Critical Role of Tripartite Fusion and LBD Truncation in Certain RARA- and all RARG-Related Atypical APL.

Atypical acute promyelocytic leukemia (aAPL) presents a complex landscape of retinoic acid receptor (RAR) fusion genes beyond the well-known PML::RARA fusion. Among these, 31 individually rare RARA and RARG fusion genes have been documented, often reported in the canonical X::RAR bipartite fusion form. Intriguingly, some artificially mimicked bipartite X::RAR fusions respond well to all-trans retinoic acid (ATRA) in vitro, contrasting with the ATRA resistance observed in patients. To unravel the underlying mechanisms, we conducted a comprehensive molecular investigation into the fusion transcripts in 27 RARA fusion gene-positive aAPL (RARA-aAPL) and 21 RARG-aAPL cases. Our analysis revealed an unexpected novel form of X::RAR::X or X::RAR::Y-type tripartite fusions in certain RARA- and all RARG-aAPL cases, with shared features and notable differences between these two disease subgroups. In RARA-aAPL cases, the occurrence of RARA 3' splices was associated with their 5' fusion partner genes, mapping across the coding region of helix 11_12 (H11_12) within the ligand-binding domain (LBD), resulting in LBD-H12 or H11_12 truncation. In RARG-aAPL cases, RARG 3' splices were consistently localized to the terminus of exon 9, leading to LBD-H11_12 truncation. Significant differences were also observed between RARA and RARG 5' splice patterns. Our analysis also revealed extensive involvement of transposable elements in constructing RARA and RARG 3' fusions, suggesting transposition mechanisms for fusion gene ontogeny. Both protein structural analysis and experimental results highlighted the pivotal role of LBD-H11_12/H12 truncation in driving ATRA unresponsiveness and leukemogenesis in tripartite fusion-positive aAPL, through a protein allosteric dysfunction mechanism.

Bone marrow-derived innate macrophages attenuate oxazolone-induced colitis.

Previous studies have shown that a subpopulation of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent F4/80high CD11bhigh innate macrophages could be derived from bone marrow cells by continuous in vitro culturing. These cells could be induced to differentiate into M1 or M2 macrophages in vitro. In the current study, we sought to determine whether bone marrow cell-derived innate macrophages (BMIMs) could be used to fulfill an anti-inflammatory purpose by intravenous transplantation in vivo after being stimulated to differentiate into M2 macrophages. Because Th2 cytokines, such as interleukin IL-4 and IL-13, can induce macrophage polarization into M2 macrophages, we treated the BMIMs with IL-4 and IL-13 in vitro. Next, the M2 macrophages were intravenously transplanted into a typical Th2-mediated inflammatory disease model, oxazolone (OXZ)-induced colitis, to assess the anti-inflammatory activity of BMIM-derived M2 macrophages (BMIM-M2Ms) in vivo. After transplantation, the severity of intestinal inflammation was attenuated. In addition, colon lengths and mouse body weights were noticeably improved. F4/80+ CD206+ double-positive cells (displaying the markers of M2 macrophages) had accumulated in the colon tissue of BMIM-M2M-transplanted mice. This evidence demonstrated that bone marrow-derived BMIM-M2Ms could be used to alleviate OXZ-induced Th2-mediated inflammation in a mouse model in vivo.

Analysis of clinical and laboratory characteristics in 42 patients with thrombotic thrombocytopenic purpura from a single center in China.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by microvascular platelet deposition and thrombus formation with resulting microangiopathic hemolytic anemia. Deficiency of the von Willebrand factor cleavage protease, also known as ADAMTS 13, has been implicated as an important etiological factor in TTP. Little studies were obtained on Chinese patients with TTP until now. Our aim was to analyze the clinical features, outcome and laboratory characteristics of Chinese TTP patients, and determine whether plasma ADAMTS 13 activity is decreased in TTP and its diagnostic value for TTP. Forty-two TTP patients (29 females; 13 males) admitted to our hospital from 1998 to 2010 were analyzed. There were 34 patients (81%) with the triad of TTP, including hemolytic anemia, thrombocytopenia and neurologic abnormalities; 7 (16.7%) had the classical pentad of TTP. Major etiologic factors were acquired autoimmunological abnormalities (31%); no familial TTP was identified in this series. The schistocytes of peripheral blood smears were present in all cases with a mean frequency of 4.6% (range from 0.3% to 13.4%). Plasma ADAMTS 13 activity was determined in 22 patients with the FRET-vWF86 assay. Only 4 idiopathic TTP patients (18.2%) had severe ADAMTS 13 deficiency (activity<10%); 9 (40.9%) had moderate decrease of ADAMTS 13 activity (activity: 10-40%); another 9 (40.91%) had normal ADAMTS 13 activity (>40%). T lymphocyte subpopulation was measured in 23 TTP patients with FACS Calibur; 14 of the 23 (60.9%) had significantly decreased CD4 cells count and CD4/CD8 ratio, suggesting cellular immune dysfunction may be involved in the pathogenesis of TTP. In the studies, plasmapheresis is the main therapeutic method. 26 of 31 patients (83.9%) accepting plasmapheresis achieved complete remission; those patients who only underwent plasma infusion had low remission rate (18.2%) and high mortality (9/11; 81.8%). Four patients with packed RBC infusion manifested transient exacerbation of neurologic or psychiatric symptoms. In conclusion, the diagnosis of TTP in China is still based on clinical features including evidence of microangiopathic hemolysis. Severe ADAMTS 13 activity deficiency might be a valuable indicator for idiopathic TTP diagnosis. Further studies are needed to determine the real value of ADAMTS 13 activity for TTP diagnosis and whether T lymphocytes subset dysregulation plays important role in TTP pathogenesis.

