[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.

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.

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.

[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.

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.

Two novel compound heterozygous mutations associated with types I and II protein C deficiency with unusual phenotypes.

INTRODUCTION: We diagnosed two Chinese hereditary PC deficiency families and identified two novel compound heterozygous mutations (p.Arg194Cys/Gly324Ser and p.Glu274X/Asp297His) in the protein C (PROC) gene. The probands were classified as types I and II PC deficiency. The aim of this article is to access the influence of the mutations on PC activity, antigen and protein structure, and to evaluate whether there is abnormal PC localization. MATERIALS AND METHODS: Genomic DNA of all family members was extracted, PCR amplified, and sequenced. The mutant PC expression plasmids were constructed. Expression assays, intracellular localization, and molecular modeling were performed. RESULTS: Proband 1, a type II PC defect, harbored a compound heterozygous mutation, p.Arg194Cys/Gly324Ser in the PROC gene, underwent two thromboembolic events. Expression assays indicated that the p.Arg194Cys mutant lead to decreased PC activity and normal PC Ag levels. Intracellular localization showed that both p.Arg194Cys and p.Gly324Ser co-localized with the endoplasmic reticuli and the Golgi apparatus. Molecular modeling suggested that the p.Gly324Ser mutation disturbed the interaction between the heavy and light chains of the PC protein. Proband 2, a type I PC defect, harbored a compound heterozygous PROC gene mutation, p.Glu274X/Asp297His, presented with recurrent spontaneous abortion and right popliteal vein thrombosis. Expression results were in accordance with the PC changes of the patient, and existed in defective PC transport. Structural model suggested p.Glu274X lead to disulfide bond between heavy and light chain cannot form. CONCLUSIONS: Our results confirm that two novel compound heterozygous PROC gene mutations are causative on the two PC deficiency families.

A TET2 rs3733609 C/T genotype is associated with predisposition to the myeloproliferative neoplasms harboring JAK2(V617F) and confers a proliferative potential on erythroid lineages.

Common germline single-nucleotide polymorphisms (SNPs) at JAK2 locus have been associated with Myeloproliferative neoplasms (MPN). And, the germline sequence variant rs2736100 C in TERT is related to risk of MPN, suggesting a complex association between SNPs and the pathogenesis of MPN. Our previous study (unpublished data) showed that there was a high frequency distribution in rs3733609 C/T genotype at Ten-Eleven Translocation 2 (TET2) locus in one Chinese familial primary myelofibrosis. In the present study, we evaluate the role and clinical significance of rs3733609 C/T genotype in JAK2V617F-positive sporadic MPN (n = 181). TET2 rs3733609 C/T genotype had a higher incidence (13.81%; 25/181) in JAK2V617F-positive sporadic MPN patients than that in normal controls (n = 236) (6.35%; 15/236), which was predisposing to MPN (odds ratio(OR) = 2.361; P = 0.01). MPN patients with rs3733609 C/T genotype had increased leukocyte and platelets counts, elevated hemoglobin concentration in comparison with T/T genotype. Thrombotic events were more common in MPN patients with rs3733609 C/T than those with T/T genotype (P < 0.01). We confirmed that rs3733609 C/T genotype downregulated TET2 mRNA transcription, and the mechanism may be involved in a disruption of the interaction between CCAAT/enhancer binding protein alpha (C/EBPA) and TET2 rs3733609 C/T locus.TET2 rs3733609 C/T genotype stimulated the erythroid hematopoiesis in MPN patients. Altogether, we found a novel hereditary susceptible factor-TET2 rs3733609 C/T variant for the development of MPN, suggesting the variant may be partially responsible for the pathogenesis and accumulation of MPN.

Thalidomide Effects in Patients with Hereditary Hemorrhagic Telangiectasia During Therapeutic Treatment and in Fli-EGFP Transgenic Zebrafish Model.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations. The efficacy of traditional treatments for HHT is very limited. The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model. METHODS: HHT was diagnosed according to Shovlin criteria. Five HHT patients were treated with thalidomide (100 mg/d). The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide. The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis. Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patient's peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-ß3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment. RESULTS: The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P = 0.009). The "telangiectatic spot" on the tongue almost vanished; CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation. The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-ß3 mRNA expression of five patients was lower after thalidomide therapy. The plasma VEGF protein expression was down-regulated in HHT patients. CONCLUSIONS: Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-ß3 and VEGF in HHT patients. It also leads to vascular remodeling in the zebrafish model.

Standardized fluorescence in situ hybridization testing based on an appropriate panel of probes more effectively identifies common cytogenetic abnormalities in myelodysplastic syndromes than conventional cytogenetic analysis: a multicenter prospective study of 2302 patients in China.

