Comparison of the anti-leukemia effect and mechanism of L-asparaginase between two different strains / 中国实验血液学杂志

This study was purposed to compare the anti-leukemic effects of E.coli-L-Asp and Erwinia-L-Asp in vitro, and to investigate their mechanism. The cell apoptosis and proliferation inhibition rate were measured by CCK-8 kit, and IC50 of two drugs was calculated by using SPSS software. Pro-apoptosis effect of E.coli-L-Asp and Erwinia-L-Asp on REH and Jurkat cell lines was also determined by flow cytometry with Annexin V/PI double staining. Concentration changes of 4 amino acids (Asn, Aspa, Gln, and Glu) before and after medication were detected by using high pressure liquid chromatography (HPLC) assay. The results showed that both REH and Jurkat cell lines were sensitive to L-Asp drugs from two different strains, and E.coli-L-Asp and Erwinia-L-Asp displayed the inhibition effect on the proliferation of Jurkat and REH cell lines in dose-dependent and time-dependent manners. The inhibition cell of proliferation and cell apoptosis in Erwinia-L-Asp group were higher than those in E.coli-L-Asp group after 24 hours (P < 0.05) . However, after treatment of REN and Jurkat cells with 2 kind of L-Asp for 48 hours, the inhibition of cell proliferation and apoptosis rates were not significantly different between the 2 L-Asp drugs (P > 0.05). The Asn in medium could be depleted by two different L-Asp drugs with low concentration. Both the two L-Asp drugs had the same capability to deplete the Asn surrounding leukemia cells (P > 0.05). The Gln in medium could be depleted by two L-Asp drugs with high concentration. The hydrolysis effect of Erwinia-L-Asp on Gln was stronger than that of E.coli-L-Asp (P < 0.05). It is concluded that in a certain range of concentrations, E.coli-L-Asp and Erwinia-L-Asp exert anti-leukemia effect in dose-dependent manner. Depletion of Gln and Asn in surrounding environment and induction of cell apoptosis are two potential mechanisms, by which leukemia cells can be killed. Erwinia-L-Asp may be chosen as the first-line drug to treat childhood ALL for its fast action and stronger hydrolysis effect on Gln.

Relation of Treg and iNKT cell reconstruction with aGVHD after allogeneic hematopoietic stem cell transplantation in children / 中国实验血液学杂志

This study was aimed to explore the relation of Treg and invariant natural killer T (iNKT) cell reconstruction with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. According to the occurrence or absence of aGVHD, 29 pediatric patients who underwent allo-HSCT were firstly divided into two groups non-aGVHD and aGVHD group,then those patients with aGVHD were divided into steroid effective group and steroid resistant group according to their reaction to the steroid treatment. Flow cytometry was used to detect the frequency of Treg cells and iNKT cells in the peripheral blood of the recipients at different time after allo-HSCT(d 15, d 30, d 60, d 90, the time of aGVHD onset and two weeks after steroid treatment). The result showed that the frequencies of Treg cells and the iNKT/T ratio on day 15 in non-aGVHD group were significantly higher than those in the aGVHD group (P < 0.05). It is concluded that a combined monitoring strategy of Treg and iNKT cell reconstruction early after allo-HSCT may facilitate the diagnosis and treatment of aGVHD in children.

Acute leukemia child with ocular Kaposi's sarcoma after hematopoietic stem cell transplantation: a case report and literatures review / 中华血液学杂志

<p><b>OBJECTIVE</b>To summarize clinical features of eye Kaposis' sarcoma ( KS ) in leukemia child after peripheral blood stem cell transplantation (PBSCT).</p><p><b>METHODS</b>One 13 years-old child with acute lymphoblastic leukemia (ALL) and negative HIV test who developed KS restricted in right conjunctiva, cornea and sclera after successful allogeneic PBSCT was reviewed retrospectively.</p><p><b>RESULTS</b>The child suffered from T cell type ALL. He received immunosuppressive treatment after PBSCT, and had once extensive herpes zoster restricted in skin. Seven months after PBSCT, he had blurred vision with right eye and slowly neoplasm formed in cornea and conjunctiva. Pathological examination confirmed KS with changes like capillary hemangioma, atypical fusiform cell, typical immunochemistry and positive immunofluorescent result of HHV8. He received excision of lump of cornea, conjunctiva, sclera and transplantation of cornea and sclera. Antiviral therapy was given together with anti-infection, prevention of cornea rejection and biotherapy. He kept right eye and hand-move eyesight, survived without GVHD or recurrence of ALL and KS.</p><p><b>CONCLUSION</b>This was the first ocular KS case in ALL child after PBSCT, without correlation with HIV infection. Complete excision combined with biotherapy was safe and effective for single ocular lesions.</p>

