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.

Effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells / 西安交通大学学报(医学版)

Objective To explore the effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells.Methods MLAA-22 gene was down-regulated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)system in U937 cells.The activity of single guide RNA (sgRNA)was detected by CruiserTMenzyme digestion assay.The mutation rate of MLAA-22 gene was analyzed by TA cloning and sequencing assay of PCR products of the gene mutation region.Cell proliferation was evaluated by CCK-8 assay.Expression of CD11b was tested by flow cytometry to evaluate cell differentiation. Results CruiserTMenzyme digestion assay showed the sgRNA of the CRISPR/Cas9 system identified the target spot efficiently.TA cloning and sequencing assay displayed the mutation rate of MLAA-22 gene was 61.3%.CCK-8 assay demonstrated that the proliferation was obviously inhibited in MLAA-22-knockdown U937 cells.In addition, flow cytometry assay indicated CD11b-positive cells significantly increased in MLAA-22-knockdown U937 cells. Conclusion MLAA-22 gene regulates the proliferation of U937 cells probably by regulating their differentiation, thus promoting the occurrence and development of 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>

Antiapoptotic Effect of the Leukemia Associated Gene MLAA-34 in HeLa Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the antiapoptotic effect of leukemia-associated gene MLAA-34 in HeLa cells.</p><p><b>METHODS</b>The MLAA-34 recombinant lentiviral expression vector was constructed, and the stably transfected HeLa cell line with high expression of MLAA-34 was set up; As(2)O(3) was used to induce apoptosis; the MTT assay, colony formation test and flow cytometry were used to detect the ability of cell proliferation, colong formation, apoptosis and cell cycle changes respectively.</p><p><b>RESULTS</b>After treatment with As(2)O(3), the survival rate of HeLa cells with MLAA-34 overexpression was significantly higher than that of the control cells, and the colony formation ability of MLAA-34 significantly increased, and the high expression of MLAA-34 gene significantly decreased the apoptosis rate of HeLa cells, and decreased the proportion of G(2)/M phase cells.</p><p><b>CONCLUSION</b>The leukemia-associated gene MLAA-34 has been comfirmed to show antiapoptotic effect in HeLa cells which are induced by As(2)O(3).</p>

Comparison of the Effectiveness and Safety of Combined Chemotherapy with PEG-Asp for Treatment of ALL and T-NHL Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effectiveness and safety of combined chemotherapy with pegasparaginase (PEG-Asp) for treatment of patients with acute lymphoblastic leukemia (ALL) and T cell non-Hodgkin's lymphoma (T-NHL) patients.</p><p><b>METHODS</b>A total of 62 ALL or T-NHL patients were diagnosed and treated in our department and were enrolled in this study. Among them, 22 patients received the combined chemotherapy with PEG-Asp, while the other 40 patients received the standard chemotherapy with L-asparaginase (L-Asp) as the control. Therapeutic effectiveness, adverse effects, duration and expense of hospitalization, treatment-related mortality and survival were evaluated and compared in 2 different groups.</p><p><b>RESULTS</b>In group of combined chemotherapy with PEG-Asp, the overall response rate was 90.91% (20 cases), among them CR rate and PR rate are 77.27% (17 cases) and 13.64% (3 cases), respectively. In the group of standard chemotherapy with L-Asp, the overall response rate was 87.5% (35 cases), among them CR rate and PR rate were 72.5% (29 cases) and 15% (6 cases), respectively. The difference neither between PEG-Asp and L-Asp chemotherapy groups nor between ALL and T-NHL subgroups was significant (P > 0.05). The 6-month and 12-month overall survival rates were not significantly different between the PEG-Asp and L-Asp chemotherapy groups, respectively (P > 0.05). The adverse effects were identified as degree 1-2 according to the WHO criteria of drug toxicity. Neither the adverse effects identified as degree 3-4 nor the treatment-related death were observed. Expect for allergy and hyperglycaemia, the difference of side-effect incidence between the two groups were not significant (P > 0.05). The treatment for all the patients in PEG-Asp chemotherapy group was completed, while the treatment with L-Asp was completed only in 29 cases. Moreover, both average duration and expense of hospitalization after the combined chemotherapy were less than the control.</p><p><b>CONCLUSION</b>With higher response rate, lower drug toxicity and allergy incidence, the combined chemotherapy with PEG-Asp can replace the standard chemotherapy with L-Asp in the treatment of ALL and T-NHL. The optimization of the combined chemotheropeutic protocols for more cases and long-term survival rates need to further and deeply explorate.</p>

