Stable Expression of Coagulation Factors by RPS6 Promoter / 中国实验血液学杂志

OBJECTIVE@#To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.@*METHODS@#Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.@*RESULTS@#The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.@*CONCLUSION@#After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

Clinical and genetic characteristics of patients with newly diagnosed acute promyelocytic leukemia: a single-center retrospective of 790 cases / 中华血液学杂志

Objective: To retrospectively analyze the data of Chinese patients with newly diagnosed acute promyelocytic leukemia (APL) to preliminarily discuss the clinical and cytogenetic characteristics. Methods: From February 2004 to June 2020, patients with newly diagnosed APL aged ≥ 15 years who were admitted to the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College were chosen. Clinical and laboratory features were retrospectively analyzed. Results: A total of 790 cases were included, with a male to female ratio of 1.22. The median age of the patients was 41 (15-76) years. Patients aged between 20 and 59 predominated, with 632 patients (80%) of 790 patients classified as low and intermediate risk and 158 patients (20%) of 790 patients classified as high risk. The white blood cell, platelet, and hemoglobin levels at diagnosis were 2.3 (0.1-176.1) ×10(9)/L, 29.5 (2.0-1220.8) ×10(9)/L, and 89 (15-169) g/L, respectively, and 4.8% of patients were complicated with psoriasis. The long-form type of PML-RARα was most commonly seen in APL, accounting for 58%. Both APTT extension (10.3%) and creatinine>14 mg/L (1%) are rarely seen in patients at diagnosis. Cytogenetics was performed in 715 patients with newly diagnosed APL. t (15;17) with additional chromosomal abnormalities were found in 155 patients, accounting for 21.7%; among which, +8 was most frequently seen. A complex karyotype was found in 64 (9.0%) patients. Next-generation sequencing was performed in 178 patients, and 113 mutated genes were discovered; 75 genes had an incidence rate>1%. FLT3 was the most frequently seen, which accounted for 44.9%, and 20.8% of the 178 patients present with FLT3-ITD. Conclusions: Patients aged 20-59 years are the most common group with newly diagnosed APL. No obvious difference was found in the ratio of males to females. In terms of risk stratification, patients divided into low and intermediate risk predominate. t (15;17) with additional chromosomal abnormalities accounted for 21% of 715 patients, in which +8 was most commonly seen. The long-form subtype was most frequently seen in PML-RARα-positive patients, and FLT3 was most commonly seen in the mutation spectrum of APL.

Clinical Analysis of Hospitalized Children with Primary Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the factors affecting the chronicity of childhood primary immune thrombo-cytopenia (ITP) and compare the efficiency of different first-line treatment regimens.@*METHODS@#Children with ITP hospitalized in our hospital from September 2013 to October 2018 were retrospectively analyzed.@*RESULTS@#Three hundred and one children (150 males and 151 females) were included in this study, with a median age of 8 (0.17-17) years old, and 110 (36.5%), 92 (30.6%), and 99 (32.9%) cases were grouped into newly diagnosed, persistent, and chronic ITP, respectively. The median of follow-up was 41.92 (1.07-74.03) months. At the end of the follow-up (October 2019), among the 202 newly diagnosed/persistent ITP children, 79 cases (59 newly diagnosed and 20 persistent ITP) achieved remission within 1 year after initial diagnosis, with a remission rate of 39.3%; 122 cases (50 newly diagnosed and 72 persistent ITP) developed chronic disease, with a chronicity rate of 60.7%; one case underwent splenectomy. In 99 cases with chronic ITP, 5 cases underwent splenectomy. Multivariable logistic regression analysis showed that, the insidious onset of symptoms (OR=3.754, 95%CI: 1.882-7.488, P=0.000) increased the risk of chronicity, while the positive antibody to anti-platelet membrane glycoprotein (OR=0.446, 95%CI: 0.224-0.888, P=0.021) might reduce the risk of chronicity. And no difference was found by the analysis of subtype of anti-platelet membrane glycoprotein (P=0.305). The efficacy of the first-line treatment of intravenous immunoglobulin (IVIG) alone or combined with steroid was better than that of steroid alone (P=0.028, 0.028), however, the efficiency was not significantly different between IVIG alone and combined with steroid (P=0.086).@*CONCLUSION@#Insidious onset of symptoms in pediatric ITP increases the risk of chronicity, while the positive titer of anti-platelet membrane glycoprotein may reduce the risk. In the first-line treatment for the newly diagnosed/persistent children. The efficacy of IVIG alone or combined with steroid is better than that of steroid alone.