Analysis of one case of adolescent blastic plasmacytoid dendritic cell neoplasm / 中国实验血液学杂志

This study was purposed to summarize the clinical characteristics and laboratorial data of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in pediatric patients in order to enhance understanding this disease in diagnosis and therapy. A rare case of BPDCN in children was enrolled in this study. The blood routine test, examination of bone marrow cell morphology, histopathology and immunophenotype of the skin lesions were performed and analysed, the single cell suspensions of the biopsied skin mass were detected by flow cytometry. The results showed that tumor cells expressed CD4, CD56, CD43 and CD123, while not expressed CD19, CD20, CD3, CD8, CD13, CD11b and myeloperoxidase (MPO). According to the clinical and laboratorial features and the results from histopathological and immunophenotype examinations, BPDCN was confirmed. It is concluded that BPDCN in children is an extremely rare hematopoietic malignancy with presenting a rapidly and fatally aggressive clinical course. The diagnosis of this disease is mainly based on the clinical presentations, pathologic and immunohistochemical features. BPDCN is a highly aggressive disease, its prognosis is very poor, its pathogenesis remans still unclear. A standard treatment protocol for BPDCN has not yet been established.

Association of miRNAs expression profiles with prognosis and relapse in childhood acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen childhood ALL related microRNAs (miRNAs), analyze association of miRNAs expression profiles with prognosis and relapse in childhood acute lymphoblastic leukemia (ALL) and explore new indicator for predicting relapse and prognosis.</p><p><b>METHODS</b>miRNAs expression profile was analyzed by gene chip in 49 newly diagnosed childhood ALL and 12 primary immune thrombocytopenia (ITP) cases (as control group). Abnormal expression of miRNAs was verified by qRT-PCR. The correlation of miRNAs expression pattern with indicators predicting early prednisone response and relapse within a year was analyzed.</p><p><b>RESULTS</b>Specific expression of miRNAs profiles associated with prednisone response and early relapse in childhood ALL was identified. Eight miRNAs (miR-18a, miR-532, miR-218, miR-625, miR-193a, miR-638, miR-550 and miR-633) could distinguish prednisone sensitive from insensitive. The early relapse of newly diagnosed patients with either high-risk or non-high-risk clinical types had some characteristics of abnormal expression of miRNAs, including miR-7, miR-216 and let-7i upregulated, while miR-486, miR-191, miR-150, miR-487 and miR-342 downregulated.</p><p><b>CONCLUSIONS</b>The initial screening reveals miRNAs differentially expressed from normal in ALL suggesting the potential roles of them in leukemogenesis. MiRNAs expression signatures may be useful for predicting prognosis and relapse in childhood ALL and directing personalized treatment.</p>

Detection of minimal residual disease in childhood acute lymphoblastic leukemia by using real-time quantitative PCR / 中国实验血液学杂志

This study was purposed to detect the minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) by using real time quantitative PCR (RQ-PCR) . The Ig and TCR gene rearrangements were amplified by using 18 primer sets in B-ALL, 8 primer sets in T-ALL; the ALL-MRD levels were quantified by using RQ-PCR with SYBR green dye staining and clone specific Ig/TCR gene rearrangements as molecular markers. The results indicated that there were 8 cases showing gene rearrangements in 9 B-ALL patients, marker detection rate for all samples was 88.8%, the MRD level on day 33 during induction treatment decreased significantly. It is concluded that Ig/TCR gene rearrangements can be used as a marker to detect MRD in childhood ALL; the technique of QR-PCR with SYBR green dye staining is reliable, relatively sensitive and easy performable method which can be used in routine detection for childhood ALL.

