Cloning of New Antigen Gene MLAA-34 Promoter and Identification of Core Region in Acute Monocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region.@*METHODS@#The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods.@*RESULTS@#The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P＜0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3.@*CONCLUSION@#The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.

Effects of Transcription Factor MZF-1 on Transcriptive Regulation of Acute Monocytic Leukemia-related Gene MLAA-34 / 中国实验血液学杂志

OBJECTIVE@#To investigate the transcriptional regulation of transcription factor MZF-1 on acute monocytic leukemia-related gene MLAA-34.@*METHODS@#The effect of MZF-1 on the transcriptional activity of MLAA-34 gene promoter was analyzed by luciferase reporter gene detection system and site-directed mutation technique. The EMSA and ChIP assay were used to verify whether MZF-1 directly and specifically binds to the core region of MLAA-34 promoter. The over-expression vector and interference vector of MZF-1 were constructed to transfect U937 cells, and RT-PCR and Western blot were used to detect the transcription and expression changes of MLAA-34 gene.@*RESULTS@#The transcription factor MZF-1 had a regulatory effect on MLAA-34 gene expression, and the relative luciferase activity was decreased after MZF-1 binding point mutation (P<0.01). EMSA and ChIP experiments demonstrated that MZF-1 could directly bind to MLAA-34 promoter and play a regulatory role. In the over-expression test, the increase of MZF-1 could up-regulate the expression of MLAA-34 (P<0.05). In the interference test, the decrease of MZF-1 could down-regulate the expression of MLAA-34 (P<0.05).@*CONCLUSION@#Transcription factor MZF-1 can bind to the transcriptional regulatory region on the promoter of MLAA-34 gene and promote the transcription of MLAA-34 gene in acute monocytic leukemia.

Detection of Novel Antigen MLAA-34 Gene Mutation in Acute Monocytic Leukemia and Its Correlation with Efficacy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of all exone mutation in MLAA-34 gene with chemotherapeutic efficacy for leukemia.</p><p><b>METHODS</b>The expression level of MLAA-34 gene in 40 patients with AML-M5 and 5 healthy volunteers as control was detected by RT-PCR and its effect on chemotherapeutic efficacy were analyzed by RT-PCR; the effect of MLAA-34 gene mutation on overall survival (OS) and progression-free survival (PFS) of AML-M5 patients was analyzed by sequencing of all 12 exoues in MLAA-34 gene, the correlation between the mutation of prognostic genes important to leukemia and the mutation of MLAA-34 gene was explored.</p><p><b>RESULTS</b>The expression level of MLAA-34 gene was significantly up-regulated as compared with that of healthy volunteers, moreover this up-regulation was related with a C59T SNP site located in second exon of MLAA-34 gene, meanswhile this SNP site is affinitive to the well-known mdecular markers of AML, inclinding Fms-like tyrosine kinase (FLT-3) and DNA methyltransferase-3A(DNAMT3A). The AML-M5 patients with high expression of MLAA-34 gene poorly responded to chemotherapy, the AML-M5 patients with MLAA-34 C59T mulation had even more high expression of MLAA-34 gene and significantly short OS and PFS in comparison with those of patients without C59T mutation.</p><p><b>CONCLUSION</b>The C59T mutation in MLAA-34 gene is a high risk factor for recurrence of AML, and may be a cadidate target for treatment of AML.</p>

Clinical Characteristics,Treatment Strategies and Prognostic Factors of 254 Cases of Non Hodgkin's Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical characteristics and prognostic factors of patients with non-Hodgkin's lymphoma (NHL) in single center of the Northwest area in China for 10 years, so as to provide the evidences for early diagnosis, stratified treatmetn, evaluation of therapeutic efficacy and prognosis, as well as early prevation and so on.</p><p><b>METHODS</b>The clinical data of 254 patients with NHL were analyzed retrospectively, the clinical characteristics were evaluated by unvariate analysis; then the single factors affecting prognosis were enrolled in multivariate analysis and the independent prognostic factors affecting the survival of patients were summarized.</p><p><b>RESULTS</b>A total of 182 cases achieved CR（71.6%）, PR 30 cases（11.8%）, SD 22 cases（8.7%）, PD 20 cases（7.9%）, and RR 212 cases（83.5%). The statistically significant unfavorable prognostic factors for NHL revealed by univariate analysis included age, invasive, Ann Arbor stage, relapse, and total course of chemotherapy. Cox regression model analysis showed that the Ann Arbor stage, IPI, ECOG, B symptoms, peripheral blood cell levels, short-term efficacy, course to achieve CR, and total course of chemotherapy all were the independent prognostic factors.</p><p><b>CONCLUSION</b>The incidence characteristics of NHL in this center displayed mainly middle and high-risk B cell type with attacks at young age, aggression and in lymph nodes. For aggressive lymphoma, the single and multiple prognostic factors may provide the significant guides for the treatment, individualized plan and evaluation of prognosis. The course number of chemotherapy is one of the important factors for survival and prognosis, possessed clinical significance, and worth further clinical research for aggressive lymphoma.</p>