Symptomatic Profiles of 173 Chinese Patients with Essential Thrombocythemia / 中国实验血液学杂志

OBJECTIVE@#To explore the symptomatic burden of patients with essential thrombocythemia (ET) and its relation with clinical characteristics including the mutation status, therapeutic protocols and sex.@*METHODS@#Total of 173 Chinese ET patients were selected and grouped on the basis of disease characteristics (mutation status, therapeutic pro to- cols, and sex).@*RESULTS@#All the groups showed low-to-high symptom burden, with the highest in the Hu (hydroxyurea)-group (total symptom score [TSS], 14.7; range, 7.6-14.7). In the JAK2V617F-positive, Hu-treated, and female groups TSS and independent symptom scores were higher than those in the control group. The CALR-positive and IFN-α-treated groups had lower overall and individual scores as compared with groups lacking the corresponding characteristics. As the number of characteristics (JAK2V617F-positive, Hu-treated, and female) increases, the severity of symptoms gradually increased.@*CONCLUSION@#The different characteristics have various effects on symptom burden in ET patients. The accumulation of certain characteristics will lead to more severe symptom burden, thus the patient＇s symptom burden should be considered comprehensively when making up the treatment schemes and prognosis.

Clinical Characteristic of "triple-negative" Essential Thrombocythaemia Patients and Mutation Analysis by Targeted Sequencing / 中国实验血液学杂志

<p><b>BACKGROUND</b>Essential thrombocythemia is a subgroup of myeloproliferative neoplasms. Previous studies identified mutations of JAK2, CALR, and MPL that are closely related with the pathogenesis of myeloproliferative neoplasms. All these mutations contribute to the hyperactivation of JAK2/STAT pathway. However, a small proportion of essential thrombocythemia patients does not display such mutations. The pathogenesis of "triple-negative" form of essential thrombocythemia remains unknown.</p><p><b>OBJECTIVE</b>To investigate the clinical characteristics of triple-negative essential thrombocythemia and related mutation genes.</p><p><b>METHODS</b>To identify the mutations associated with triple-negative essential thrombocythemia, next-generation sequencing was used to conduct targeted sequencing of 360 genes in samples from 68 patients.</p><p><b>RESULTS</b>At least one missense mutation was detected in all the patients and all the detected genes. After screening the data, it was observed that 10 genes with the 10 highest mutation were follows: FLT3, SH2B3, ASXL1, ADAMTS1, TET2, TP53, EGFR, CUX1, GATA2, and MPL.When only rare genes (i.e., with a frequency in Asian populations lower than 5%, as estimated by the 1000 Genomes Project) were analyzed, the most frequently mutated genes in the patients were TET2 (33.82%), SH2B3(29.41%), and ASXL1 (23.53%). Our study identified some mutations that did not previously reported. Although all these mutations need further validation, high incidence rates may indicate relevance of the respective mutations to essential thrombocythemia pathogenesis. Some of the detected mutations have been previously reported; these mutations were also found in a large proportion of our subjects.</p><p><b>CONCLUSION</b>whole-exon sequencing can provide a higher level of accuracy for gene mutation analysis and assist in identifying mutations that contribute to illustrate the pathogenesis of essential thrombocythemia.</p>