Distribution and interaction of bone marrow mesenchymal stem cells and cord blood CIK/NK cells infused via different ways at different time periods in NOD/SCID mice / 中国实验血液学杂志

The study was aimed to explore the distribution and interaction mechanism of human bone marrow mesenchymal stem cells (MSC) and cord blood cytokine-induced killer (CIK)/natural killer (NK) cells infused via different ways at different times in NOD/SCID mice. 5 microl 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) dye(red) was added in suspension of MSC per ml, and 1 microl carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) dye(green) was added in suspension of CIK/NK cells per ml. The amounts of MSC and CIK/NK cells infused in each 6 NOD/SCID mouse were 1 x 10(6) (0.1 ml) and 1 x 10(7) (0.1 ml) respectively. All mice were divided into 4 groups, each group consisted of 6 mice. Group A: MSC (intravenous infusion, iv) + CIK/NK cells (iv) at the same time, group B: MSC (iv) + CIK/NK cells (iv) at 48 hours after infusion of MSC; group C: MSC (intramedullary infusion, im) + CIK/NK cells (iv) at the same time; group D: MSC (im) + CIK/NK cells (iv) at 48 hours after infusion of MSC. 3 NOD/SCID mice were sacrificed per batch at 24 hours and 48 hours after infused CIK/NK cells. Frozen sections of liver, spleen, lung and kidney were prepared, and then followed by counting the amounts of red and green fluorescence cells under fluorescence microscope, and calculating the ratio of MSC to CIK/NK cells for reflecting the interaction of MSC and CIK/NK cells in mice, and for showing the suppressive intensity of MSCs on CIK/NK cells. The results showed that the sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen of group A and B were higher than that in group C and D at 24 hours and 48 hours respectively after infusing CIK/NK cells. The sum of average ratios of MSC to CIK/NK cells in group A was slightly higher than that in group B at 24 hours and 48 hours after infusing CIK/NK cells, but there was no significant difference between them. The sum of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group C was slightly lower than that in group D at 24 hours after infusing CIK/NK cells, but reversed at 48 hours later and there was no significant difference between them. The sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group A, B, C and D were all higher than those in kidney at 24 and 48 hours respectively after infusing CIK/NK cells. It is concluded, the MSC and CIK/NK cells may interact if they are infused via the same way and at the same time, the location where the suppression of MSC on CIK/NK cells occur in vivo may be reticulo-endothelial systems in lungs and livers.

Effect of (-)-epigallocatechin-3-gallate and 5-AZA-CdR on expression of X-linked inhibitor of apoptosis protein-associated factor 1 in human leukemia HL-60 and K562 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression of X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (Xaf1) in human leukemia HL-60 and K562 cells treated with interferon alpha (INFalpha) and the demethylating agent 5-AZA-CdR, and observe the synergetic antitumor effect of Xaf1 inducer and (-)-epigallocatechin-3-gallate (EGCG).</p><p><b>METHODS</b>Human leukemia HL-60 and K562 cells were treated for 48 h with 1000 U/ml INFalpha and different doses of 5-AZA-CdR, and the mRNA expressions of both Xaf1 and XIAP were measured by RT-PCR. The leukemia cells were also treated with the optimal Xaf1 inducer in combination with EGCG, after which flow cytometry was employed to examine the changes in the members of the Bcl-2 family, mitochondrial transmembrane potential and apoptosis.</p><p><b>RESULTS</b>As the dose increased, 5-AZA-CdR dose-dependently up-regulated the mRNA expression of Xaf1 in HL-60 and K562 cells; INFalpha treatment also resulted in increased Xaf1 expression, but 5 micromol/L 5-AZA-CdR showed the most potent effect. Neither INFalpha nor 5-AZA-CdR caused significant changes in XIAP expression. Combined treatment with 5-AZA-CdR and EGCG altered the expressions of Bcl-2 (Bcl-xl) and Bax in HL-60 and K562 cells, decreased the mitochondrial transmembrane potential and induced cell apoposis, and the two agents exhibited obvious synergistic effect.</p><p><b>CONCLUSION</b>INFalpha and 5-AZA-CdR can induce Xaf1 mRNA expressions in HL-60 and K562 cells, and the effect of 5-AZA-CdR was dose-dependent. 5-AZA-CdR and EGCG induces apoptosis of leukemia cells in vitro, and they exhibits obvious synergetic effects.</p>