In an attempt to establish the advantages of fluorescence in situ hybridization (FISH) studies over conventional cytogenetic (CC) analysis, a total of 2302 de novo MDS patients from 31 Chinese institutions were prospectively selected in the present study for both CC and standardized FISH analysis for +8, -7/7q-, -5/5q-, 20q- and-Y chromosomal abnormalities. CC analysis was successful in 94.0% of the patients; of these patients, 35.9% of the cases were abnormal. FISH analysis was successful in all 2302 patients and detected at least one type of common cytogenetic abnormality in 42.7% of the cases. The incidences of +8, -7/7q-, -5/5q-, 20q- and-Y chromosomal abnormalities by FISH were 4.1% to 8.7% higher than those by CC. FISH identified abnormalities in 23.6% of the patients exhibiting normal CC results and revealed that 20.7% of the patients with adequate normal metaphases (≥20) had abnormal clones. FISH identified cytogenetic abnormalities in 50.4% of the patients with failed CC analysis. In summary, our multicenter studies emphasised and confirmed the importance of applying standardized FISH testing based on an appropriate panel of probes to detect common cytogenetic abnormalities in Chinese de novo MDS patients, particularly those with normal or failed CC results.

Dramatic response to itraconazole in central nervous system aspergillosis complicating acute promyelocytic leukemia.

Abstract Fungal infection is a rare complication of acute leukemia, in which a combination of voriconazole and amphotericin B is a first-line regimen of treatment. Here administration of itraconazole plus caspofungin resulted in a dramatic response in a patient with acute promyelocytic leukemia (APL).

Cytotoxic effect of icaritin and its mechanisms in inducing apoptosis in human burkitt lymphoma cell line.

Icaritin (ICT), a hydrolytic product of icariin from Epimedium genus, exhibits antitumor activities in several human solid-tumor and myeloid leukemia cells with extensive influence on various cell signal molecules, such as MAPKs being involved in cell proliferation and Bcl-2 participating in cell apoptosis. However, the effect of icaritin on Burkitt Lymphoma has not been elucidated. In the present study, we first screened the potential effect of icaritin on Burkitt lymphoma Raji and P3HR-1 cell lines and found that icaritin showed cytotoxicity in both cell lines. We further found that icaritin could significantly inhibit Raji cells proliferation with S-phase arrest of cell cycle and induced cell apoptosis accompanied by activation of caspase-8 and caspase-9 and cleavage of PARP. We also observed that icaritin was able to decrease Bcl-2 levels, thus shifting the Bcl-2/Bax ratio, and it could obviously reduce c-Myc, a specific molecular target in Burkitt lymphoma. Our findings demonstrated that icaritin showed cytotoxicity, inhibited cell growth, caused S arrest, and induced apoptosis in Burkitt lymphoma cells and provided a rationale for the further evaluation of icaritin for Burkitt lymphoma therapy.

Differential diagnosis between AML infiltration, lymphoma and tuberculosis in a patient presenting with fever and mediastinal lymphadenopathy: A case report.

The diagnosis of tuberculosis in immunocompromised hosts is often difficult as the hosts have atypical tuberculosis symptoms. The current study presents a case of scrofula and pulmonary tuberculosis with acute myelocytic leukemia (AML). As the disease became aggravated, the patient presented with fever, hemophagocytosis in the bone marrow, lymphadenopathy of the supraclavicular fossa, and mediastinal and nodular shadow in the chest by computed tomography. The symptoms presented successively or were coexistent, which made differentiation between tuberculosis, lymphoma, AML infiltration or other infections challenging. The diagnosis of tuberculosis was based on clinical and radiographic observations, morphological observation of the biopsies and the positive effect of antituberculosis drugs, while Ziehl-Neelsen stainings for acid fast bacilli were negative. The patient was treated with antituberculosis drugs, while receiving chemotherapy for AML. It is important to distinguish tuberculosis in adults with AML from other causes of fever, mediastinal masses in radiographic observations and hemophagocytosis in the bone marrow.

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.

An intron mutation in the ACVRL1 may be associated with a transcriptional regulation defect in a Chinese family with hereditary hemorrhagic telangiectasia.

PURPOSE: To identify a novel pathogenic gene mutation present in a Chinese family with hereditary hemorrhagic telangiectasia (HHT) and to determine if an intron mutation may influence the transcriptional activity of the ACVRL1 gene. METHODS: HHT family members were ascertained following the presentation of proband and involved subjects. All family members (n = 5) and 113 healthy individuals were genotyped for the variant in intron 6 c.772+27G>C of ACVRL1 gene. The genomic structure of ACVRL1 in affected HHT patients and healthy individuals was determined by long range PCR and sequencing. The expression of ACVRL1 mRNA and protein in patients with HHT was evaluated using real-time polymerase chain reaction and immunoblot analysis. Luciferase activity assay and electrophoretic mobility shift assay (EMSA) were performed to uncover the mechanism of intron-related transcriptional regulation. RESULTS: Only one novel mutation in intron 6 (c.772+27G>C) of ACVRL1 gene, no other mutation, abnormal splice, gross genomic deletion or rearrangement was found in this HHT2 family. Compared with healthy individuals, ACVRL1 mRNA and protein were significantly decreased in affected HHT2 individuals. Luciferase activity assay demonstrated that the transcriptional activity of the mutated ACVRL1 was significantly lower than that of the wild-type of intron 6; EMSA results showed that intron 6 c.772+27G>C mutation was able to inhibit the binding of transcriptional factor Sp1. CONCLUSIONS: A novel intron mutation in ACVRL1 gene is associated with familial HHT2. The mechanisms may be involved in the down-regulation of ACVRL1 gene transcription.