Clinical Characteristics and Long-Term Outcome of 125 Chinese Young Patients with Essential Thrombocythemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical characteristics and long-term outcome of Chinese young patients (≤40 years) with essential thrombocythemia(ET), and to develop a thrombosis predicting model specific for young patients with ET, so as to provide a new evidence for risk stratification and treatment.</p><p><b>METHODS</b>Medical records of 125 Chinese young patients with newly diagnosed of ET were retrospectively analyzed.</p><p><b>RESULTS</b>The median age at diagnosis was 32 (18-40) years old, with 37 males and 88 females. During follow-up, 18 patients (14.4%) experienced major thrombotic events. JAK2 V617F (HR=8.895, P=0.001), history of thrombosis (HR=8.001, P<0.001) and WBC≥12.0×10/L (HR=5.225, P=0.002) were independent risk factors for thrombosis. The incidence of thrombosis and risk factors in young patients were different from that in general ET population, so a thrombosis predicting model specific for young patients with ET was developed. In this model, JAK2 V617F (score 2), history of thrombosis (score 2) and WBC≥12.0×10/L (score 1) were used to divide the patients into low risk (score 0), intermediate risk (score 1-2) and high risk (score≥3) groups. These 3 groups exhibited significantly different thrombosis-free survival (χ=32.223, P<0.001). Antiplatelet treatment could prevent the occurrence of thrombosis (HR=0.081, P<0.001), while cytoreductive agents significantly decreased the risk of thrombosis only in intermediate and high risk groups (14.3% vs 36.4%, χ=4.416, P=0.036). Seven patients (5.6%) evolved to myelofibrosis, and one of them finally progressed in to acute leukemia. The only risk factor for evolution was WBC≥15.0×10/L (χ=5.434, P=0.020). Neither antiplatelet treatment nor cytoreductive agents could prevent disease progression.</p><p><b>CONCLUSION</b>The incidence of thrombosis and risk factors in young patients with ET are different from that in general ET population. The thrombosis-predicting model specific for young patients with ET is useful for guiding therapeutic decisions.</p>

Role of Toll-like receptor 2 in primary immune thrombocytopenia / 中国实验血液学杂志

The aim of this study was to explore the role of Toll-like receptor (TLR) 2 in primary immune thrombocytopenia (ITP) by detecting TLR2 expression in the peripheral blood lymphocytes of patients with ITP and evaluating the role of TLR2 activation on inflammatory cytokine secretion. A total of 39 ITP patients and 21 normal controls were enrolled in this study. The expression of TLR2 was detected by real-time PCR and flow cytometry, and the concentration of IL-6 and TNF-α in culture supernatant of PBMNC treated with pam3CSK4 for 48 hours were detected by ELISA. The results showed that the expression of TLR2 mRNA in active ITP patients (3.561 ± 0.741) was significantly higher than that in normal controls (1.750 ± 0.314) (P < 0.05), but there was no statistically significant difference between remission ITP patients (2.333 ± 0.448) and normal controls (P > 0.05) . Flow cytometry analysis found that the TLR2 was not expressed on T and B cells, but expressed on all monocytes both from ITP patients and normal controls. Further activation experiment showed that TLR2 activation in vitro could induce the expression of IL-6 (1644 ± 634.0 vs 4111 ± 525.2 pg/ml) and TNF-α (75.37 ± 22.31 vs 326.0 ± 109.9 pg/ml) in PBMNC from ITP patients (both P < 0.05), but just could promote IL-6 expression in normal controls (2119 ± 636.9 vs 4671 ± 315.9 pg/ml)(P < 0.05). It is concluded that the expression of TLR2 mRNA is up-regulated in PBMNC of ITP patients, and this increased TLR2 maybe participate in ITP through inducing secretion of inflammatory cytokines.

