Adverse effect of oxidized cholesterol exposure on colitis is mediated by modulation of gut microbiota.

Both cholesterol and oxidized cholesterol (OXC) are present in human diets. The incidence of inflammatory bowel diseases (IBDs) is increasing in the world. The present study was to investigate the mechanism by which OXC promotes colitis using C57BL/6 mice as a model. Results shown that more severe colitis was developed in OXC-treated mice with the administration of dextran sulfate sodium (DSS) in water. Direct effects of short-term OXC exposure on gut barrier or inflammation were not observed in healthy mice. However, OXC exposure could cause gut microbiota dysbiosis with a decrease in the relative abundance of short-train fatty acids (SCFAs)-producing bacteria (Lachnospiraceae_NK4A136_group and Blautia) and an increase in the abundance of some potential harmful bacteria (Bacteroides). OXC-induced symptoms of colitis were eliminated when mice were administered with antibiotic cocktails, indicating the promoting effect of OXC on DSS-induced colitis was mediated by its effect on gut microbiota. Moreover, bacteria-depleted mice colonized with gut microbiome from OXC-DSS-exposed mice exhibited a severe colitis, further proving the gut dysbiosis caused by OXC exposure was the culprit in exacerbating the colitis. It was concluded that dietary OXC exposure increased the susceptibility of colitis in mice by causing gut microbiota dysbiosis.

[Expression of IGLL1 Gene and Its Clinical Significance in Pediatric T-ALL].

OBJECTIVE: To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients. METHODS: A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed. RESULTS: The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients. CONCLUSION: In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.

[A study on allele frequencies and mismatching proportion of HLA-A, B, Cw, DRB1 and DQB1 on high-resolution donor-recipient typing in Chinese Han population].

OBJECTIVE: To analyze the allele frequencies and polymorphism of human leukocyte antigens (HLA) -A, B, Cw, DRB1 and DQB1 between donors-recipients on high-resolution typing; and to analyze the matching and mismatching proportion between donors and recipients. METHODS: HLA high-resolution types were determined by sequence based typing (SBT), sequence specific oligonucleotide probe (SSOP) and sequence specific primer (SSP) on 2540 unrelated Chinese Han individuals including 1168 recipients and 1372 donors, then statistical analyses were carried out. RESULTS: Forty-four HLA-A alleles were detected, and among them the frequencies of A*1101, A*2402, A*0201, A*0207, A*3303, A*0206 and A*3001 exceeded 0.05, and accounted for 80.4%. Eighty-one HLA-B alleles were detected, and the frequencies of B*4001, B*4601, B*5801, B*1302 and B*5101 exceeded 0.05, and accounted for 43.0% of total. There were 44 HLA-Cw alleles, among them the frequencies of Cw*0702, Cw*0102, Cw*0304, Cw*0801, Cw*0602, Cw*0303, Cw*0302 and Cw*0401 exceeded 0.05, and were 80.3% of total. There were 61 HLA-DRB1 alleles, the frequencies of DRB1*0901, DRB1*1501, DRB1*1202, DRB1*0803, DRB1*0701, DRB1*0405, DRB1*0301 and DRB1*1101 exceeded 0.05, and were 70.1% of total. Finally, 22 HLA-DQB1 alleles were detected, the frequencies of DQB1*0301, DQB1*0303, DQB1*0601, DQB1*0602, DQB1*0202, DQB1*0302, DQB1*0401, DQB1*0502 and DQB1*0201 exceeded 0.05, and they were 87.4% of total. All the five loci were of heterozygote deficiency. The HLA-A, B and DRB1 loci conformed to Hardy-Weinberg equilibrium (HWE) (P > 0.05); but HLA-Cw and HLA-DQB1 loci did not (P < 0.05). Except several particular genotypes, all the five loci conformed to HWE. After comparing data between donors and recipients, only 22.4% of recipients found HLA matched donors (10/10); 24.6% of recipients found single HLA allele mismatched donors (9/10); 26.3% of recipients had two HLA alleles mismatched donors (8/10). CONCLUSION: The characteristics of allele frequencies and polymorphism of HLA-A, B, Cw, DRB1 and DQB1 on high-resolution typing in Chinese Han population is valuable for donor searching in unrelated hematopoietic stem cell transplantation, and it provides genetic basis for donor registry and usage of donor resource for Chinese Marrow Donor Program.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

The significance of detecting WT1 expression in childhood acute leukemias.