Screening of Serum Markers in Primary Immune Thrombocytopenia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the serum protein fingerprints of immune thrombocytopenia (ITP) patients and healthy controls by using weak cation exchange nanometer magnetic beads and MALDI-TOF-MAS technology, to identify the proteins with different expression, to establish the diagnostic model for ITP and to explore the pathogenesis of ITP.</p><p><b>METHODS</b>A total of 40 patients with ITP and 40 healthy controls were selected, the serum protein components were captured by using weak cation exchange nanaometer magnetic beads, the protein spectra of all specimens were detected by Autoflex II matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF-MS) and then the data were analyzed by CliprotoolsTM2.2 software, by which the distinct protein molecules were screened to set up ITP diagnostic model. To identify the established model, the sera of 20 ITP patients and 20 healthy controls were selected to make category and cross validations.</p><p><b>RESULTS</b>The detection of Clinprot system and the analysis of CliprotoolsTM2.2 software showed that about 55 protein peaks were detected with the range of 700 Da to 10 000 Da of molecular weight in the protein spectrum of serum speciments from 40 ITP patients and 40 healthy controls. Compared with healthy controls, 19 protein expression peaks with statistically significant difference were found in ITP patients (P < 0.05), among them 5 expressions were up-regulated and 14 expressions were down-regulated. The diagnostic model on basis of Supervised Neural Network Algorithm (SNN) was established through 10 MS peaks with strongest capability in ITP group and control group automatically distinguished by software, and it is expected that the sensitivity of model group reached to 100%, and the specificity to 100%. The category validation showed that this diagnostic model correctly identificed all 20 ITP patients and 20 healthy controls, and in cross validation, the model sensitivity was 100% and the specificity was 100%.</p><p><b>CONCLUSION</b>The ITP SNN model ertablished by using ChinProt System with high flax and good repetition is composed of 10 protein peaks with significant difference, this model can effectively distinguish ITP patients and healthy controls.</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>

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>

Short and long term efficacy of immunosuppressive therapy and survival in adult severe aplastic anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the short and long term therapeutic efficacy of the immunosuppressive therapy(IST) for adult severe aplastic anemia(SAA), and to analysis the relationship between the clinical factors(age, typing, lymphocyte percentage, reticulocyte percentage, neutrophil count) and the response to IST.</p><p><b>METHODS</b>The response rate of 39 patients received the IST between September 2009 and September 2013 in our hospital was assayed, the effective time in which all patients had hematologic response, and the survival rate at the first year were analyzed. The survival rates, the average amounts of the RBC and Plt transfusion per month in the first year were compared by using χ2 test between the IST group and the non-IST group; the multinomial logistic regression was used to analyze the relationship between the clinical factors and the response to IST.</p><p><b>RESULTS</b>The response rates of the 39 SAA patients at the first month, the third month, the sixth month and the first year were 29.73%, 70.27%, 75.68%, 86.49%, respectively. The median effective time of hematologic response in all patients had was 61.5 d(10 d-344 d). The survival rate of the IST group was 92.31%, which was much higher than that of the non-IST group (P<0.05). The average amounts of the RBC and Plt transfusion per month at the first year in the IST group were 1.04(0.13-2.78)×400 ml and 1.38(0.17-5.10)×200 ml, respectively, which were much lower than those in the non-IST group (P<0.01). Among the five clinical factors, the age, lymphocyte percentage and neutrophil count related to the response of IST (P<0.05).</p><p><b>CONCLUSION</b>The response rate of the 39 SAA patients received IST is 86.49% at the first year, and their long term survival is better than that of non-IST group. The age, lymphocyte percentage and neutrophil count relate to the response of IST.</p>