Relationship between the expression of autoantibodies against platelet membrane glycoprotein and therapeutic effect in primary immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of specific anti- platelet glycoprotein autoantibodies GP II b/III a, GP I b/IX and GP I a/II a in primary immune thrombocytopenia (ITP), and to evaluate the relationship between the therapeutic effect and the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa.</p><p><b>METHODS</b>Anti-GPIIb/IIIa, GPIb/ IX and GP I a/II a antibodies were assayed by ELISA for patients with ITP. Total 442 patients in our hospital, who were retrospectively investigated from December 2010 to November 2012, were divided into newly diagnosed ITP, persistent and chronic ITP. The expression of specific anti- platelet glycoprotein antibody in each group was measured separately. The newly diagnosed ITP patients were treated with intravenous IgG (IVIG) and corticosteroids. The relationship between the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa and the complete response (CR) was studied.</p><p><b>RESULTS</b>Positive rates of anti- platelet glycoprotein antibodies were 59.09%, 26.97% and 37.35% respectively in newly diagnosed ITP, persistent and chronic ITP, the difference was statistical significant (P<0.05). In newly diagnosed ITP, positive rate of antibody against GPIIb/IIIa was 38.64%, double positive rate of antibodies against both GP II b/III a and GP I a/II a was 15.91%, there was statistical significance (P<0.05) compared with that of persistent and chronic ITP. The complete response (CR) rate in newly diagnosed ITP patients with positive antibody against GP II b/III a was 80.39% after treatment with IVIG and corticosteroids. There was statistical significance compared with that in patients having no antibodies (P<0.05).</p><p><b>CONCLUSION</b>The expression of antibodies against GP II b/III a and double positive for both GP II b/III a and GP I a/II a autoantibodies increased in newly diagnosed ITP patients. Patients with anti-GP II b/III a autoantibody had good response to medication with IVIG and corticosteroids.</p>

Clinical characteristics of 520 children with hemophilia in a single medical center / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical characteristics, diagnosis and treatment of pediatric hemophilia in single center over the decade.</p><p><b>METHODS</b>A retrospective study was conducted with 520 hemophilic children hospitalized in our medical center between January 2002 and December 2012.</p><p><b>RESULTS</b>All the patients were male including 438 hemophilia A (HA) and 82 hemophilia B (HB). There were significant differences in APTT between severe and mild- to moderate hemophilia (P<0.05). In pediatric HA and HB, delay time of diagnosis were 1.42 and 1.17 year, respectively. Children of 7-12 years were the largest population of visiting a doctor, and the spontaneous bleeding episode was the main cause. The most common hemorrhage site was soft tissue in early childhood, but joint was increasingly affected with age as children growth. All bleeding sites and frequencies were not associated with plasma factor level of patient (P>0.05). Knee and anKle were mainly involved in early child, while elbow and shoulder were involved increasingly in later childhood. Additionally, in HA and HB, inhibitor occurrence were 8.9%(19/214) and 12.8%(5/39), inducing 78.9%(15/19) and 40.0%(2/5) of high titer inhalator, and antiHCV-positive rate were 2.8%(11/397) and 2.5%(2/79), respectively.</p><p><b>CONCLUSION</b>Our data highlights that delay in diagnosis and blood-borne infections were significantly reduced over the decade, but the development of inhibitor still remains a major challenge with wide-scale usage of factor in replacement therapy.</p>

Effect of mesenchymal stem cells on human Th1 cells by flow cytometry / 中国实验血液学杂志

This study was aimed to investigate the effect of fetal bone marrow-derived mesenchymal stem cells (FBM-MSC) on the development of human Th1 cells. FBM-MSC were isolated, cultured and expanded in vitro. The cells were identified by their phenotype profiles and differential capacity. Human CD4(+) T cells from healthy donors were cultured alone or co-cultured with FBM-MSC (FBM-MSC/CD4). In these two cultures, the quantities of Th1 cells (interferon-γ(+)) were analyzed by flow cytometry. The results indicated that the immunophenotype and multilineage differentiation of FBM-MSC satisfied the generally accepted criteria. FBM-MSC played an inhibitory role in the development of Th1 cells. Flow cytometry analysis showed that the percentage of Th1 cells in FBM-MSC/CD4 was significantly lower than that in CD4(+) T cells cultured alone. The protein level of IFN-γ in FBM-MSC/CD4 detected by ELISA was also lower than that in CD4(+) T cells cultured alone. It was also demonstrated that the expression level of IL-6 in FBM-MSC/CD4 was much higher than that in CD4(+) T cells cultured alone or FBM-MSC. The neutralizing antibody of IL-6 could increase the quantities of Th1 cells and the expression levels of IFN-γ. It is concluded that FBM-MSC may play an inhibitory role in the development of human Th1 cells, and the IL-6 pathway may be one of mechanisms involved in the inhibitory role.