WT1 (Wilms' tumor gene 1) overexpression is implicated in the prognosis of acute leukemia. The purpose of this study was to investigate WT1 expression and its clinical implication in childhood acute leukemia (AL) in Chinese population. Bone marrow specimen from 200 children at different stages of acute leukemia and from 21 children without leukemia were studied. The WT1 expression at diagnostic marrow specimen in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) was higher than control group, whereas WT1 expression in AML was higher than in ALL, and WT1 expression level in relapse in ALL increased more significantly than in AML. The WT1 expression level showed positive correlation with the hypodiploidy and BCR-ABL fusion gene in acute leukemia. A rapidly decrease of WT1 expression level predicted a good response to the induction therapy and low expression of WT1 correlates with remission status. This study suggested that WT1 expression levels in acute leukemia can potentially be a marker for evaluating therapeutic efficacy, correlating with monitoring minimal residue disease, and predicting hematological relapse in children acute leukemia.

[Analysis of Factors Influencing Overall Survival of MDS Patients Transplanted with HSCs].

OBJECTIVE: To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT). METHODS: 121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method. RESULTS: Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference. CONCLUSION: age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

[Detection and clinical significance of JAK2 mutation in 412 patients with chronic myeloproliferative neoplasms].

OBJECTIVE: To investigate the frequency of JAK2V617F mutation in Chinese patients with chronic myeloproliferative neoplasms (MPN) and to study the relationship between JAK2V617F mutation and clinical characteristics. METHODS: JAK2V617F mutation was screened by allele-specific polymerase chain reaction (AS-PCR). RESULTS: JAK2V617F mutation was detected in 277 of the 412 patients with MPN. The frequency of JAK2V617F mutation was similar among essential thrombocythemia (ET), idiopathic myelofibrosis (IMF) and chronic myeloproliferative disorders-unclassified (MPD-U) (P > 0.05), but it was significantly lower than that in polycythemia vera (PV) (P < 0.05). The presence of JAK2V617F was found to be significantly correlative with advanced age at diagnosis (P < 0.01) and with higher hemoglobin levels and higher leukocyte counts (P < 0.05). Significant difference was found in complication of vascular events between JAK2V617 positive and negative patients (P < 0.05). JAK2V617F positive MPD-U patients were more prone to progress into typical MPN compared with JAK2V617F negative MPD-U patients. The association between abnormal karyotype and JAK2V617F was not found in cytogenetical analysis of 301 patients. CONCLUSION: The presence of JAK2V617F in MPD-U is associated with the disease development. There is a correlation between JAK2V617F mutation in MPN and advanced age, higher leukocyte counts, hemoglobin level and vascular events.

[Clinical and laboratory features of patients with CD34(+) acute promyelocytic leukemia].

OBJECTIVE: To explore the expression of CD34 in patients with acute promyelocytic leukemia (APL) and investigate the clinical and laboratory features of CD34(+) APL patients. METHODS: 262 APL patients diagnosed by chromosome analysis and/or fusion gene examination in the last five years were retrospectively analyzed in this study. To survey the expression of CD34 in those patients, all the cases were divided into two groups (CD34(+) APL vs. CD34(-) APL). The clinical features including age, gender, abnormal values of the peripheral hemogram before treatment, the complete remission (CR) rate and the incidence of DIC and laboratory data such as the results of morphology, immunology, cytogenetics and molecular biology (MICM) between those two groups were compared. RESULTS: Of the 262 APL patients, 38 (14.5%) cases were positive for CD34 expression. There were no statistically significant differences between CD34(+) APL and CD34(-) APL groups in gender and age (P > 0.05). Before treatment, the median level of WBC in CD34(+) APL was 25.92 x 10(9)/L, which was significantly higher than that of CD34(-) APL (5.3 x 10(9)/L, P < 0.05). CD34(+) APL by morphology classification were mostly of the subtypes M3b and M3v (65.8%), while these subtypes in CD34(-) APL (40.3%) were significantly less (P < 0.01). There were no statistically significant differences between the two groups compared in respect of complete remission (CR) rate and the incidence of DIC (P > 0.05). The expression level of CD34 in APL had correlation to the expression level of CD2, CD7 and CD117; the latter three phenotypes in CD34(+) APL were significantly higher than those in CD34(-) APL (P < 0.01). No significant difference was found between those two groups by chromosome analysis, but there was more PML-RAR-alpha transcript short form in CD34(+) APL than that in CD34(-) APL (P < 0.05). CONCLUSION: CD34(+) acute promyelocytic leukemia is a unique subtype of APL with different biological characteristics.

