OBJECTIVETo investigate the quantity and function of circulating dendritic cells (DC) in patients with chronic idiopathic thrombocytopenic purpura (ITP).

METHODSHigh dose dexamethasone (HD-DXM) at a dose of 40 mg orally per day for four consecutive days was the initial treatment for chronic ITP patients. Flow cytometry was used to analyze the number of myeloid DC (mDC), plasma cytoid DC (pDC) and CD4+FOXP3+ T cells in patients before and after the treatment, meanwhile the co-stimulatory molecules on circulating DCs were assayed as well. Monocyte-derived DCs and CD4+ T cells were co-cultured with autologous or allogeneic normal fresh platelets and after 6 days of incubation H-TdR was used to assay the proliferation of CD4+ T cells.

RESULTSThe absolute numbers of circulating mDC and pDC were not significantly different between pre-treatment patients and healthy controls (P > 0.05 and P >0.05). However, percentage of CD4+ FOXP3+ T cells was decreased (P < 0.01), and their percentage was inversely correlated with the number of pDC and mDC (r = -0.396, P =0.045 and r = -0.410, P =0.037). The initial response rate to HD-DXM was 92.3%. After 4-days treatment, CD4 FOXP3+ Treg cells increased (P <0.01) while pDCs decreased (P <0.01). Although mDCs increased after HD-DXM (P <0.05), their CD11c expression level was decreased (P < 0.01), the mean fluorescence intensity (MFI) decreased from 340 +/- 30 before treatment to 199 +/- 21 after treatment. The inverse correlation between pDCs and CD4+ FOXP3+ Treg cells remained (r= -0.524, P =0.006) while that between mDCs and Treg cells disappeared (r = - 0.360, P =0.071). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P <0.05), while the proportions of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P > 0.05). DCs from chronic ITP patients co-cultured with autologous or allogeneic platelets were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to those derived from healthy donors (P < 0.05 and P <0.05).

CONCLUSIONDCs may play a role in the pathogenesis of chronic ITP in relation with CD4+CD25+ Treg cells.

Adult ; CD4-Positive T-Lymphocytes ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology

Objective To investigate the effect of all-trans retinoic acid (ATRA) on expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in glioma stem cells (GSCs). Methods GSCs were isolated from human glioblastoma cell line U87 and identified by detecting the expressions of CD133 and nestin with immunofluorescence staining. The obtained GSCs were divided into control group,empty vector group (cultured with dimethyl sulfoxide [DMSO]) and ATRA treatment group (cultured with 10 nmool/L ATRA).After 10 d of differentiation; the proliferation of the treated GSCs was evaluated using CCK8 assay; the expressions of glial fibrillary acidic protein (GFAP),β-tubulin Ⅲ and galactoeerebroside (GralC) in the cells were detected by immunofluorescence.VEGF and bFGF levels in cultured supernatant were measured by ELISA; the mRNA expressions of VEGF and bFGF were detected by RT-PCR. Results The target antibodies of neural stem cells (NSCs), CD133 and nestin,positively expressed in the GSCs; differentiated GSCs can differentiate several kinds of homologous daughter cells,which expressed the cell markers of astrocytes,neurons and oligodendrocytes: GFAP, β-tubulin LⅢ and GalC, respectively. The percentage of GFAP-positive differentiated GSC s in the ATRA treatment group was significantly higher as compared with that in the other 2 groups after 10 d of differentiation (P＜0.05); the speed of proliferation of GSCs in ATRA treatment group was obviously slower than that in the other 2 groups 3-7 d after differentiation (P＜0.05).The VEGF and bFGF levels and the mRNA expression levels of VEGF and bFGF in GSCs of the ATRA treatment group 24 h after differentiation were also significantly lower than those in the other 2 groups (P＜0.05). Conclusion ATRA can induce the differentiation of GSCs and inhibit its proliferation.It may exerts its anti-glioblastoma effect through the VEGF and bFGF signaling pathways.

BACKGROUNDEndogenous hydrogen sulfide (H(2)S) plays an important role in hypertension. The aim of this study was to investigate the role of erythrocyte and serum H(2)S in patients with untreated essential hypertension.

METHODSWe recruited 62 patients (age 22 - 74 years) with untreated prehypertension or hypertension, and 64 normotensive subjects (age 18 - 64 years). We assessed the 3-mercaptopyruvate sulphurtransferase (MPST) protein expression in erythrocytes and measured the H(2)S production from erythrocytes and serum H(2)S levels, then analyzed the association of erythrocytic or serum H(2)S content and blood pressure or cardiovascular risk factors (e.g., age, body mass index (BMI) and dyslipidemia). A stepwise regression analysis was used to evaluate the possible relationship of erythrocytic H(2)S in hypertension.