Preparation of osteogenic growth peptide nasal spray / 中国药学杂志

OBJECTIVE: To prepare osteogenic growth peptide(OGP) nasal spray and investigate the absorption characteristics. METHODS: The absorption enhancer was selected by orthogonal experiment, using the declining quantity of osteogenic growth peptide in rat nose as evalution index with rat nasal perfusion model, and further the dose of protease inhibitor was selected. Finally, the acceletated and long term test were made to investigate the stability of osteogenic growth peptide nasal spray according to Chinese Pharmacopoeia 2010. The rat nasal mucosal shape were inspected by electron microscope after perfusion of osteogenic growth peptide nasal spray. RESULTS: The optimal prescription of osteogenic growth peptide nasal spray is composed of osteogenic growth peptide 2 mg, citric acid 50 mg, HP-β-CD 100 mg, Tween-80 300 mg, and bacitracin zinc 50 mg in 10 mL patossium hydrogen phthalate buffer (pH 3.0). Osteogenic growth peptide nasal spray is much lower irritation to the nasal mucosa cilium. CONCLUSION: The stablity of osteogenic growth peptide nasal spray was good, safe and had very good absorption in rat nose.

Effect of artemisinin combined with glucocorticoid on the expressions of glucocorticoid receptor α mRNA, glucocorticoid receptor β mRNA and P300/CBP protein in lupus nephritis mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic effects and mechanisms of using artemisinin (Art) combined with glucocorticoid (GC) to treat lupus nephritis (LN) mice.</p><p><b>METHODS</b>Forty hybrid female mice were randomly and equally divided into four groups with the method of random number table: control group, model group, prednisone group administrated with 6.45 mg/(kg·d) prednisone suspension, and Art+prednisone group administrated with 150 mg/(kg·d) Art suspension and 3.225 mg/(kg·d) prednisone suspension. A mice model of LN was established by injection with living lymph cell suspension. The changes of urine protein/24h, the expressions of GC receptor α (GRα) mRNA, GC receptor β (GRβ) mRNA in peripheral blood mononuclear cells (PBMCs), and transcriptional coactivator P300/CBP protein in renal tissue were measured.</p><p><b>RESULTS</b>Compared with the model group, the treatment groups had significant decrease in urine protein/24 h, and renal pathological lesion (P<0.01). In the same groups, the expression of transcriptional coactivator P300/CBP protein in renal tissue and GRα mRNA were significantly increased, and GRβ mRNA expression was significantly decreased (P<0.01). And the Art+prednisone group has a better therapeutic effect than the prednisone group (P<0.01).</p><p><b>CONCLUSIONS</b>Art has therapeutic sensitization effects on GC in the LN mice. The underlying mechanism could be correlated with the effect of Art on the increase of the expressions of GRα mRNA and transcriptional coactivator P300 300/CBP protein in renal tissue and on the decrease of the expression of GRβ mRNA in PBMC.</p>

Efficiency of GHA priming therapy on patients with acute monocytic leukemia and its mechanism / 中国实验血液学杂志

The aim of this study was to explore the clinical efficiency and side effects of GHA-priming therapy on patients with acute monocytic leukemia, and to analyze its mechanism. 37 patients with refractory, relapse, hypocellular acute monocytic leukemia and elderly patients with AML-M(5) were treated with GHA-priming therapy (G-CSF, homoharringtonine and low dosage of cytarabine). Clinical efficiency, side effects, and therapy-relevant mortality were observed. By using U937 cell line as in vitro model, effect of G-CSF on cell cycle was determined by propidium iodide staining method. The inhibition rate, apoptosis rate of U937 cell line treated with various combination of G-CSF, homoharringtonine and cytarabine were detected by flow cytometry. The expression of MLAA34 on U937 before or after treating with chemotherapy was analyzed by immunohistochemical method. The results showed that in all the 37 patients, the total remission rate was 62.2% [complete remission rate was 45.95% (17/37) and partial remission rate was 16.2% (6/37)]. The incidence of granulocyte deficiency was 18.92% (2/37) with median time of 4 days. The severe infection occurred in 2 cases. No severe bleeding, no mild digestive effect occurred. Other non-hematological toxicities were low in vitro when incubated with G-CSF for 24 hours, the S-phase cells obviously increased. The inhibition rate, apoptosis rate and expression of MLAA34 of U937 cells treated by GHA significantly decreased as compared with cells treated with HA. It is concluded that the GHA priming therapy can be used to treat patients with refractory, relapse, senile and hypocellular acute monocytic leukemia with satisfied response rate and low hematological and non-hematological toxicities. G-CSF can enhance cytotoxicity of drugs such as Ara-C and HHT by promoting G(0) phase cells into the reproductive cycle. GHA and HA therapy can inhibit cell proliferation, induce apoptosis, and the former has a more significant function. GHA priming therapy can down regulate the expression of MLAA 34. MLAA-34 is a novel anti-apoptotic factor of acute monocytic leukemia.

