Analysis of Clinical Features and Prognosis of Patients with Chronic Neutrophil Leukemia / 中国实验血液学杂志

OBJECTIVE@#To provide clinical basis for the diagnosis and treatment of chronic neutrophilic leukemia (CNL) and to provide possible molecular targets for the treatment.@*METHODS@#By summarizing the clinical data of 14 patients with CNL, the clinical characteristics, gene mutation types and possible prognostic factors were analyzed.@*RESULTS@#Among the 14 patients with CNL, males (9 cases) were more than females (5 cases), with a median age of 57 years old. The detection rate of CSF3R mutation was 92.86% (13/14), including 12 cases (85.71%) with T318I mutation and 1 case of Y799X mutation, and only 1 case was not detected for mutation of CSF3R. The ASXL1 mutation was detected in 42.86% (6/14) of the patients, all of which were nonsense mutations, including 4 cases with R693X and 2 cases with E705X, and 14.29% (2/14) of the patients was detected for SETBP1 mutation, all of which were with D868N mutation. No patients with simultaneous ASXL1 and SETBP1 mutations were found, and JAK2 and CALR mutations were not detected. All of the patients had normal karyotypes. These patients' median survival time was 30 months (95%CI 13.19-46.80), and the influence of age over 60 years old was statistically significant (21.83 months vs 35.35 months) (P＜0.05).@*CONCLUSION@#It is difficult to diagnose CNL. CSF3R T618I mutation is its specific mutation, and ASXL1 mutation and SETBP1 mutation have auxiliary diagnostic significance for CNL. The age＞60 years old at diagnosis is a factor of unfavourable prognosis.

Analysis of Phenotype and L12R Mutation in Signal Peptide and 3' Non-translation Region c11814-insAA Mutation of F7 Gene in a Family with Hereditary Coagulation Factor VII Deficiency / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To examine one young female patient with hereditary FVII deficiency and her family members, to observe the gene mutation and clinical phenotype, and to investigate the molecular mechanism of the dysfunction.</p><p><b>METHODS</b>Prothrombin time (PT), activated partial thromoploastin time (APTT), fibrinogen (Fg) and FVII activity (FVII:C) and FVII antigen (FVII:Ag) were tested. The gene mutations were sought by DNA sequencing for all of the exons and flanks, 5' and 3' non-translation region of F7 gene. To confirm the role of the found gene mutation, the reverse sequence were determined with Chromas software. To infer the influence of the mutation on the synthesis and function of FVII protein, the FVII protein molecule model containing the found mutation was constructed and the function prediction was performed by the signal peptide prediction database.</p><p><b>RESULTS</b>Compared with the normal population, the proband's PT value was significantly prolonged, and the ratio % FVII:C and that of FVII:Ag were significantly decreased by 1.1% and 0.9%, respectively. The PT, APTT, FVII:C and FVII:Ag of the proband's parents were both normal. Heterozygous 556th nucleotide mutations T/G were found in the proband's and his father's exon lA of F7 gene, with codon CTG turning into CGG, corresponding leucine (L) into arginine (R), i.e Leu12Arg. Function prediction showed that L12R mutations affected the segmentation of different parts of the signal peptide and its corresponding function, which could result in the decline in the mature protein synthesis and its activity obviously. In addition, a spontaneous 3' untranslated region c11814-insAA heterozygous mutation was detected in the proband's F7 gene, while her parents didn't possess this mutation.</p><p><b>CONCLUSION</b>A new hererozygous mutation (L12R) located in signal peptide of F7 gene is the primary molecular basis of the case with hereditary FVII deficiency. At the same time, the proband's spontaneous 3' non-translation region c11814-insAA mutation may lead to the further reduetion of the FVII synthesis.</p>

IL-11 promotes the treatment efficacy of hematopoietic stem cell transplant therapy in aplastic anemia model mice through a NF-κB/microRNA-204/thrombopoietin regulatory axis