[Effect of glucocorticoid on dendritic cells in children with chronic immune thrombocytopenia].

OBJECTIVE: To investigate the change in dendritic cells (DCs) in children with chronic immune thrombocytopenia (cITP) and the effect of glucocorticoid on DCs in children with cITP. METHODS: Fifteen children with cITP and 20 healthy controls were included in the study. Flow cytometry was used to measure the DC subsets count in the 15 children with cITP before and after glucocorticoid treatment as well as the corresponding values in the 20 healthy controls. The DCs derived from peripheral blood monocytes in children with cITP were cultured in vitro and collected, and their immunophenotypes were determined by flow cytometry. RESULTS: Before glucocorticoid treatment, the children with cITP showed no notable change in the absolute count of myeloid DCs (mDCs) but showed decreased absolute count of plasmacytoid DCs (pDCs) and increased mDC/pDC ratio compared with the healthy controls (P<0.05). After glucocorticoid treatment, the children with cITP demonstrated increased absolute count of pDCs and decreased absolute count of mDCs and mDC/pDC ratio compared with before treatment (P<0.05). Before glucocorticoid treatment, the children with cITP had significantly higher positive rates of HLA-DR, CD80, CD83 and CD86 on peripheral blood DCs than the healthy controls (P<0.01). All the positive rates were significantly decreased after glucocorticoid treatment (P<0.01), so that there was no significant difference from the healthy controls (P>0.05). CONCLUSIONS: Disproportion and functional disturbance of DC subsets is associated with the pathogenesis of cITP in children. Glucocorticoid can strengthen the immunosuppression of DCs in children with cITP, which may contribute to the effectiveness of glucocorticoid as a treatment.

Use of all-trans retinoic acid in combination with arsenic trioxide for remission induction in patients with newly diagnosed acute promyelocytic leukemia and for consolidation/maintenance in CR patients.

In the present study, 90 patients with newly diagnosed acute promyelocytic leukemia (APL) were studied for all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) combination treatment in remission induction and postremission therapy. In addition, 20 APL patients who had achieved complete remission (CR) with an ATRA-based regimen received ATRA/As(2)O(3) combination for consolidation and maintenance were also enrolled. The results showed that ATRA/As(2)O(3) combination therapy yielded a high CR rate of 93.3% and a significantly shorter time to enter CR (median: 31 days; range: 18-59 days) compared to the ATRA-based regimen (n = 72; median: 39 days; range: 25-62 days). With the ATRA/As(2)O(3) combination for CR maintaining, regardless of the way by which CR was attained, the relapse-free survival was significantly better than with an ATRA plus cytotoxic chemotherapy regimen (92.9 +/- 3.2% vs. 72.4 +/- 7.6%, for the 3-year Kaplan-Meier estimate of relapse-free survival). The drug toxicity profile showed that with the use of As(2)O(3), the incidence of hepatotoxicity was obviously high during remission induction but decreased significantly during postremission treatment. We conclude that APL patients may benefit from the early use of the combination of ATRA and As(2)O(3), in either remission induction or consolidation/maintenance.

[Relationship between TET2 Gene SNP rs3733609 C/T and JAK2V617F Allele Burden in Patients with Myeloproliferative Neoplasms].