Therapeutic effects of porcine versus rabbit antithymocyte globulins for treatment of severe aplastic anemia / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the efficacy of porcine and rabbit antithymocyte globulins (ATG) in the treatment of severe aplastic anemia (SAA).</p><p><b>METHODS</b>We reviewed the clinical data of 43 SAA patients receiving porcine ALG treatment and 32 patients receiving rabbit ATG treatment between 2004 and 2013 in our hospital. The overall response rates of the patients at 6 month were compared, and the patients' survival in the two groups was analyzed using Kaplan-Meier survival curves.</p><p><b>RESULTS</b>The overall response rates at 6 months was significantly higher in porcine ALG group than in rabbit ATG group (79.07% vs 56.25%, P=0.034). The 5-year overall survival was also higher in porcine ALG group than in rabbit ATG group, but this difference was not statistically significant (86.047% vs 72.878%, P=0.190).</p><p><b>CONCLUSIONS</b>Porcine ALG is superior over rabbit ATG in terms of hematological response but is comparable with rabbit ATG in view of the patients' survival and safety.</p>

Effects of Thalidomide on Peripheral Blood Th17 Cells of Patients with Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the change of T help cell 17 (Th17) in the peripheral blood of patients with multiple myeloma (MM) before and after treatment with thalidomide.</p><p><b>METHODS</b>A total of 35 MM patients treated with thalidomide and 35 healthy controls were enrolled in this study. The percentage of Th17 cells were detected by flow cytometry. The mRNA levels of retinoid-related orphan receptor gamma-t (RORγt) were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and the plasm IL-17 levels were measured by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The percentage of Th17 cells, the mRNA expression of RORγt and the plasm IL-17 levels in patients with MM were statistically higher than those in normal controls (P < 0.05). The percentage of Th17 cells was not correlate with the sex, age, disease type, globulin, immune globulin, light chain, M-protein and the proportion of plasmocytes (P > 0.05), but correlated with ISS stage, the level of β2-microglobulin and the plasm IL-17 levels (P < 0.05). The percentage of Th17 cells, the mRNA expression of RORγt and the plasm IL-17 levels in patients with response to thalidomide were statistically lower than those in patients before treatment (P < 0.05).</p><p><b>CONCLUSION</b>The Th17 cells increase in the peripheral blood of patients with MM, the Th17 cells may participate in the occurrence of MM. Thalidomide may exert anti-MM through down-regulating Th17 cells.</p>