Hematopoietic stem cell (HSC) transplantation could be of therapeutic value for aplastic anemia (AA) patients, and immunosuppressants may facilitate the efficiency of the procedure. As anti-inflammatory cytokine interleukin-11 (IL-11) has a thrombopoietic effect, its use in cases of chronic bone marrow failure, such as AA, has been proposed to induce HSC function. However, the putative mechanisms that may support this process remain poorly defined. We found that decreased miR-204-5p levels were coincident with increased proliferation in mouse HSCs following exposure to IL-11 in vitro. Through inhibiting NF-кB activity, miR-204-5p repression was demonstrated to be a downstream effect of IL-11 signaling. miR-204-5p was shown to directly target thrombopoietin (TPO) via sequence-dependent 3′-UTR repression, indicating that this microRNA-dependent pathway could serve an essential role in supporting IL-11 functions in HSCs. Increased TPO expression in HSCs following IL-11 exposure could be mimicked or blocked by inhibiting or overexpressing miR-204-5p, respectively. Consistent with these in vitro findings, IL-11 promoted HSC engraftment in a mouse model of AA, an effect that was attenuated in cells overexpressing miR-204-5p. The reduction in miR-204-5p levels is an integral component of IL-11 signaling that may play an essential role in treating AA.

Effect of Artesunate on Proliferation, Cell Cycle and Apoptosis of SKM-1 Cells and Its Underlying Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of artesunate (ART) on proliferation, cell cycle and apoptosis of SKM-1 cells in vitro and to explore the underlying mechanisms.</p><p><b>METHODS</b>After SKM-1 cells were treated with different concentrations of ART, the cell proliferation was determined by CCK-8 method. Apoptosis and distribution of cell cycle were detected by flow cytometry. Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species (ROS) and calcium ion concentration. Western blot was used to measure the protein levels of BCL-2, BAX, BAD, P-BAD, survivin and XIAP.</p><p><b>RESULTS</b>ART obviously inhibited the growth of SKM-1 cells in time and dose-dependent manners (r = -0.841; r = 0.-786). The antioxidant trolox-pretreatment significantly decreased the growth inhibition effect of ART on SKM-1 cells. Caspase inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART on SKM-1 cells. After treatment with ART for 24 hours, the apoptosis of SKM-1 cells was found, the cell cycle of SKM-1 was arrested in G0/G1 phase, ART could elevate the levels of calciumion and reactive orygen. ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-1 cells, and showed a highly negative correlation with ART dose (r = -0.909; r = -0.849). On the contrary, ART had no significant effect on expression levels of BAD and XIAP in SKM-1 cells, and after ART treatment, although BCL-2 protein expression was not significantly different when compared with control group, but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose (r = -0.866).</p><p><b>CONCLUSION</b>The ART significantly suppresses the cell proliferation, induces the apoptosis and promoted cell cycle arrest at G0/G1 phase in SKM-1 cells. The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels. In addition, the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.</p>

Magnetic Nanoparticles of Fe3O4 Enhance Artesunate-inducing Apoptosis of SKM-1 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of the copolymer of magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)) and artesunate (ART) on myelodysplastic syndromes (MDS) cell line SKM-1 cells and the potential mechanisms.</p><p><b>METHODS</b>The protein expression levels of BCL-2, BAX, Caspase-3, and Survivin in SKM-1 cells treated with or without the co-polymer were measured by Western blot. The co-polymer-induced apoptosis rate of SKM-1 cells was measured by flow cytometry.</p><p><b>RESULTS</b>The apoptosis rate of SKM-1 cells in the copolymer groups was higher than that in both MNPs-Fe(3)O(4) and artesunate groups alone. The MNPs-Fe(3)O(4) may enhance ART-induced cell apoptosis. Western blot assay showed that the expression of survivin and BCL-2 protein were down-regulated in the ART group, and this down-regulation was even more significant in the group of copolymer of ART with MNPs-Fe(3)O(4). The levels of BAX were increased both in ART group and the copolymer of ART with MNPs-Fe(3)O(4) group, as compared with control group and MNPs-Fe(3)O(4) group. The levels of active-caspase-3 were obviously up-regulated when the ART was combined with the MNPs-Fe(3)O(4). The copolymer of ART with MNPs-Fe(3)O(4) could trigger changes in the expression levels of apoptosis-related genes in SKM-1 cells, among which up-regulation of BAX and down-regulation of survivin and BCL-2 are the 2 major alterations.</p><p><b>CONCLUSION</b>Artesunate can induce the apoptosis of SKM-1 cells, and MNPs-Fe(3)O(4) may enhance the cell apoptosis induced by ART.</p>