Construction of Microvessel Angiogenesis Model in Nonobese Diabetic/Severe Combined Immunodeficient Disease Leukemia Mice / 实用儿科临床杂志

Objective To construct microvessel angiogenesis model in nonobese diabetic/severe combined immunodeficient disease(NOD/SCID) leukemia mice.Methods Divided NOD/SCID mice into group A:blank group;group B:endothelial cell(EC) injection group;group C:leukemia model mice with EC injection group.Microvessel density(MVD) in bone marrow was calculated with cnemis slice after immunohistochemistry dyeing.Evaluated leukemia burden in leukemia model mice.Results Bone marrow MVD in group C was significantly more than group B.Microvessels in leukemia mice had disordered branches and irregular cavity.The morphism of vessels was immature.Conclusions Microvessel angiogenesis model in NOD/SCID leukemia mice has been constructed successfully.It can be used in the study of anti-angiogenesis in leukemia and relative researches.

Experimental study on the mechanism of the apoptosis of leukemic cells induced by valproic acid / 中华儿科杂志

<p><b>OBJECTIVE</b>To overcome the drug-resistance of tumor cells is one of the methods of improving the therapeutic results. Histone deacetylase inhibitors (HDACIs) is a novel class of chemotherapeutic agents which can induce apoptosis of tumor cells. Valproic acid (VPA) is a common drug used in the treatment of epilepsy. It has been shown that VPA has a marked HDACIs effect at the pharmaceutical level, and can induce the differentiation and apoptosis of transformed cells. But the mechanism of its effect has not been clarified. The aim of this study was to investigate the mechanism of VPA in inducing the apoptosis of leukemic cells at molecular level.</p><p><b>METHODS</b>The cell lines U937, Jurkat clone E6 - 1 (Jurkat) and BALL-1 were cultured in RPMI 1640 medium containing 20% calf serum, then divided into three groups (control group, 1 mmol/L VPA group and 1 mmol/L VPA + 1 micromol/L Pan-caspase inhibitor zVAD-fmk group). At 72 hours after the treatment, the cells were double stained with Aunexin and PI (propidium iodide) and then were analyzed with the flow cytometry (FCM) to detect apoptosis. Before and after treatment with VPA the mean fluorescence index (MFI) of Bcl-2, Bax, Bcl-xl and the levels of caspase 8, 9 and 3 were also detected with the FCM. The changes of P(44/42) mitogen activating protein kinase (MAPK) and phosphorylated P(44/42) MAPK were determined by Western blotting.</p><p><b>RESULTS</b>Seventy-two hours after the treatment, 1 mmol/L VPA induced apoptosis of U937 and Jurkat. The apoptotic rate of U937 was (75.78 +/- 4.20)% and that of Jurkat was (53.50 +/- 5.87)% (P < 0.01, vs. control group); zVAD-fmk could fully inhibit the apoptosis of U937, and the apoptotic rate was (2.89 +/- 0.36)%; while it could partly inhibit the apoptosis of Jurkat, and the apoptotic rate was (15.38 +/- 1.40)% (P < 0.01). 1 mmol/L VPA could not induce the apoptosis of BALL-1 which had a high expression level of Bcl-2. The MFI of Bcl-2, Bax and Bcl-xl in these three cell lines did not change significantly with VPA (P > 0.05). After treatment with VPA, the level of caspase 3 in U937 increased from (14.09 +/- 1.19)% to (32.30 +/- 2.47)%, and caspase 8 from (4.58 +/- 1.41)% to (86.47 +/- 3.26)% (P < 0.01), but there was no significant change in caspase 9 [(13.25 +/- 3.11)% and (10.95 +/- 1.30)%]. In Jurkat, the level of caspase 3 increased from (12.01 +/- 1.63)% to (35.56 +/- 0.27)%, and caspase 9 from (13.89 +/- 1.71)% to (75.89 +/- 4.08)% (P < 0.01 for both); no significant change was observed for caspase 8 [(5.94 +/- 1.38)% and (5.44 +/- 0.72)%]. In BALL-1, there was a slight decline in caspase 3 (P < 0.05). With the effect of VPA, levels of P(44/42) MAPK and phosphorylated P(44/42) MAPK decreased in all three cell lines (P < 0.01).</p><p><b>CONCLUSION</b>VPA could induce apoptosis of U937 through the activation of caspase 3 and 8; and it induced the apoptosis of Jurkat involving the activation of caspase 3 and 9. P(44/42) MAPK pathway also plays an important role in this course. VPA induced apoptosis of these cell lines without the alteration of Bcl-2, Bax and Bcl-xl. High level of Bcl-2 could antagonize the effect of VPA.</p>