OBJECTIVE: To investigate the relationship between the polymorphism of TET2 gene SNP rs3733609 and JAK2V617F allele burden in patients with myeloproliferative neoplasms (MPN). METHODS: The exon 9 of TET2 gene was amplified by RT-PCR, and the nucleotide sequence of SNP rs3733609 site was analyzed by gene sequencing. The MGB Taqman probe PCR method was used to detect the JAK2V617F allele burden. The correlation of TET2 gene SNP rs3733609 C/T with the JAK2V617F allele burden and clinical parameters was analyzed. RESULTS: TET2 gene rs3733609 C/T heterozygosity (normal T/T) could be detected in 19 cases of 85 cases of JAK2V617F positive MPN (22.4%) patients, while the TET2 gene rs3733609 C/T heterozygosity could be detected only in 9 of the 106 healthy volunteers, and the incidence was only 8.5% (9/106). Compared with the negative group (TET2 rs3733609 T/T), there was no significant difference in the median age, hemoglobin level and platelet count in the patients with TET2 gene SNP rs3733609 (CT/TC) positive, but the WBC count of peripheral blood and JAK2V617F allele burden significantly increased. In JAK2V617F high allele burden group, TET2 gene SNP rs3733609 was positive in 7 cases (36.8%, 7/19), the ratio was higher than that in the low allele burden group(18.2%, 12/66). CONCLUSION: TET2 SNP rs3733609 C/T may be a new susceptible allelee, which affects the clinical characteristics and clonal evolution of MPN patients.

Arsenic trioxide, thalidomide and retinoid acid combination therapy in higher risk myelodysplastic syndrome patients.

OBJECTIVE: To evaluate the clinical efficacy and safety of arsenic trioxide, retinoic acid and thalidomide combination therapy in higher risk MDS. METHODS: Twenty-one patients diagnosed with higher risk MDS were administered 10mg/day arsenic trioxide intravenously for 10 days, 40mg/day retinoic acid orally for 2 weeks and 100mg/day thalidomide orally for 4 weeks per cycle. RESULTS: After at least two treatment cycles, 10 patients showed hematologic responses. One achieved CR, one achieved PR, three patients achieved major hematological improvements. The efficacy rate was 24% (5/21), and the response rate was 48% (10/21). The schedule was tolerated well by all patients and toxicities were moderate and reversible. CONCLUSION: The combination of arsenic trioxide, retinoic acid and thalidomide could have therapeutic benefit in higher risk MDS with safety.

Dup-697, a specific COX-2 inhibitor, suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.

This investigation was designed to assess the effect of DuP-697 on growth and apoptosis in a human chronic myeloid leukemia (CML) cell line (K562 cells) and primary CML cells from CML patient bone marrow. DuP-697 significantly suppressed K562 cells and primary CML cells growth and induced apoptosis in a concentration-dependent manner and the growth-inhibiting effect was independent on Philadelphia chromosome. The IC50 of DuP-697 at 36 h was 31.7 muM. It arrested G1-S phase transmit on cell cycle and its apoptosis activity was partially abrogated by pretreating K562 cells with Z-IETD-fmk, a specific inhibitor of caspase-8. This study suggested that Dup-697 suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.

Fms-like tyrosine kinase (FLT) 3 and FLT3 internal tandem duplication in different types of adult leukemia: analysis of 147 patients.

AIM: To assess the expression level of fms-like tyrosine kinase 3 (FLT3), the incidence of FLT3/internal tandem duplications (ITD) mutation, and prognostic value of FLT3 changes in different types of adult leukemia. METHODS: Bone marrow mononuclear cells were isolated from 147 adult patients with leukemia. Reverse transcriptase polymerase chain reaction (PCR) was used to screen FLT3/ITD mutation and quantitative PCR was performed to evaluate the expression of the FLT3 transcript. Flow cytometry was used for detection of FLT3 receptor protein expression on bone marrow mononuclear cells. Pearson correlation analysis was performed to estimate the significance of FLT3. RESULTS: FLT3 expression was higher in acute myeloid leukemia and B-acute lymphoid leukemia than in T-acute lymphoid leukemia (P=0.006, P=0.001) and chronic myelogenous leukemia (P<0.001). In chronic myelogenous leukemia, FLT3 expression in blast transformation phase was higher than in acceleration phase (P=0.023). Surface expression of FLT3 protein was correlated with high percentage of bone marrow blasts and with FLT3 mRNA expression (r=0.366, P<0.001) in acute leukemia. FLT3/ITDs in the juxtamembrane domain were found in 25% of patients with acute myeloid leukemia and 7% of patients with acute lymphoid leukemia. FLT3/ITD positive sequences had 36, 42, and 57 nucleotides. FLT3/ITD mutation was associated with a higher white blood cell count, higher marrow blast percentage, and elevated serum lactate dehydrogenase (P=0.045, P=0.014, P<0.001, respectively) and not associated with a higher FLT3 mRNA and FLT3 protein expression, and lower complete remission (P=0.091, P=0.060, P=0.270, respectively). CONCLUSION: FLT3 expression levels differed in different types of adult leukemia. Overexpression of FLT3 and presence of a positive FLT3/ITD mutation in acute leukemia were associated with unfavorable clinical characteristics and poor prognosis.

[Molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin].

OBJECTIVE: To determine the molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin. METHODS: Effects of ADR and puerarin on NF-kappaB activity of K562,K562/AO2 were tested by immunofluorescence. The expression of survivin of K562,K562/AO2 was examined by immunocytochemistry. The p-gp expression was detected by flow cytometry. RESULTS: The NF-kappaB activity of K562 was significantly higher than that of K562/AO2. The NF-kappaB activity of K562 treated by ADR was significantly higher than untreated. The NF-kappaB activity of K562 which was pretreated by puerarin and then treated by ADR was much lower than that treated by ADR alone. The NF-kappaB activity of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin.The p-gp and survivin expression of K562/AO2 was significantly higher than K562. The p-gp and survivin expression of K562 treated by ADR was higher than that untreated by ADR. But the p-gp and survivin expression of K562 which was pretreated by puerarin and then treated by ADR was much lower than that not pretreated by puerarin.The p-gp and survivin expression of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin. The expression was negatively correlated to the duration of intervention. The inhibition effect demonstrated time dependence. CONCLUSION: The activation of NF-kappaB can increase the expression of p-gp and survivin, which may be part of the molecular mechanism of multi-drug resistance of K562. Puerarin can prevent and stop the multi-drug resistance in K562 and reverse the multi-drug resistance of K562/AO2 to ADR by inhibiting the activity of NF-kappaB and the expression of p-gp and survivin.

Chinese guidelines for treatment of adult primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is a bleeding disorder commonly encountered in clinical practice. The International Working Group (IWG) on ITP has published several landmark papers on terminology, definitions, outcome criteria, bleeding assessment, diagnosis, and management of ITP. The Chinese consensus reports for diagnosis and management of adult ITP have been updated to the 4th edition. Based on current consensus positions and new emerging clinical evidence, the thrombosis and hemostasis group of the Chinese Society of Hematology issued Chinese guidelines for management of adult ITP, which aim to provide evidence-based recommendations for clinical decision making.

[Effect of Icaritin on Proliferation and Apoptosis of THP-1 Cell and Its Mechanism].

OBJECTIVE: To investigate the effect of icaritin on the proliferation and apoptosis of THP-1 cells and its mechanism. METHODS: After treated with various concentrations of icaritin, cell proliferation was detected by MTS method, and apoptosis was measured with flow cytometry and Hoechst 33258 staining. Expression of BCL-2, BAX and Caspase-3 protein in THP-1 cell was detected by Western blot. RESULTS: After treatment with various concentrations (4-32 µmol/L) of icaritin for 24, 48, 72 h, the inhibition rate of cell growth significantly increased (P<0.05) in time-dose dependent manner(r=0.946); and the apoptotic rate of cells significantly increased (P<0.05) in time-dose dependent manner(r= 0.924). The expression of BCL-2 protein at 48 h decreased significantly in icaritin-treated group, compared with that in control group (P<0.05), while the expression of BAX and Caspase3 protein at 48 h increased significantly in icaritin-treated group, compared with that in control group (P<0.05). CONCLUSION: Icaritin can inhibit proliferation and induce apoptosis of THP-1 in vitro, Icaritin may induce apoptosis in THP-1 cells through the mitochondrial pathway.

Rejuvenation of Cardiac Tissue Developed from Reprogrammed Aged Somatic Cells.