Research advance on hemophagocytic lymphohistiocytosis / 中国实验血液学杂志

Hemophagocytic lymphohistiocytosis (HLH) is named as hemophagocytic syndrome (HPS) and is a complicated disease with reactive hyperplasia of mononuclear/macrophagocytic system. This disease characterised by release of massive cytokines and severe functional destruction of visceral organs, which results from immune function disturbance causing by various pathogenic factors. The cardinal clinical symptoms of HLH are prolonged fever, hepatosplenomegaly, cytopenia, elevated ferritin and triglycerides, low fibrinogen, symptom in nerve system and so on. Nevertheless, impaired function of natural killer cells and cytotoxic T-cell is characteristic for HLH. HLH has of two different types that may be difficult to distinguish from one another: a primary and a secondary form. The combined immunochemotherapy of dexamethasone, etoposide and cyclosporin A and hematopoietic stem cell transplantation are considered as the effective therapies for HLH. In this article, the recent advance in research on the etiological factors, pathogenesis, clinical manifestations, laboratory examination, diagnosis as well as recommended therapy of HLH were reviewed.

Preliminarily screening of serum characteristic markers in acute myeloid leukemia and clinical significance / 中国实验血液学杂志

This study was purposed to preliminarily screen characteristic tumor markers of acute myeloid leukemia (AML) and to investigate the serum proteomics characteristics of patients with AML and their significance in pathogenesis. 14 patients with AML and 28 healthy controls were enrolled in this study. The serum protein components were captured by weak cation exchange nanometer magnetic beads, the protein mass-spectra of all samples were detected by Autoflex II matrix-assisted laser desorption/ionization time of flight mass spectrometer, and the detection data were analyzed by means of CliprotoolsTM2.2 software, then the differential protein molecules were screened and the diagnostic model was established. Sera of 7 AML patients and 14 healthy controls were selected to verify the established model by using blind test. The results indicated that about 69 protein peaks could be detected within the range of 0.7-10 kD in protein spectra of serum samples from AML patients and controls. Compared with healthy controls, there were 44 statistically differential expression peaks in AML group (p<0.0001). Among them, 10 protein peaks were upregulated protein peaks and 34 protein peaks were downregulated. Diagnostic model was established on the basis of Quick Classifier Algorithm (QC), and the three mass peaks had the strongest power for software to automatically distinguish AML group from control group. Mass charge ratios (m/z) were 3216.57, 4089.7, and 7762.87 respectively. Sensitivity was expected as 86.4% while 82.8% in this established model group. Category validation showed that this diagnostic model correctly identified all 6 cases out of AML and 12 cases out of 14 healthy controls. In cross validation, the model sensitivity and specificity both were 85.7%. It is concluded that the AML QC model is composed of three protein peaks, which can effectively distinguish AML patients from healthy controls. Owing to higher sensitivity and specificity, they may act as serum tumor markers of AML. Among the three proteins, the one with m/z 7762.87 is the platelet-derived protein chemokine (PF4) protein. This finding will probably provide significant experimental evidence for understanding pathogenesis, molecular type, prognosis and treatment effect of AML.

Expression of genes psma6 and slc25a4 in patients with acute monocytic leukemia / 中国实验血液学杂志

The aim of this study was to investigate the expression levels of genes psma6 and slc25a4 in bone marrow of patients with acute monocytic leukemia and their correlation with clinical features and prognosis. The expression levels of genes psma6 and slc25a4 in AML-M5 leukemia cells, normal blood cells and non-leukemia cells were detected by real-time quantitative RT-PCR and compared each other. The expression levels of psma6-encoding protein P27K was assayed by using immunohistochemistry method. The results showed that the expression levels of psma6 mRNA in AML-M5 leukemia cells was lower than that in non AML-M5 leukemia cells, non-leukemia cells and normal blood cells. The results obtained by immunohistochemistry assay were consistent with above-mentioned results. The expression level of psma6 in AML-M5 patients with complete remission was higher than that in AML-M5 patients without remission. The expression level of P27K protein in AML-M5 and AL correlated to leukocyte count in peripheral blood and LDH content. The overexpression of slc25a4 mRNA was found in AML-M5, but there was no significant difference in slc25a4 mRNA expression between the patients with complete remission and those without remission. It is concluded that the expression level of psma6 is probably a new prognostic indicator of acute monocytic leukemia, slc25a4 may be a novel gene of antigen associated with acute monocytic leukemia.