Clinical Study of Non Hodgkin's Lymphoma Treated with Enhanced Chemotherapy Regimen and Increased Treatment Courses / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the non-Hodgkin's lymphoma treated with enhanced chemotherapy regimen and increase of treatment courses, including number of treatment courses, short-term efficacy, long-term survival and safety.</p><p><b>METHODS</b>All the 254 cases of NHL in our hospital from January 2004 to February 2014 received a variety of intensive enhanced chemotherapy regimen, such as CHOPE, MAED, MMED and TAED. The median number of treatment course was 14, including 8 in the 1st year, 4 in the 2nd and 2 in the 3rd.</p><p><b>RESULTS</b>(1) In 254 assessable patients, 182 patients (71.7%) achieved complete responses (CR), 30 patients (11.8%) achieved partial responses (PR), 22 patients (8.7%) achieved stable disease (SD), 20 patients (7.9%) achieved progressive disease (PD), 212 patients (83.5%) achieved response rate (RR). The median time of following-up was 56.5 months, the overall survivals (OS) of 1, 3 and 5 years were 90.1%, 74.5% and 61.1% respectively, the median survival time was 69 months, and the disease-free survivals (DFS) were 81.8%, 65.4% and 54.7% respectively, the median DFS was 65 months. (2) In therapeutic effects at early phase, the 3-year OS of patients who achieved CR, PR, SD and PD were 92.2%, 56.0%, 20.2% and 0% respectively; The 5-year OS of patients who achieved CR through ≤4 cycle treatments and the 5-year OS of patients who achieved CR through >4 cycles treatments were 83.1% and 6.8%, their DFS were 72.4% and 0% respectively. (3) The relapse rates of patients who received < 6, 6-8, 9-10, 11-13, 14, 15 and 20 cycle treatments were 82.5%, 78.9%, 71.9%, 65.8%, 41.8%, 30.4% and 16.7%. The response rate (RR) of patients who received 6-8 traditional chemotherapy cycle as CHOP or CHOP-like regimen were 50%-60% and relapse rate > 70%.</p><p><b>CONCLUSION</b>Compared with traditional chemotherapy regimens, the dose-escalated, intensive and modified chemotherapy regimen can significatly improve the therapeutic efficiency for patients with NHL, including CR, long-term survival rate, and a good tolerance for patients. The chemotherapy intensity has been confirmed to be an important factor that associated with therapeutic efficiency. On the conditions tolerated by patients, the number of treatment cycles for NHL patients can be increased at lest 14, with 8 in the first year, 4 in the second year and 2 in the third year. The increase of chemotherapy cycle can obviously reduce the relapse rate and improve the long-term prognosis of patients. It is worth to further explore.</p>

Cohort Study on GHA and New Combined Priming Chemotherapeutic Regimens in Treatment of Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical efficacy and adverse effects of GHA(G-CSF+homoharringtonin+cytarabine C) and new combined priming chemotherapeutic regimens(GHAA/GHTA) with high efficacy and low toxicity for treatment of relapsed and refractory acute myeloid leukemia(AML) and myelodysplastic syndrome(MDS), and to analyze the relation of above-mentioned regimens with the expression of co-stimuolating molecule B7.1.</p><p><b>METHODS</b>Standard GHA regimen consisting of G-CSF: 100 µg/(m2·d) subcutaneous injection, d 0-14; homoharringtonine: 1.0 mg/(m2·d) intravenous drip, d 1-14; Ara-C: 7.5-10 mg/(m2·d) subcutaneous injection, q12h, d 1-14. Other regimens as GHAA/GHTA were combined respectively with aclarubicin 20 mg d 1-7, or pirarubicin 20 mg d 1-7. 74 patients with refractory AML and 46 patients with MDS received these priming chemotherapy. The clinical efficacy and toxicity of above-mentioned priming chemotherapy were compared with 56 patients received routine chemotherapy (MA/TAE) respectively. And the expression of costimulatory molecule B7.1 on leukemia cells in patients of different subtypes was also detected by immunofluoressence and its relationship with clinical efficiency was explored.</p><p><b>RESULTS</b>(1) for AML patients treated with priming chemotherapy, the total remission was 67.56% (CR 54.05%, PR 13.51%), which was much higher than that of patients received routine chemotherapy (P<0.05). The CR rate of AML-M2 and AML-M5 group (65.51%, 61.90% respectively) was much higher than that of AML other subtypes (P<0.05), and the longest remission period lasted for 4 years; (2) for MDS patients treated with priming chemotherapy, the total remission was 60.87% (CR 45.65%, PR 15.22%), which was also significantly higher than that of patients received routine chemotherapy (P<0.05); (3) in comparison with patients received standard GHA priming regimen, the remission rate of combined priming chemotherapy GHAA/GHTA was significantly higher both in patients with AML (85.18%) and MDS (81.25%); (4) side effects after chemotheropy, including granulocyte deficiency, thrombocytopenia and anemia etc, lasted for 7-14 days; the severe infection rate was 1%, there were no severe bleeding, digest effect and damage of function in heart, liver and kidney. The therapy-related mortality was zero. Compared with routine chemotherapy, priming chemotherapy proved significantly safe and effective (P<0.05); (5) the expression rate of costimulatory molecule B7.1 showed large variance between AML and MDS, it was higher in AML-M2/AML-M5 and lower in AML of other subtypes (P<0.05). In the same case, B7.1 expression was positive correlated with efficiency of priming chemotherapy.</p><p><b>CONCLUSION</b>GHA priming chemotherapy, as well as other combination regimens GHAA/GHTA, are well-tolerated, effective regimens for refractory AML and advanced MDS, without severe side effects and therapy-related mortality. Especially the new regimens GHAA/GHTA has better efficacy, which are proved more efficient than conventional GHA. Efficiency of priming chemotherapy is positive correlated with B7.1 expression, its mechanism will be further explored.</p>