Clinical analysis of 14 patients with thrombotic thrombocytopenic purpura / 中国实验血液学杂志

In order to enhance the understanding of thrombotic thrombocytopenic purpura (TTP), the clinical features, laboratory characteristics, treatment and outcome of 14 patients with TTP were retrospectively analyzed and investigated. The results showed that 7 out of 14 patients with TTP had predisposing factors, such as pregnancy in 4 cases, infection in 3 cases, systemic lupus erythematosus (SLE) in 1 case and hematopoietic stem cell transplantation (HSCT) in 1 case. Fourteen patients all had neuropsychological symptoms, hemolytic anemia with negative-Coombs test, and decreased platelet counts. Eight patients had irregular fever with different degree. There were 8 patients with kidney damage including proteinuria in 8 cases and renal function abnormalities in 4 cases. The von Willebrand factor-cleaving protease (VWF-CP, ADAMTS13) activity of 13 cases out of 14 patients significantly decreased (less than 10%). At same time, plasma ADAMTS13 inhibitors were detected in 12 cases out of these 13 patients with decreased ADAMTS13 activity. After treatment with plasma exchange, glucocorticoid and rituximab so on, 12 cases achieved complete remission, in which 8 cases relapsed in two years. Two patients died at last, in which one case was secondary to HSCT. It is concluded that TTP is a kind of thrombotic microangiopathy due to platelet microthrombosis involved in multiple systems and multiple organs dysfunction with dangerous clinical process. The mortality of TTP patients is very high. Early diagnosis and early treatment with plasma exchange as the main means can greatly improve the prognosis of patients with TTP.

Effects of DNA methylation on expression of TIG1 gene in acute leukemia / 中国实验血液学杂志

This study was purposed to investigate the expression and methylation status of TIG1 in acute leukemia (AL). The TIG1 expression of 53 cases of AL and 20 cases of normal control (NC) were measured by using real-time quantitative PCR (RT-QT-PCR) and methylation-specific PCR(MS-PCR). The leukemia KG-1a, U937 and K562 cells were treated with 5-Aza-CdR. The results indicated that TIG1 gene expressed at a high level in cases of NC, but expressed at a low level in patients with AL. TIG1 gene was unmethylated in NC, but frequently methylated in AL. Aberrant methylation rate of TIG1 in AL was 75% (40/53). The expression of TIG1 in unmethylated patients was higher than that in methylated patients. Hypermethylation of TIG1 promoter CpG islands was detected in all the cell lines. 5-Aza-CdR treatment led to the hypomethylation of TIG1 promoter CpG islands. After the treatment with 5-Aza-CdR of different concentration, the expression of TIG1 was restored, and the effect of 5-Aza-CdR displayed dose-dependency. It is concluded that the reduced expression of TIG1 may play an important role in the pathogenesis of AL, and methylation may be responsible for the decreased transcription of TIG1 gene.