Antileukemic effects in vitro of new co-stimulatory molecule CD137 / 中国实验血液学杂志

This study was aimed to investigate the expression characteristics of new co-stimulatory molecule CD137 (4-1BB) on human T lymphocytes and antileukemic effects of monoclonal antibody hCD137mAb in stimulating the T lymphocyte proliferation, promoting the cytokine secretion, enhancing the cell killing effect and so on. The expression of CD137 on normal T lymphocytes treated with phytohemagglutinin (PHA) was detected by FACS and indirect immunofluorescence. In HL-60 and T lymphocyte system in vitro, the effect of hCD137mAb and PHA on T lymphocyte proliferation was tested by MTT colorimetric assay. The IFN-gamma and IL-4 expression levels on the surface of T cells were detected by FACS and indirect immunofluorescence. In vitro mixed lymphocyte tumor cell culture (MLTC) system, the function of hCD137mAb enhancing toxicity killing leukemic cells at different effect-target ratio were studied. The results showed that almost no expression of hCD137 was found in T cells without PHA stimulation, but after activation of T cells by PHA, the expression gradually increased with a peak at 7th day (FACS 56.4%+/-1.98%, indirect immunofluorescence 52.8%+/-2.01%). CD137mAb alone could not stimulate T cell proliferation (proliferation index 1.002+/-0.011), but could enhance PHA stimulating activity (proliferative index of 2.161+/-0.102) about 2-folds (proliferation index 4.705+/-0.133). Moreover, hCD137mAb increased expression of IFN-gamma high by about 3-fold in presence of PHA, but did not effect on IL-4. The hCD137mAb markedly enhanced T cell killing activity on HL-60 cell line and its co-stimulatory effect was best at the effect-target ratio of 40:1 with increasing of killing percentage by about 2-fold. It is concluded that the new co-stimulatory molecule CD137 has significant antileukemic effect, use of hCD137mAb is an effective, safe and simple immunization strategy for leukemia therapy, this study provides some experimental basis for clinical immunotherapy with CD137 mAb.

Expression of co-stimulatory molecules in peripheral blood of patients with idiopathic thrombocytopenic purpura in relation with platelet antibodies / 中国实验血液学杂志

This study was aimed to detect the expression of co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood of patients with idiopathic thrombocytopenic purpura (ITP), the content of platelet antibodies in serum (PAIgG), and to analyze the relationship between them and their correlation with the number of platelet in peripheral blood, so as to clarify the roles of co-stimulatory molecules in pathogenesis of idiopathic thrombocytopenic purpura and evaluation of disease status. The co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood mononuclear cells (PBMNCs) of 48 ITP patients and 40 normal persons were detected by immunofluorescence and flow cytometry (FACS), PAIgG content in serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that CD80, CD86 and CD137 expression levels in ITP patients were (4.92 +/- 2.02)%, (8.68 +/- 4.25)%, (5.32 +/- 2.67)% respectively, PAIgG content was 210 +/- 3.02 ng/10(7) PA, all these of which were significantly higher than these in normal control group (2.01 +/- 0.75)%, (4.56 +/- 2.06)%, (1.37 +/- 1.25)%, PAIgG 20 +/- 1.13 ng/10(7) PA (p < 0.01). Correlation of co-stimulatory molecule expression with PAIgG content were positive (r = 0.302, p < 0.05), but correlation of co-stimulatory molecule expression with platelet number was negative (r = -0.369, p < 0.05). It is concluded that the co-stimulatory molecules CD80, CD86 and CD137 are involved in immune response and the incidence of ITP. Their over-expression closely associates with the pathogenesis of ITP and clinical status, so that correcting the abnormal expression and regulating the immune status may be one therapeutic strategy and have important clinical significance.