Induced pluripotent stem cells (iPSCs) derived via somatic cell reprogramming have been reported to reset aged somatic cells to a more youthful state, characterized by elongated telomeres, a rearranged mitochondrial network, reduced oxidative stress, and restored pluripotency. However, it is still unclear whether the reprogrammed aged somatic cells can function normally as embryonic stem cells (ESCs) during development and be rejuvenated. In the current study, we applied the aggregation technique to investigate whether iPSCs derived from aged somatic cells could develop normally and be rejuvenated. iPSCs derived from bone marrow myeloid cells of 2-month-old (2 M) and 18-month-old (18 M) C57BL/6-Tg (CAG-EGFP)1Osb/J mice were aggregated with embryos derived from wild-type ICR mice to produce chimeras (referred to as 2 M CA and 18 M CA, respectively). Our observations focused on comparing the ability of the iPSCs derived from 18 M and 2 M bone marrow cells to develop rejuvenated cardiac tissue (the heart is the most vital organ during aging). The results showed an absence of p16 and p53 upregulation, telomere length shortening, and mitochondrial gene expression and deletion in 18 M CA, whereas slight changes in mitochondrial ultrastructure, cytochrome C oxidase activity, ATP production, and reactive oxygen species production were observed in CA cardiac tissues. The data implied that all of the aging characteristics observed in the newborn cardiac tissue of 18 M CA were comparable with those of 2 M CA newborn cardiac tissue. This study provides the first direct evidence of the aging-related characteristics of cardiac tissue developed from aged iPSCs, and our observations demonstrate that partial rejuvenation can be achieved by reprogramming aged somatic cells to a pluripotent state.

Myelodysplastic syndrome with transformation to acute monocytic leukemia with FLT3-ITD mutation following orthotopic liver transplantation.

A rare case of a 46-year-old man who underwent myelodysplastic syndrome, acute monocytic leukemia with FLT3-ITD mutation and splenic disruption following orthotopic liver transplantation is reported. The study of this case may be helpful to understand both the pathogenesis of acute leukemia and new complication of liver transplantation.

[Cytotoxic activities of Celecoxib on leukemic cells and the synergistic effects of Celecoxib with Imatinib thereupon].

OBJECTIVE: To explore the effects of Celecoxib on cell growth, early apoptosis, PRb and P27(Kip) protein expression of human leukemic cells of the line K562 and to determine its synergistic effects in combination of Imatinib. METHODS: K562 cel1s were incubated with Celecoxib of the concentrations of 0, 10, 20, 40, 80, and 160 micromol/L for 36 hours, the cell vitality was determined by MTT assay, the early apoptosis was examined by flow cytometry (FC). The percentage of annexin V positive was detected by FC so as to evaluate the early apoptosis. The expression of PRb and P27(Kip) protein was detected by Western blotting. Another K562 cells were incubated with Celecoxib of the concentration of 40 micromol/L, Imatinib of the concentration of 0.2 micromol/L, or Celecoxib 40 micromol/L + Imatinib 0.2 micromol/L for 36 hours. Then the cell vitality and early apoptosis of the cells were detected so as to evaluate the synergistic effects of the combination of Celecoxib and Imatinib. RESULTS: Celecoxib inhibited the K562 cells vitality significantly in a dose-dependent manner. The apoptotic rate of K562 cells was elevated with the increasing Celecoxib concentration. Celecoxib down-regulated the expression of PRb protein and up-regulated the expression of P27(Kip) protein in the K562 cells. The combination of Celecoxib and Imatinib exhibited evidently synergistic effects in terms of anti-proliferation on K562 cells. CONCLUSION: Celecoxib inhibits the proliferation and induces apoptosis on leukemic cells in a dose-dependent fashion. The anti-proliferation effect of Celecoxib may be associated with the down-regulation of PRb expression and up-regulation of P27(Kip) expression. Combination of Celecoxib and Imatinib has obviously synergistic effects in terms of anti-proliferation on K562 cells.

[Specific cell immune response mediated by dendritic cells in multiple myeloma patients in vitro].

OBJECTIVE: To explore KM3 multiple myeloma (MM) cell line specific cytotoxic T lymphocyte (CTL) response in vitro mediated by autologous dendritic cells (DCs) aroused by KM3 cells' lysates and acid-eluted peptides. METHODS: Monocytes were isolated from the peripheral blood of healthy individuals and MM patients, and were cultured in serum medium with IL-4, GM-CSF, and TNF-alpha to generate DCs. These DCs were pulsed by KM3 cells' lysates or the acid-eluted peptides, then incubated with autologous T lymphocytes for 3 days to induce KM3 cell antigen specific CTL. MTT assay was performed to examine the specific KM3 cells' lysing ability of CTL. RESULTS: DCs were generated in peripheral blood monocytes cultured in the serum medium containing GM-CSF, IL-4, and TNF-alpha. Autologous T lymphocytes induced by IL-2 and DCs pulsed with KM3 cells' lysates or the acid-eluted peptides showed strong killing effect on KM3 cells. CONCLUSION: The DCs pulsed by KM3 cells' lysates or the acid-eluted peptides incubated with autologous T lymphocytes can induce KM3 cell antigen specific CTL.