Effect of G-CSF on induction of ENA-78 and IL-8 in the patients with malignant lymphoma / 中国实验血液学杂志

Granulocyte colony stimulating factor (G-CSF) restores neutrophil count in patients with chemotherapy-induced neutropenia. G-CSF can also induce production of epithelial neutrophil activating protein-78 (ENA-78) and interleukin-8 (IL-8), chemotactic factors from neutrophils in vitro. This study was purposed to investigate whether this effect is also observed in vivo. 10 lymphoma patients were selected who received chemotherapy and G-CSF (nartograstim) administration. Blood was obtained before chemotherapy [Time Point 1 (TP1)], at neutropenic phase before G-CSF administration (TP2), and at neutrophil recovery phase after G-CSF (TP3). ENA-78 and IL-8 mRNA in neutrophils were quantified by real-time PCR. Phagocytosis and reactive oxygen species (ROS) generation were examined by flow cytometry. The results showed that ENA-78 and IL-8 mRNA expression at TP2 increased in 5 and 8 patients, respectively. The ENA-78 mRNA expression at TP3 was increased in 3 and decreased in 6 patients, and IL-8 mRNA expression at TP3 decreased in 7 patients. G-CSF did not affect phagocytosis and normalized ROS generation in all of the patient. It is concluded that increase of ENA-78 and IL-8 expression in neutrophils is common in chemotherapy-induced neutropenic patients. G-CSF administration does not significantly increase ENA-78 and IL-8 expression.

Anti-tumor effect of two adjuvants of CpG ODN on leukemic tumor in mouse models / 中国实验血液学杂志

To investigate the anti-tumor and side effect of CpG ODN 1826 and CpG ODN2006 as an adjuvants on leukemic tumor in mouse-models, an acute lymphocytic leukemic tumor in mouse model was established, then inoculated inactivated L1210 cells alone or with different vaccine adjuvants were injected subcutaneously into each DBA/2 model mouse at different times. The activities of mice, the tumor formation rate and the growth status of leukemic tumor were observed. The tumor was examined by pathologic section. The results showed that the vaccine of inactivated L1210 cells and CpG ODN 1826 could decrease the leukemic tumor formation rate, slow down the growth of leukemic tumor mass in mice and obviously cause necrosis of tumor cells, but it could not prolong the life spans of the tumor-burden mice; while CpG ODN2006 could not only decrease the tumor formation rate, slow down the growth of tumor mass in mice and result in obvious necrosis of tumor cells, but also could eliminate the existing tumor mass in mice, and prolong the life spans of the tumor-burden mice. It is concluded that using CpG ODN2006 as an adjuvant enhances the anti-tumor effect against the leukemic tumor, prolong the life span of tumor-burden mice without obvious side effect.

Indoleamine 2, 3-dioxygenase expression in cells of human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia and therapeutic effect of its inhibitor 1-methyl tryptophan / 中国实验血液学杂志

The objective of this study was to investigate the expression and function of indoleamine 2, 3-dioxygenase (IDO) in leukemia. The IDO expressions in human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) were detected by immunofluorescence staining. Constructed leukemia mouse model was used to observe whether the IDO inhibitor, 1-methyl tryptophan (1-MT), has any effect in treating leukemia. The experimental group were fed with 1-MT solution every day while the mice in control group had no further treatment. The results showed that the average ratios of IDO expression were 29.4 +/- 11.2% in M(5) patients and 24.7 +/- 7.96% in ALL patients respectively. After statistical test, IDO expression level in leukemia cells was significantly higher than that of normal mononuclear cells. The tumor decreased gradually in mice treated with 1-MT. At the terminal point of the experiment (88 days after vaccination), the average survival time in the experimental group was 42.3 days while the mice in control group only lived 15.1 days in average, which difference was statistically significant (P < 0.05). Some of the leukemia mice in the experimental group long-term survived without tumor (more than three months after vaccination). It is concluded that human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) express IDO, and both can be treated by 1-MT in mice.