[Epigenetic Regulation and Epigenomic Landscape in Normal Physiological Hematopoiesis--Editorial].

Abstract　　The physiological hematopoiesis depends on the programmed expression of a series of gene regulated by mechanisms at various levels. Currently, the epigenetic regulation has been considered as the most important mechanism during hematopoietic differentiation, resulting in a specific epigenomic landscape in the hematopoietic stem/progenitor cells. We try to concisely review the epigenetic mechanisms, including the genomic methylation, the histone modifications and the expression profiles of noncoding RNA, illustrating briefly the differentiation from the hematopoietic stem/progenitor cells to to the erythroid, myeloid and lymphoid cells.

Methylation status of fragile histidine triad (FHIT) gene and its clinical impact on prognosis of patients with myelodysplastic syndrome.

The fragile histidine triad (FHIT) gene plays an important role in anti-cancer and the abnormal methylation of FHIT gene is found in many carcinomas. The epigenetic changes of tumor suppressor genes (TSG) are now recognized as an abnormal mechanism contributing to the development of myelodysplastic syndrome (MDS). To clarify the role of FHIT in MDS, we examined the methylation status of FHIT in patients with MDS. Methylation-specific polymerase (MSP) chain reaction was performed to detect the aberrant promoter methylation of FHIT gene in 55 patients with MDS. The abnormal methylation of the FHIT gene was found in 26 of 55 (47.2%) MDS cases, but it was not in normal control. No relationship was found between FHIT gene methylation and sex, hematologic parameters, chromosomal abnormalities of MDS patients. However, the significant difference was observed in the frequencies of FHIT gene hypermethylation among patients with RA/RARS (7/25, 28.0%), RAEB (11/18, 61.1%) and RAEBt (8/11, 72.7%) (chi2 value=7.938, P=0.019). Furthermore, there was a positive correlation between the frequency of FHIT gene hypermethylation and different IPSS groups (chi2 value=10.110, P=0.018). The MDS patients with FHIT gene methylation had significantly shorter survival time than those without FHIT methylation (20.0 months vs. 40.0 months, P=0.025). These results suggested that aberrant methylation of the FHIT gene might be one of molecular events involved in the disease progression of MDS and be an adverse prognostic factor in MDS.

Prognostic analysis of refractory anaemia in adult myelodysplastic syndromes.

BACKGROUND: Patients with myelodysplastic syndrome (MDS) display a very diverse pattern. In this study, we investigated prognostic factors and survival rate in adult patients with MDS refractory anaemia (MDS-RA) diagnosed according to French-American-British classification and evaluated the International Prognostic Scoring System (IPSS) for Chinese patients. METHODS: A multi-center study on diagnosis of MDS-RA was conducted to characterize the clinical features of Chinese MDS patients. The morphological criteria for the diagnosis of MDS-RA were first standardized. Clinical data of 307 MDS-RA patients collected from Shanghai, Suzhou and Beijing from 1995 to 2006 were analyzed using Kaplan-Meier curve, log rank and Cox regression model. RESULTS: The median age of 307 MDS-RA cases was 52 years. The frequency of 2 or 3 lineage cytopenias was 85.6%. Abnormal karyotype occurred in 35.7% of 235 patients. There were 165 cases (70.2%) in the good IPSS cytogenetic subgroup, 44 cases (18.7%) intermediate and 26 cases (11.1%) poor. IPSS showed 20 (8.5%) categorized as low risk, 195 cases (83.0%) as intermediate-I risk and 20 cases (8.5%) as intermediate-II risk. The 1-, 2-, 3-, 4- and 5-year survival rates were 90.8%, 85.7%, 82.9%, 74.9% and 71.2% respectively. Fifteen cases (4.9%) transformed to acute myeloid leukaemia (median time 15.9 months, range 3 - 102 months). Lower white blood cell count (< 1.5 x 10(9)/L), platelet count (< 30 x 10(9)/L) and cytogenetic abnormalities were independent prognostic factors by multivariate analysis, but age (= 65 years), IPSS cytogenetic subgroup and IPSS risk subgroup were not independent prognostic factors associated with survival time. CONCLUSIONS: Chinese patients were younger, and had lower incidence of cytogenetic abnormalities, more severe cytopenias but a more favourable prognosis than Western patients. The major prognostic factors were lower white blood cell count, lower platelet count and fewer abnormal karyotypes. The international prognostic scoring system risk group was not an independent prognostic factor for Chinese myelodysplastic syndrome patients with refractory anaemia patients.

