant. This combinant was transfected into human ovarian cancer cell line A2780 by lipofectamine. Stably-transfected cells A2780/TF were screened. A2780 and A2780/TF cell lines were stimulated by FVIIa respectively, and the transcriptional levels of u-PA and u-PAR were examined by RT-PCR.</p><p><b>RESULTS</b>(1) The constructed product was identified as TF-pcDNA3 combinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780/TF cell (transfected cell 3.91 +/- 0.28, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. (3) The u-PA and u-PAR mRNA levels in A2780 cell line did not change significantly after stimulated by FVIIa; (4) While stimulated by FVIIa, the u-PAR mRNA levels in A2780/TF cells increased significantly in both dose-dependent and time-dependent manner, while the u-PA mRNA levels did not change significantly; (5) In the A2780/TF cell line the enhanced expression of u-PAR mRNA by FVIIa was significantly inhibited by coincubated with anti-TF antibody.</p><p><b>CONCLUSION</b>TF/FVIIa complex could up-regulate the transcription of u-PAR in human ovarian cancer cells so as to enhance tumor invasion and metastasis.</p>

The effects of tissue factor/activated factor VII complex on the invasion and metastasis of human ovarian cancer / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of tissue factor/activated factor VII (TF/FVIIa) complex in human ovarian cancer invasion and metastasis.</p><p><b>METHODS</b>(1) Constructed an expression vector of TF, pcDNA3-TF and established a human ovarian cell line A2780/TF expressing high level TF by using molecular cloning and gene transfection techniques. (2) By Boyden chamber assay to count the numbers of A2780 and A2780/TF cells that penetrated the matrigel to the back of PVPF membrane after FVIIa stimulation. (3) BALB/c nude mice were used to establish experimental model of metastasis with A2780 or A2780/TF and the lung tissue sections were examined by microscopy for cancer metastasis.</p><p><b>RESULTS</b>(1) Compared with their parental A2780 cells, A2780/TF cells expressed high level of TF mRNA (3.99 +/- 0.15 vs 0.97 +/- 0.23, P < 0.01) and TF antigen on cell surface \[(48.56 +/- 9.53)% vs (2.73 +/- 1.15)%, P < 0.01\]. (2) After stimulation, the A2780/TF cell number on the back of PVPF membrane increased from basal level 157.3 +/- 19.2 to 447.7 +/- 39.4 (P < 0.01), which could decreased to basal level when coincubated with anti-TF antibody. (3) Cancer metastasis was found in 22.2% of nude mice transplanted with A2780 cells, while in 88.9% of those transplanted with A2780/TF cells.</p><p><b>CONCLUSION</b>TF could promote the invasion and metastasis of human ovarian cancer cells through TF/FVIIa pathway.</p>

In vitro infection of human megakaryocyte precursors by human cytomegalovirus (HCMV) and the antiviral effect of HCMV antisense oligonucleotides / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the suppression effect of human cytomegalovirus (HCMV) on megakaryocytes and their precursors and study the antiviral effect of antisense phosphorothioate deoxyoligonucleotide (ASON) against HCMV.</p><p><b>METHODS</b>CD34(+) cells were induced to proliferate and differentiate committedly to megakaryocytes in a semi-solid CFU-MK culture system. Cultured cells and ASON pretreated CD34(+) cells were infected by HCMV of AD169 strain. HCMV immediate early protein (IEP) DNA and mRNA and UL36 mRNA were detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Cytotoxicity was evaluated by MTT assay.</p><p><b>RESULTS</b>HCMV AD169 suppressed the proliferation of megakaryocytes significantly. Compared with the mock group, the CFU-MK yields were decreased by 21.6%, 33.8%, and 46.3%, respectively, in 3 different titers of virus infected groups (P < 0.05). The suppression was virus titer dependent. HCMV IEP DNA, HCMV IEP mRNA and UL36 mRNA were detected in the colony cells of viral infection group. Compared with the infected group by HCMV AD169, UL36Anti treatment at 0.08 micromol/L could recover the CFU-MK yields significantly (P < 0.05). In the infected MK, which was pretreated with UL36Anti at 0.08 micromol/L, HCMV UL36 mRNA was undetectable by RT-PCR. The oligonucleotide MM(1) containing a G-to-C substitution in UL36Anti was inactive at 0.08 micromol/L but active at 0.40 micromol/L. The concentration of UL36Anti necessary to significantly affect cell growth was 90.00 micromol/L.</p><p><b>CONCLUSIONS</b>HCMV AD169 infection inhibits the proliferation and differentiation of megakaryocytes and their precursors. There are early transcriptions of HCMV IE and UL36 protein in infected CFU-MK. The specific ASON has a definite anti-HCMV activity.</p>

Experimental study on activating antileukemic T cells by vaccination with dendritic cells pulsed with survivin / 中国实验血液学杂志

The objective of this study is to investigate the effect of vaccination with dendritic cells pulsed with survivin antigen on activation of antileukemic T cells, and inhibiting proliferation of leukemic cells. The expression of survivin on acute leukemic cells were detected by cofocal microscopy and immunoprecipitation-Western blot. DCs collected from peripheral blood mononuclear cells were pulsed with survivin purified proteins. Stimulation index (SI) and antileukemia CTL induction were analyzed with (3)H-TdR incorporation and (51)Cr releasing assay, respectively. The phenotype of T cells and DCs were identified by flow cytometry. By immunofluorescence of bone marrow and peripheral blood mononuclear cells, survivin expression was detected in 16 out of 19 AML cases (84.2%). The results showed that survivin fluorescence distribution was in cytoplasm. DCs from peripheral blood mononuclear cells were successfully induced, with typical DC morphologic characteristic. The vaccination with dendritic cells pulsed with survivin antigen dramatically stimulated the proliferation of T cells. The DCs loading survivin activated T cells with higher CD4(+) T(H) ratio as compared with DCs group, T cells activated with DCs expressed CD8 and CD56. Survivin DCs significantly inhibited the growth of leukemic cells in vitro. In conclusion, survivin antigen expressed in the cytoplasm of leukemic cells, leukemic vaccination with DCs pulsed with survivin antigen in vitro inhibited the proliferation of leukemic cells, that may be a pathway for therapy of leukemia.