The activity levels and prevalence of deficiency of protein C, protein S and antithrombin in Chinese Han population / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the distribution and influence factors of protein C (PC), protein S (PS) and antithrombin (AT) activities and to determine the prevalence of their deficiencies in the Chinese Han healthy population.</p><p><b>METHODS</b>Healthy volunteers including blood donors and individuals for routine check-up were recruited from 4 Chinese medical centers. The plasma levels of PC, PS and AT activities were measured. The plasma levels of activities were measured by chromogenic substrate assay (AT and PC) and clotting assay (PS).</p><p><b>RESULTS</b>A total of 3493 healthy Chinese adults had been recruited in this study. Males had higher PS and PC activities than females, especially for PS (P < 0.01). PC activities increased with age in both sexes but decreased in men after 50 years old. There was no significant change with age were of PS in 50 years old, while there was a decline in males and a rise in females above 50 years old. AT tended to increase with age in women but decreased with age in men after 50 years old. Based on the age and gender, the general prevalence of PC, PS and AT deficiencies in the general Chinese Han population were 1.15%, 1.49% and 2.29%, respectively.</p><p><b>CONCLUSION</b>PC, PS and AT activities have correlation with age and gender in Chinese Han population. Reference range should be laid down and deficiencies should be identified</p>

A novel diagnostic measure of platelet-specific antibody in immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the platelet glycoprotein-specific antibodies in serum of thrombocytopenia patients and evaluate its diagnostic value for immune thrombocytopenia.</p><p><b>METHOD</b>Anti-GPIIb/IIIa, GPIb/IX and GPIa/IIa antibodies were assayed by ELISA kit (PAKUTO) in patients with thrombocytopenia.</p><p><b>RESULTS</b>The sensitivity and specificity of PAKAUTO in immune thrombocytopenia were 44.0% and 95.7%, respectively. The values of positive and negative predictions were 98.0% and 26.2%, respectively. Among those PAKAUTO positive patients, positive rates of GPIIb/IIIa, GPIa/IIa and GPIb/IX were 87%, 35% and 10%, respectively. The positive rate of patients not received immune suppressive agents (58.5%) was significantly higher than those received immune suppressive agents (26.9%) (P < 0.01). The positive rate of patients with platelet count ≤ 20 × 10(9)/L (51.6%) was significantly higher than those with platelet count > 20 × 10(9)/L (27.8%) (P < 0.01). The positive rate of patients with secondary immune thrombocytopenia (66.7%) was significantly higher than those with primary immune thrombocytopenia (41.7%) (P < 0.05).</p><p><b>CONCLUSION</b>The highly specific method (PAKAUTO) could effectively differentiate immune or non-immune thrombocytopenia and be applied to diagnosis of immune thrombocytopenia.</p>

Acquired factor V inhibitor: 3 cases report and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To better understand the acquired factor V (FV) inhibitors.</p><p><b>METHODS</b>The clinical features, laboratory manifestations, treatment options and prognosis of 3 cases were reported and related literature were reviewed.</p><p><b>RESULTS</b>All the 3 patients were older than 50 years without family history and related disease. Their clinical manifestations included spontaneously mucous bleeding, hematuria, epistaxis and encephalic bleeding. Laboratory test showed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT). The FV levels decreased and the presence of FV inhibitor was confirmed by Bethesda method. All patients were treated with glucocorticoid and immunosuppressive agents. The haemorrhages of two patients stopped but their coagulation test and FV level recovered slowly. One patient died from encephalic bleeding.</p><p><b>CONCLUSIONS</b>Acquired FV inhibitor is a rare coagulation disorder with variable clinical symptoms. Immunosuppressive agents are effective to eliminate the inhibitors. The prognosis of acquired FV inhibitors seemed to be strictly related to the basic disease.</p>