[Alteration of methylation status of fragile histidine triad gene promoter in patients with myelodysplastic syndrome].

OBJECTIVE: To study the methylation status of fragile histidine triad (FHIT) gene promoter in patients with myelodysplastic syndrome (MDS) and its clinical relevance. METHODS: Methylation-specific PCR (MSP) was used to detect FHIT promoter methylation in bone marrow samples from 54 MDS cases. RESULTS: Hypermethylation of FHIT promoter was detected in 26 cases (48.1%). Association was not found between FHIT gene hypermethylation and sex, hematologic parameters and chromosomal abnormalities of MDS patients, but found between FHIT gene hypermethylation and age of the MDS cases. Although significant difference was not observed in the frequencies of FHIT gene hypermethylation among patients with refractory anemia/refractory anemia with ringed sideroblasts (RA/RAS) (1/6, 16.7%), refractory anemia/refractory anemia with ringed sideroblasts (RCMD) and refractory cytopenia with multilineage dysplasia with ringed blasts (RCMD-RS) (6/19, 31.6%), refractory anemia with excess blasts-1 (RAEB-1) (7/11, 63.6%), refractory anemia with excess blasts-2 (RAEB-2) (4/7, 57.1%) and refractory anemia with excess blasts in transformation/acute myeloid leukemia (RAEBt/AML) (8/11, 72.7%)(chi-square=8.417, P=0.077), it was observed in patients in early stages (RA/RAS and RCMD) (7/25, 28.0%), advanced stages (RAEB-1 and RAEB-2)(11/18, 61.1%) and RAEBt/AML (8/11, 72.7%) (chi-square=7.938, P=0.019). Furthermore, there was a positive correlation between the frequency of FHIT gene hypermethylation and different IPSS groups (chi-square=10.110, P=0.018). CONCLUSION: FHIT gene hypermethylation might be one of the molecular events involved in the disease progression of MDS.

[The clinical and laboratory features of acute promyelocytic leukemia: an analysis of 513 cases].

OBJECTIVE: To investigate the clinical and laboratory features of acute promyelocytic leukemia (APL). METHODS: 513 APL patients in the last two decades were retrospectively analyzed in this research. We investigated the clinical features including age, sex, abnormality of peripheral hemogram before treatment, therapeutic effect and follow-up and laboratory data such as morphology, immunology, cytogenetics and molecular biology (MICM). RESULTS: The median age of the APL patients was 33 years old and the ratio of male and female was 1.21:1. Before treatment, the median level of WBC was 4.3 x 10(9)/L and the detection rate of abnormal promyelocyte on blood film was 85.8%; with immunophenotypic detection, the expression levels of CD117, CD34, HLA-DR, CD7, CD14 and CD19 in APL were found to be lower and the expression levels of CD2, CD33 and MPO higher than those in other subtypes of acute myelocytic leukemia (AML) (both P < 0.01). Specific abnormal chromosome t (15;17) was detected in 91.7% of the patients, of whom 75.9% had standard translocation of t (15;17), being the most common one and 15.8% of the patients had t (15;17) with additional abnormal chromosome. There was only 7.5% of the patients with normal karyotype. However, the presence of both simple translocation and complex translocation was seldom seen. With molecular biological detection, PML/RARalpha fusion gene positive rate was 99.6%. In a relatively long clinical follow-up, we found that the complete remission (CR) rate in APL patients was 84.7%, incidence of DIC was 13.4% and five-year survival rate was 30.7%. The median count of WBC in CR group was lower than that non-remission group (P < 0.01). There were no significant differences on expressions of CD34 and CD2 and changes of cytogenetics between the two groups (P > 0.05). CONCLUSIONS: Comprehensive evaluation of MICM could be of important significance in the diagnosis and prognosis judgment for APL patients. The CR rate in these patients with high WBC count was considerable low.

[Elimination of leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes specifically targeting WT1-derived peptide: an experimental study in vitro].