Inhibiting Effect of Vascular Endothelial Growth Factor Antisense Oligonucleotides on HL60 Cell and HL60/VCR Multidrug Resistance Cell / 实用儿科临床杂志

Objective To explore the effect of vascular endothelial growth factor antisense oligonucleotides(AS-VEGF)on HL60 cell and HL60/VCR multidrug resistance cell and analyze the function of P-gp and the expression of related multidrug resistance genes including Bcl-2,Mcl-1,MDR1,MRP,GST?,TopoⅡ? and TopoⅡ?.Methods A vector AS-VEGF which expressed in eukaryotic cell was established,then transfected the vector into HL60 and HL60/VCR by limposome transfection technology,observed and drew the growth curve by Tapanlan taining,RT-PCR was used to detect the expression of Bcl-2,Mcl-1,MDR1,MRP,GST?,TopoⅡ? and TopoⅡ? in mRNA level after transfected 24 h and 48 h.Western Blot was used to detect the expression of P-gp in proteinum level after transfected 24 h and 48 h.Results The growth of HL60 and HL60/VCR was inhibited by AS-VEGF(1.25 mmol/L).Between HL60 and HL60/VCR,AS-VEGF decreased the expression of MDR1,MRP,GST? and TopoⅡ? but could not influence the expression of Bcl-2,Mcl-1 and TopoⅡ?,and the expression of P-gp was also obviously decreased in 48 h compared with that in 24 h.Conclusions AS-VEGF can inhibite the growth of HL60 and HL60/VCR and reverse multidrug resistance by changing cell microenvironment and the cell membrane correlated protein transportating channel,reduce the cell disintoxicating and the self-repair ability.

Diagnostic values of serum levels of HA, PC III, C IV and LN to the liver fibrosis in children with beta-thalassemia major / 中华儿科杂志

<p><b>OBJECTIVE</b>The presence of liver fibrosis in patients with beta-thalassemia major has been demonstrated to be an important negative prognostic factor. Identification of liver fibrosis in early stage would be of great value. Hyaluronic acid (HA), type III pre-collagen (PC III), collagen IV (C IV) and laminin (LN) as serum markers were widely used in the diagnosis of liver fibrosis in patients with chronic viral infections or alcoholic liver diseases. However, their values in thalassemic liver fibrosis have not been studied. This work was to determine the serum HA, PC III, C IV and LN levels in children with beta-thalassemia major and evaluate the diagnostic utility.</p><p><b>METHOD</b>Serum HA, PC III, C IV and LN in 49 hospitalized children with beta-thalassemia major (aged 1 - 15 years with the media age of 6.27 years) and 41 healthy children served as controls (aged 1 - 13 years with media age of 6.40 years) were detected by radioimmunoassay (RIA). Forty-five of 49 cases were performed percutaneous liver biopsies, and the histopathological fibrosis was compared with the four serum markers. The correlation and discriminate analysis were used.</p><p><b>RESULTS</b>All the serum levels of HA, PC III, C IV and LN in beta-thalassemia were significantly higher than those in controls (P < 0.01). In 36 of 45 cases, the histopathology showed liver fibrosis including stage I and stage II by biopsies with a positive rate of 80%. The serum levels of four markers increased successively with the aggravation of liver fibrosis from stage 0 to stage II, and significant correlation was observed between the level of HA or PC III and the stage of fibrosis (HA, r = 0.379, P = 0.017; PC III, r = 0.455, P = 0.04). While there was no difference between the level of C IV or LN and fibrosis (C IV, r = 0.312, P = 0.053; LN, r = 0.310, P = 0.055). Using discriminate analysis, the discriminate function of co-detection of the four markers for the diagnosis of fibrosis was 0.002 HA + 0.003 PC III + 0.002 C IV + 0.006 LN-1.859, which had a sensitivity of 93.88%, specificity of 68.29%, predictive value of positive test and negative test of 77.97% and 90.32%, respectively. Moreover, there was a significant correlation between the serum level of HA or PC III and the liver iron concentration (HA, r = 0.318, P = 0.035; PC III, r = 0.305, P = 0.044).</p><p><b>CONCLUSION</b>The results suggest that, in beta-thalassemia major with chronic liver disease, HA and PC III showed more practical value in diagnosing liver fibrosis than the levels of C IV and LN. The combination of the four serum markers could improve the accuracy and reliability of the diagnosis. A validation study is necessary before introducing into the prediction function during the clinical practice.</p>