Study on activated protein C resistance and disordered coagulation in patients with myeloproliferative neoplasms / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the correlation of activated protein C (APC) resistance, coagulation factors and inhibitors abnormality and JAK2V617F mutation burden in patients with myeloproliferative neoplasms (MPN).</p><p><b>METHODS</b>The APC resistance was defined as the ratio of activated partial thromboplastin time (APTT) in the presence and absence of APC, i.e. APC sensitivity ratio (APCsr). Plasma protein C (PC), protein S (PS), prothrombin (FII), factor V (FV), factor VIII levels and CD11b expression on neutrophils were measured. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real time polymerase chain reaction (qRT-PCR).</p><p><b>RESULTS</b>Expression of CD11b on neutrophils was significantly elevated in MPN patients compared with that of the control group. APCsr, PS and FV levels were reduced in patients with MPN. The APCsr level was decreased mainly in patients with thrombosis and JAK2V617F mutant burden higher than 75%. APCsr was not only positively correlated with PS levels but also inversely correlated with JAK2V617F allele burden in JAK2V617F mutant gene carriers.</p><p><b>CONCLUSION</b>The neutrophil was activated and PS, FV level were reduced in MPN patients. The APCsr level was decreased and the occurrence of relatively acquired APC resistance was found in MPN patients with thrombosis. The APCsr is correlated with the PS level and JAK2V617F mutational furden.</p>

Efficacy and safety of two different low-dose rituximab regimens for Chinese adult patients with immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the efficacy of two different regimens of low doses rituximab for the treatment of adult patients with immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Fifty-one patients were enrolled in this study and was non-randomly assigned to receive 100 mg rituximab weekly for 4 weeks (group A, 31 cases) or a single dose of 375 mg/m2 rituximab (group B, 20 cases).</p><p><b>RESULTS</b>For group A: Overall and complete response (OR and CR) rates were 58% and 29% , respectively. In responders, the median time to response was 42 (10 -101) days, with a median follow-up time of 15 (10 - 16) months, 3 of 18 responders (17%) relapsed. For group B: OR and CR rates were 50% and 35% , respectively. In responders, the median time to response was 35 (18 - 108) days, with a median follow-up time of 13 (6 -17) months, 1 of 9 responders (11%) relapsed. No significant difference in the OR, CR, the relapse rate and relapse free survival was observed in patients between the two groups.</p><p><b>CONCLUSION</b>The low dose rituximab regimen (100 mg weekly for 4 weeks or a single close of 375 mg/m2) may be a useful alternative therapy in patients with ITP.</p>

The role of Th17 cells in immune thrombocytopenia mice models / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of Th17 cells in immune thrombocytopenia (ITP) mice model.</p><p><b>METHOD</b>ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD41 antibody (MWReg30) into BALB/c mice, the mRNA expressions of Th17 cell associated transcription factors and cytokines in peripheral blood and spleen mononuclear cells were measured by real-time PCR, and the proportion of Th17 cells by FCM analysis.</p><p><b>RESULTS</b>The percentage of Th17 cell was significantly elevated in ITP mice both in splenocyte and peripheral blood as compared with that in normal controls (P<0.01). ITP mice had elevated mRNA expressions of IL-17F, IL-17A and IL-6 in splenocyte (P<0.05), and of IL-21 in peripheral blood (P<0.05). There was a positive correlation between IL-17A and IL-17F (r = 0.934, P = 0.000), and between IL-17A/IL-17F and IL-6 (r = 0.598, P = 0.001; r = 0. 619, P = 0.000).</p><p><b>CONCLUSIONS</b>Th17 cell might play an important role in the pathogenesis of ITP, at least involving in the clearance of platelets.</p>

Interferon-alpha-2b induces molecular responses of patients with polycythemia vera and its post-polycythemic myelofibrosis / 中国实验血液学杂志