OBJECTIVE: To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide. METHODS: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls. RESULTS: The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B. CONCLUSIONS: The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.

Methylation level of Rap1GAP and the clinical significance in MDS.

Previous studies on the pathogenesis of myelodysplastic syndrome (MDS) have identified multiple associated gene mutations, including mutations of tetmethylcytosinedioxygenase 2, isocitrate dehydrogenase [NADP(+)] 1 cytosolic, isocitrate dehydrogenase [NADP(+)] 2 mitochondrial and additional sex combs like 1 transcriptional regulator, all of which may be considered epigenetic regulators. Furthermore, mutations of RAS type GTPase family genes have been identified in 10-15% patients with MDS. The authors' previous study on the gene expression profile of cluster of differentiation 34+ cells using microarray analysis identified elevated expression of RAP1GTPase activating protein 1 (Rap1GAP) in patients with MDS compared with that in non-malignant blood diseases (NM) control group. To further investigate the mechanism of increased Rap1GAP expression, the methylation pattern of the promoter of this gene was determined in 86 patients with MDS (n=29), acute myeloid leukemia (AML) (n=31) or NM (n=26) using bisulfite-specific polymerase chain reaction and DNA sequencing. The results demonstrated that the methylation of Rap1GAP occurred in all 29 patients with MDS at multiple CpG sites. The methylation level of Rap1GAP in patients with MDS was decreased compared with that in patients with NM. Significant differences at 4CpG sites (5,7,8 and 12) of Rap1GAP promoter were identified between MDS and NM. Furthermore, based on the present clinical records of the patient cohort, the methylation status of Rap1GAP promoter did not appear to be associated with the clinicopathological characteristics of patients with MDS, including age, gender and International Prognosis Score System. The difference in methylation level at CpG site 8 of Rap1GAP promoter was identified to be significantly increased in patients with MDS-refractory anemia with ring sideroblasts compared with that in the MDS-refractory cytopenia with multilineage dysplasia or MDS-unclassified groups. The results of the present study suggest that patients with MDS exhibit a lower overall methylation level within Rap1GAP promoter compared with patients with NM or AML. In addition, the methylation level at the four identified CpG sites can distinguish between MDS and NM.

[Bone marrow WT1 gene expression and clinical significance in chronic myelogenous leukemia].

OBJECTIVE: To investigate the clinical significance of Wilms tumor gene (WT1) expression levels in the bone marrow (BM) of chronic myelogenous leukemia patients. METHODS: Real-time quantitative retroversion polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in the BM of 109 CML and 23 non-leukemic patients with Light Cycler. Normalized WT1 expression level (WT1(N)) was determined as a ratio between WT1 and GAPDH expression times 10(4). RESULTS: The median expression levels of WT1(N) in 23 CML patients of accelerate phase (CML-AP) and 22 CML patients of blast crisis (CML-BC) were statistically higher than those of 64 CML patients of chronic phase (CML-CP) and 23 non-leukemic controls (103.71 and 129.44 vs 3.44 and 1.47). No statistic differences were found between the CML-CP group and control group, nor between the CML-AP group and CML-BC group. Furthermore, the WT1(N) levels were correlated to the BCR/ABL fusion gene expression levels. Dynamic change of WT1(N) levels in 7 CML patients before or after allogeneic bone marrow transplantation showed that WT1 expression levels could predict relapse of leukemia. CONCLUSION: WT1 expression levels in CML patients in accelerate phase or blast crisis were strikingly higher than those in non-leukemic patients or CML patients in chronic phase, in whom WT1 expression was undetectable or showed low expression. WT1 gene could be an useful marker for detecting MRD and evaluating therapeutic efficacy in CML.

[Effect of AML1-ETO Fusion Protein on Transcriptional Regulation of p14ARF].

OBJECTIVE: To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14ARF. METHODS: P14ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14ARF promoter in AML1-ETO positive leukemia cell line. And the p14ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza. RESULTS: AML1-ETO-expressing cell subclone displayed low level of p14ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14ARF promoter. 5-Aza could increase the expression of p14ARF. CONCLUSION: P14ARF is a possible target gene of AML1-ETO. The p14ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M2b subtype of acute myeloid leukemia.

Coexistence of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia: A report of 4 cases.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17-25% of patients with acute lymphocytic leukemia and 0.9-3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-ß-myosin heavy chain 11 (CBFß-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFß-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFß-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFß-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFß-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFß-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer.