BMMSC from blastic phase CML down-regulate leukemia cell apoptosis / 中国实验血液学杂志

The purpose of this study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSC) from patients with chronic myeloid leukemia (CML) in blastic phase (Bp) on K562 cells and the primary CML-Bp cells, and to explore its potential mechanisms. K562 cells and primary CML-Bp cells were co-cultured with BMMSC of different groups; the cell proliferation was detected by MTT method, the cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry, the expression levels of Caspase-8, Caspase-9, and activated Caspase-3 in cells were measured by Western blot. The results showed that the CML-Bp BMMSC could enhance the survival rate of K562 cells treated with adviamycin (ADM) and display protective effect on K562 cells and primary CML-Bp mononuctear cells, inhibited ADM-induced leukimia cell apoptosis (P < 0.05); as compared with CML-chronic phase (CML-Cp) BMMSC and normal BMMSC, the CML-Bp BMMSC showed the highest protective effect on leukemic cells, the mitochondrial membrane potential of co-cultured cells slightly droped (P < 0.05). In the CML-Bp BMMSC cultured with K562 cells, the expression level of caspase-3 was more down-regulated than that in K562 alone plus ADM group, while the expression of caspase-9 significantly increased (P < 0.05). It is concluded that the CML-Bp BMMSC down-regulates ADM-induced leukemia cell appoptosis, its mechanism may relate with the inhibition of mitochondrial membrane potential drop, the stabilization of unactive expression of caspase-9 and down-regulation of caspase-3 expression.

Fechtner syndrome, a nonmuscle myosin heavy chain 9 gene mutation related disease: a case report and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To improve the recognition of Fechtner syndrome.</p><p><b>METHODS</b>The clinical and laboratory data and family survey of a patient with Fechtner's syndrom was reported.</p><p><b>RESULTS AND CONCLUSION</b>Giant platelets, thrombocytopenia and characteristic granulocyte inclusion bodies (Döhle-like bodies) were found in both peripheral blood and bone marrow smears of the patient. Clinically the patient had renal damage, nervous deafness, and vitreous lesions. There was a family genetic tendency on family survey the diagnosis of Fechtner syndrome is established.</p>

Expression and mutation of myc antagonist genes Mad1, Mxi1 and Rox in leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression and mutation of Mad1, Mxi1 and Rox genes in leukemia cells.</p><p><b>METHODS</b>Expression and mutation of Mad1, Mxi1 and Rox genes in bone marrow mononuclear cells (BMMNC) from 26 de novo acute leukemia (AL) patients, and in peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, as well as in 7 human leukemic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.</p><p><b>RESULTS</b>RT-PCR showed that all the above cells expressed Mad1, Mxi1 and Rox mRNA. SSCP revealed four polymorphisms: two in Mad1, one each in Mxi1 and Rox. DNA sequencing detected nine missense mutations: two in Mad1 in AL patients, four in Mxi1 (three in AL patients and one in KG-1 cell line), and three in Rox in AL patients. The mutations of Mad1, Mxi1 and Rox mRNA were detected in 2, 3 and 3 patients, respectively.</p><p><b>CONCLUSION</b>It is for the first time to demonstrate the mutations of Mad1, Mxi1 and Rox genes in AL patients suggesting these mutated genes involve in the pathogenesis of leukemia.</p>

Effects and mechanisms of cholecystokinin octapeptide on hippocampal injury during endotoxic shock / 中国应用生理学杂志

<p><b>AIM</b>To study the effects and the mechanisms of cholecystokinin octapeptide(CCK-8) on hippocampal injury during endotoxic shock (ES).</p><p><b>METHODS</b>Rabbits were injected intravenously with lipopolysaccharide (LPS, 8 mg/kg) to establish ES model. Thirty-two Rabbits were divided into 4 groups at random (n = 8): control (saline, iv), LPS, CCK-8 + LPS (CCK-8 pre-administrated 30 min before LPS, iv), proglumide (Pro, nonspecific antagonist of CCK receptors) + LPS (Pro pre-administrated 30 min before LPS, iv) group. The changes of mean arterial pressure (MAP) were measured. The morphologic changes in the hippocampus were observed through light microscope (LM) and transmission electron microscope (TEM). The alterations of activities of nitric oxide synthase (NOS) and superoxide dismutase (SOD), contents of nitric oxide (NO) and malondialdehyde (MDA) in the hippocampus were assayed. Twelve Sprague-Dawley rats, grouped as that of the rabbits, were used to detect the expression of inducible NOS (iNOS) and neuronal NOS (nNOS) protein by immunohistochemistry staining.</p><p><b>RESULTS</b>LPS administration resulted insignificant reduction in MAP (P < 0.01 vs control group) and hydropic degeneration of neurons in the hippocampus. Compared with those of control group, the NOS activity, NO level and MDA content were increased significantly (P < 0.05, P < 0.01 and P < 0.05), while SOD activity was reduced (P < 0.01) in the hippocampus of ES rabbits. LPS administration induced the expression of iNOS protein in the cytoplasm of hippocampus neurons, and lead to stronger positive signals of nNOS than that of control group. CCK-8 pre-administration could alleviate the changes induced by LPS, while Pro pre-administration aggravated those alterations.</p><p><b>CONCLUSION</b>CCK-8 could protect hippocampus neurons against the injury induced by LPS during ES, which might be associated with its effects of suppressing the over production of NO and free radicals.</p>