To evaluate the efficacy and safety of interferon-alpha-2b (IFN-α-2b) in polycythemia vera patients(PV patient) with or without post-polycythemic myelofibrosis (post-PV MF), 30 patients with mutated JAK2V617F were enrolled in this study, from which 29 patients were evaluable. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real-time polymerase chain reaction (RT-PCR) before and after treatment with IFN-α-2b. The correlation of V617F allele burden with the major clinical outcomes was studied. Adverse effects appeared in patients was observed. The results showed that the median follow-up was 24 (12 - 42) months for 29 evaluable patients. Complete hematologic response was achieved in 10%, 48%, 72% and 78% of patients after treatment for 6, 12, 24 and 36 months respectively. The detection of V617F allele burden revealed that the molecular remission of patients (V617F%) was achieved in 41%, 76%, 89% and 89% after treatment for 6, 12, 24 and 36 months respectively. Molecular complete remission (JAK2V617F undetectable) was achieved in 4 patients, lasted from 6 to 12 months after IFN-α-2b discontinuation. The decrease of V617F% in patients with post-PV MF was significantly higher than that in patients without post-PV MF (53 ± 18% vs 32 ± 22%, respectively; p = 0.031) after treatment for 12 months. PV patients had a good tolerance to IFN-α-2b. It is concluded that IFN-α-2b can decrease the mutated V617F allele burden. Patients with PV, especially with post-PV MF, can achieve molecular remission after treatment with IFN-α-2b.

Immunoregulatory function of interleukin-6 on human Th17 cells / 中国实验血液学杂志

The aim of this study was to explore the regulatory function of interleukin-6(IL-6) on human Th17 cells. Human peripheral blood CD4(+) T cells were purified from healthy donors by anti-CD4 monoclonal antibody (mAb) conjugated microbeads. The experiment was divided into 2 groups. Test group in which CD4(+) T cells (1 × 10(6)/ml) were stimulated by human recombined IL-6 (20 ng/ml) for 4 days; control group in which CD4(+) T cells did not stimulated by IL-6. The concentrations of IL-17 protein in the supernatants were assayed by enzyme-linked immunosorbent assay (ELISA), and quantity of Th17 cells were detected by flow cytometry. The results showed that as compared to control group, IL-17 protein level in the supernatants of CD4(+) T cells significantly increased in IL-6 stimulated group: (337.05 ± 189.09 pg/ml; vs 15.07 ± 12.70 pg/ml) (p < 0.05). Furthermore, the percentage of Th17 cells in cultures of CD4(+) T cells stimulated by IL-6 was significantly higher than that in control group (4.05% ± 0.30% vs. 2.81% ± 0.44%)(p < 0.01). It is concluded that IL-6 promotes the expansion of Th17 cells in vitro.

Clinical and laboratory investigation of patients with hematologic malignancies harboring der(1;7)(q10;p10) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical and laboratory characteristics of patients with various hematological malignancies harboring der(1;7)(q10;p10).</p><p><b>METHODS</b>Bone marrow samples were collected and undergone short-time unstimulated culture and R-banding, and karyotyped by conventional cytogenetic assay (CCA). Megalokaryocytes were detected by streptavidin-AKP (SAP). Retrospective analyses including the clinical and laboratory data were performed.</p><p><b>RESULTS</b>Nineteen of the 21 patients were male. Most of the patients are of older age. Thirteen cases (61.9%) were der(1;7)(q10;p10) without additional aberrations, 8(38.1%) patients had additional aberrations. Sixteen out of the 18 cases (88.9%) who underwent SAP analysis had diminutive megalokaryocyte, and lymphoid megalokaryocyte was found in 10 cases (55.6%). The der(1;7) patients manifested poor response to treatment.</p><p><b>CONCLUSION</b>The der(1;7) patients demonstrated distinct male predominance, older age at diagnosis, and some clinically distinctive features. These patients showed poor prognosis. The cytogenetic abnormality, i.e., der(1;7)(q10;p10), can be used as a prognostic indicator.</p>

Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase.</p><p><b>METHODS</b>The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR.</p><p><b>RESULTS</b>The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes.</p><p><b>CONCLUSION</b>Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.</p>