BACKGROUND: Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein (IAP) family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA (siRNA) targeting survivin on the biological behaviour of bladder cancer cells. METHODS: A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations (50200 nmol/L), the transfectant cells were intratumourally injected at different doses (5 microg or 50 microg). The effects were measured in vitro and in vivo. RESULTS: The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% (P < 0.01) and the markedly increased apoptotic rate was 45.70% (P < 0.01). In vivo intratumoural injection of 50 microg siRNA-survivin could notably prevent the growth of bladder cancer (P < 0.01) in xenografted animals. CONCLUSION: The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.

[Overexpression of tissue inhibitor of metalloprotease-2 promotes proliferation and infiltration of human monocytic leukemia cells].

OBJECTIVE: To investigate the functional role of human tissue inhibitor of metalloprotease (TIMP)-2 gene on the proliferation and infiltrating capability of human monocytic leukemic cells. METHODS: Human monocytic leukemic cells of the line SHI-1 were cultured and the TIMP-2 expression on the cell membrane was detected by flow cytometry (FCM). The SHI-1 cells were transfected with human TIMP-2 gene (SHI-1/TIMP-2 cells) or blank vector (SHI-1/MSCV cells). The TIMP-2 expressed on the surface of the cell membranes of SHI-1/TIMP-2 and SHI-1/MSCV cells was detected by FCM. The SHI-1/TIMP-2 and SHI-1/MSCV cells were inoculated into the 96-well plate, 24, 48, 72, and 96 hours later MMT method and ELISA were used, and the cell growth curve was drawn to detect the proliferation ability of the cells. Matrigel was put into the upper layer of Transwell chamber. Human bone marrow matrix cells (BMMC) of leukemia patient and SHI-1/TIMP-2 cells or SHI-1/MSCV cells were put into the upper layers as experimental groups, and SHI-1/TIMP-2 cells or SHI-1/MSCV cells only, without human BMMC, were put into the upper layers as control groups. 72 hours later blood cell counting plate was used to measure the number of cells migrating through Matrigel. SHI-1/TIMP-2 cells or SHI-1/MSCV cells were injected intravenously into pre-treated BALB/c nu/nu mice. Thirty days later 8 mice from each group were killed to observe the tumorigenesis in the organs, especially the central nervous system leukemia (CNL). The survival of the other mice was observed. RESULTS: The expression level of TIMP-2 on the cell membrane of the SHI-1/TIMP-2 cells was 99.3% +/- 0.1%, significantly higher than that of the SHI-1/MSCV cells (85.9% +/- 2.6%, P < 0.05). The A values 24, 48, 72, and 96 hours later of the SHI-1/TIMP-2 cells were 0.34 +/- 0.05, 0.6 +/- 0.05, 0.97 +/- 0.12, and 1.28 +/- 0.06 respectively, all significantly higher than those of the SHI-1/MSCV cells (0.28 +/- 0.03, 0.36 +/- 0.03, 0.54 +/- 0.09, and 0.99 +/- 0.03 respectively, all P < 0.05). The SHI-1/TIMP-2 cells and SHI-1/MSCV cells only could not migrate through Matrigel basically. 24 - 48 hours after co-cultivation Shi-1 cells began to appear in the lower layer of Transwell chambers, 72 hours later the trans-Matrigel SHI-1/TIMP-2 cells accounted for 24.7% +/- 6.9% of the inoculated SHI-1/TIMP-2 cells, a proportion significantly higher than that in the case of the SHI-1/MSCV cells (12% +/- 1.4%, P < 0.05). 24 days after the inoculation the mean body weight of the mice inoculated with SHI-1/TIMP-2 cells was 21.5 g +/- 0.4 g, significantly higher than that of the mice inoculated with SHI-1/MSCV cells (17.4 g +/- 0.6 g, P < 0.01). The mice inoculated with SHI-1/TIMP-2 cells showed much more tumors in different organs and much more severe infiltration in the CNS in comparison with the mice inoculated with SHI-1/MSCV cells The mean survival time of the mice inoculated with the SHI-1/TIMP-2 cells was 33.7 days, significantly shorter than that of the mice inoculated with SHI-1/MSCV cells (40 days). CONCLUSION: TIMP-2 expressed on the cell membrane is critical to promote the proliferation and infiltration of SHI-1 cells.