Effects of meisoindigo on the expression of globin gene in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>The pathophysiology of beta-thalassemia is the imbalance of the alpha and non-alpha globin chain which leads to a series of clinical symptoms of hemolytic anemia. Scientists continuously try to explore gene-activated drugs to increase the level of non-alpha globin chain or decrease the level of alpha globin chain in the treatment of beta-thalassemia. To probe into the effects on globin-gene expression of meisoindigo (Me) in cultured erythroid cells derived from peripheral blood, so as to provide the theoretical basis for applying Me in the treatment of beta-thalassemia.</p><p><b>METHODS</b>By using the two-step liquid culture of erythroid progenitor cells and reverse transcription polymerase chain reaction (RT-PCR), and by using alpha mRNA as an inner control, the level of gamma mRNA and beta mRNA in cultured erythroid cells derived from peripheral blood of 11 patients with severe beta-thalassemia and 6 normal volunteers were measured under the effect of different concentration (2.5 micro mol/L, 5 micro mol/L and 10 micro mol/L) of Me.</p><p><b>RESULTS</b>(1) No statistic significance was found in the ratio of beta/alpha mRNA by Me in cultured cells from both normal individuals and beta-thalassemia. (2) Me can significantly increase the ratio of gamma/alpha mRNA and (beta + gamma)/alpha mRNA (that is non-alpha/alpha mRNA) in cultured cells from normal individuals and beta-thalassemia. The ratio of gamma/alpha mRNA was increased 0.31 - 0.45 times and the ratio of non-alpha mRNA/alpha mRNA increased 0.21 - 0.32 times in Me induced cells from normal individuals. No significant result was observed among the different concentrations of Me (2.5 micro mol/L, 5 micro mol/L and 10 micro mol/L) in normal individuals. With the increasing of Me concentrations, the ratios of gamma/alpha mRNA and alpha/alpha mRNA were increased in cultured cells from beta-thalassemia. The ratio of gamma/alpha mRNA was increased 0.33 - 1.17 times and the ratio of non-alpha/alpha mRNA increased 0.25 - 0.89 times in Me induced cells from beta-thalassemia. There was no significant difference between the concentrations of 2.5 micro mol/L and 5 micro mol/L concentration in beta-thalassemia. However, there was significant difference between the concentrations of 10 micro mol/L and the concentrations of 2.5 micro mol/L and 5 micro mol/L in beta-thalassemia. (3) The increase of the ratio of gamma/alpha mRNA and non-alpha/alpha mRNA in beta-thalassemia was higher than that in normal individual with induction by Me with a higher concentration (10 micro mol/L).</p><p><b>CONCLUSION</b>Me can raise the ratio of gamma/alpha mRNA and non-alpha/alpha mRNA in cultured erythroid cells derived from peripheral blood of both normal individual and beta-thalassemia in the level of transcription, which can improve the imbalance of the alpha and non-alpha globin chain. So Me has a latent value in the therapy of beta-thalassemia.</p>