Immunological screening for multiple myeloma-associated antigens and their bioinformatics analysis / 中国实验血液学杂志

This study was aimed to screen the cell cDNA expression library of multiple myeloma HMy2 (MM HMy2) by using "serological analysis of cDNA expression library (SEREX)" technique. The obtained 30 positive clones were all sequenced, and analyzed by BLAST (basic local alignment search tool). The results indicated that 6 known genes and 12 new MM-associated genes were obtained, part of which sequences were spliced by EST (expressed sequence tag) splicing. 6 known genes such as for ring finger protein 167, KLF10, TPT1 protein, p02 protein, cDNA FLJ46859 fis, DNMT1 methyltrasferase etc. have been demonstrated a certain relationship with other tumor's formation, progress and prognosis. The structures and functions of the new genes preliminarily analyzed and predicted by means of bioinformatics showed that MMSA-3, MMSA-8 and MMSA-11 encoding 215, 160 and 122 amino acid residues respectively had the full open reading frames (ORF). All the new genes might be located at euchromosomes but MMSA-1 at sex chromosome. MMSA-4 was highly similar to the protein controlling the transcription of tumor antigen, MMSA-5 might take part in cell phagocytosis, MMSA-7 might inactivated NF-kappaB, and MMSA-12 might be a lymphocytic cytoplasmic protein. The specificity of new genes such as MMSA-3 and MMSA-7 were higher, by a preliminary analysis using CrELISA. It is concluded that tumor antigens screened by this study can be used for early immunological diagnosis, surveillance of minor residual foci, assessment of prognosis, and preparation of tumor vaccine and so on.

Retrospective observation of curative effects on MDS refractory anemia with combination of all-trans retinoic acid, 1, 25-dihydroxyvitamin D3 and androgen / 中国实验血液学杂志

This study was aimed to examine whether a combination of all-trans retinoic acid (ATRA), 1, 25-dihydroxyvitamin D(3) and androgen possesses the therapeutic value for the MDS-refractory anemia (MDS-RA), and to analyze the mechanisms in detail. 62 cases receiving a scheme of combination of ATRA, 1, 25-dihydroxyvitamin D(3) and androgen (group A) were monitored. The remaining 33 cases (group B) were provided with vitamin supplementation, chalybeate drugs, and one or two of the combination. Bone marrow aspiration and biopsy were performed for collecting the specimens at the baseline and afterwards. The conditions of the patients were monitored by means of weekly complete blood counts and the monthly examination, including toxicity test, physical examination, electrocardiography, and biochemistry panel. The results showed that after treating for 8 weeks in group A, 4 out of 62 patients showed complete remission and 12 patients showed partial remission according to the defined response criteria, and 43 patients (69.35%) showed hematological improvement (HI). The further treatment for 16 out of 62 patients (25.81%), 13 failures (10 deaths, 2 RAEB and 1 RAEB-T) and 3 transformations (M(2), M(3), M(5)) with a median survival interval of 26.25 months, were observed and interrupted for some reasons. However, partial remission was observed only in 3 patients in group B, and HI amounted to 51.51%. Furthermore, the disease progression was observed in 12 out of 33 patients (36.36%) with a median survival interval of 16 months, 9 failures (including 6 deaths, 2 RAEB and 1 RAEB-T) and 3 transformations (M(2), M(3), M(4)). The overall ratios of survival for 3 and 5 years in group A, which received the combination, reached to 69.24% and 53.72% respectively, in comparison with 52.23% and 31.34% in the patients of group B (log-rank, P = 0.016). The following requirements, if were met, would be significant for prognosis: the combination regiment, no transformation, children, no complication, female, 90-120 g/L of hemoglobin concentration, normal cellular bone marrow and uni-cytopenias (P < 0.05). Moreover, Cox regression showed that therapy, transformation and age are all the independent factors (P < 0.05). It is concluded that the combination of above mentioned 3 drugs may be effective and safe treatment for the patients with MDS-RA. Its relevant mechanisms can be involved in the combination, that elicits a wide range of pharmacological effects, such as differentiation, anti-tumor-promotion, anti-apoptosis, anti-angiogenesis, anti-cachexia and immunoregulation.

Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937 / 中国实验血液学杂志

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.