WT1 gene expression lowered by IL-12 In vitro in peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes / 中国实验血液学杂志

Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

Effects of integrin beta1 on phycocyanin inhibiting proliferation of K562 cells / 中国实验血液学杂志

This study was purposed to investigate the effect of phycocyanin at different concentration on proliferation of K562 cells, to detect the changes of integrin beta1 expression and intracellular focal adhesion kinase (FAK) gene expression on the surface K562 cells treated with phycocyanin, and to explore the possible mechanism of integrin beta1 effect on phycocyanin inhibiting proliferation of K562 cells. The expression level of integrin beta1 on the surface of K562 cells was evaluated by flow cytometry (FCM); the growth of K562 cells treated with phycocyanin was measured by MTT assay; the expression level of FAK mRNA was analyzed by relatively quantitative RT-PCR after four-day culture of K562 cells with phycocyanin of 40 microg/ml, 80 microg/ml and 160 microg/ml, respectively. The results showed that integrin beta1 expression on the surface of K562 cells was significantly higher than that in bone marrow mononuclear cells (BMMNC) from normal subjects. Phycocyanin could not change the level of integrin beta1 expression. Phycocyanin could increase the expression of FAK gene on K562 cells and inhibit the proliferation of K562 cells. It is concluded that phycocyanin can inhibit the proliferation of K562 cells through enhancing the conjunction of cell stroma with integrin beta1 on K562 cell surface, up-regulating the expression level of FAK gene in K562 cells, restoring the signaling pathway of proliferation inhibition mediated by integrin beta1. The possible mechanism of phycocyanin in the proliferation inhibition of K562 cells is to increase the expression of FAK gene. The phycocyanin may be considered as a potential agent for inhibition of cancer cell proliferation.

Effects of cholecystokinin-octapeptide on the tension of pulmonary artery in rabbits with endotoxic shock / 生理学报

For investigation of the regulatory mechanism of cholecystokinin-octapeptide (CCK-8) on pulmonary circulation in rabbits with endotoxic shock (ES) induced by lipopolysaccharides (LPS), mean arterial pressure (MAP) and pulmonary arterial pressure (PAP) were evaluated for 5 h in five groups of rabbits: group of LPS (8 mg/kg, i.v.)-induced ES, group of CCK-8 pretreatment (15 microg/kg, i.v.) 15 min before LPS administration (8 mg/kg, i.v.), group of proglumide pretreatment (1 mg/kg, i.v.) 15 min before LPS administration (8 mg/kg, i.v.), group of CCK (15 microg/kg, i.v.) only, and normal saline (control) group. The pulmonary arterial tension was measured with isolated vascular ring technique. The results showed that LPS-induced pulmonary arterial hypertension was abolished by CCK-8. In contrast, proglumide, a nonspecific antagonist of CCK-8 receptor, potentiated the deleterious effect of LPS. The contractile response of isolated pulmonary artery to alpha-adrenoceptor agonist phenylephrine (PE) was enhanced and the relaxation response to acetylcholine (ACh) was depressed significantly after LPS was injected, but the effect could be reversed by CCK-8. These results suggest that pulmonary circulation is improved by CCK-8 in ES, and the regulatory effects of CCK-8 may be brought about by modulating the pulmonary arterial tension.