RESULTSIn hypertensive patients, erythrocyte H(2)S production ((111.04 ± 29.20) nmol/min per 10(8) erythrocytes) was higher than that in controls ((78.85 ± 19.38) nmol/min per 10(8) erythrocytes), and serum H(2)S was also higher. The erythrocytic H(2)S production was associated with increased systolic blood pressure (sBP), diastolic blood pressure (dBP), age, BMI, level of C-reactive protein (CRP), as well as triglycerides (TG) and high density lipoprotein cholesterol (HDL-C). Serum H(2)S was not associated with age or CRP. Stepwise regression analysis showed that erythrocytic H(2)S production was correlated with sBP, TG, HDL-C, low density lipoprotein cholesterol (LDL-C) and blood urea nitrogen (BUN) and serum H(2)S was correlated with dBP and TG. Results of receiver-operating characteristic curve analysis suggested that erythrocytic H(2)S production was a more sensitive predictor of hypertension development than serum H(2)S.

CONCLUSIONErythrocytic or serum H(2)S production is sensitive predictor of hypertension.

Adolescent ; Adult ; Aged ; Erythrocytes ; metabolism ; Female ; Humans ; Hydrogen Sulfide ; blood ; Hypertension ; blood ; metabolism ; Male ; Middle Aged ; Young Adult

Objective: To clarify the characteristics of the A20 regulatory changes by analyzing mutations in the non-coding region of the A20 gene in patients with T-cell lymphoma leukemia (T-LCL) . Methods: PCR and nucleotide sequence analysis were used to detect mutations in the non-coding region of the A20 gene, and DNA samples from PBMCs of 52 cases of T-LCL and 99 healthy controls. Results: A missense mutation (c.-672T>G) was detected in the A20 gene promoter from one T-LCL patient, which has been registered as a SNP (rs139054966) in gene bank. Meanwhile, a new mutation was detected in the 3' UTR mRNA (3916 (C>G) ) . These two mutations were absent in other T-LCL samples and controls. Conclusion: The rs139054966 (c.-672T>G) and 3916 (C>G) mutations in the A20 gene were detected in T-LCL patients for the first time. There was also rs139054966 located on the binding region of the transcription factor P53, and its significance remained to be further clarified.
3' Untranslated Regions ; Humans ; Leukemia ; Lymphoma, T-Cell ; Mutation ; Promoter Regions, Genetic

OBJECTIVETo evaluate the in-hospital outcome of patients with calcified chronic total occlusion (CTO) lesion underwent percutaneous coronary intervention (PCI).

METHODSThe clinic and lesion characteristics as well as acute PCI outcome were analyzed in 726 patients with calcified CTO [624 detected by coronary angiography (CAG) and 102 detected by intravascular ultrasound (IVUS)] and received PCI therapy from June 1995 to February 2007 in our department.

RESULTSThere were 728 diseased vessels with 732 lesions in these patients. Total procedure success rate (80.6% vs. 89.2%, P < 0.05) and the lesion success rate (80.2% vs. 88.2%, P < 0.05) were significantly lower in calcified CTO detected by CAG compared to that detected by IVUS. The causes of procedure failures in CAG detected patients were as follows: 87 guide failure, 21 balloon failure, 8 procedure related complications and 5 low TIMI blood flow (grade 2) at the end of PCI procedure. The causes of procedure failures in IVUS detected patients were as follows: 7 guide wire failure, 2 balloon failure, 1 procedure related complications and 1 patient with low TIMI blood flow (grade 2). The in-hospital major adverse cardiac events (MACE) rate was 1.1% in CAG detected calcified CTO and 1.0% in IVUS detected calcified CTO (P > 0.05).

CONCLUSIONPCI therapy resulted in satisfactory procedure success rate and in-hospital outcome for patients with calcified CTO and IVUS is helpful for further increasing the PCI procedure success rate in patients with calcified CTO.

Adult ; Aged ; Aged, 80 and over ; Angioplasty, Balloon, Coronary ; Calcinosis ; therapy ; Coronary Angiography ; Coronary Artery Disease ; pathology ; therapy ; Coronary Occlusion ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Ultrasonography, Interventional

Hemophilia B (HB) is a recessive X-linked inherited disorder, the pathogenesis of HB is deficiency or functional abnormalities of coagulation factor IX, which is caused by F9 gene mutations. To explore the mechanism of its molecular pathology, 40 patients with HB were studied with polymerase chain reaction (PCR) and direct sequencing. The diagnosis of HB patients were based on clinical manifestation and deficient factor IX activity in plasma. DNA was routinely extracted from peripheral blood cells of the patients and their relatives, all the 8 exons and their flanking boundaries were amplified by PCR, and the PCR products were screened by direct sequencing. Mutations which were found in study need to exclude polymorphism. The results showed that 34 mutations were confirmed in 40 HB patients, including 6 nonsense mutations, 24 missense mutations, 2 splice site mutations and 2 frame mutations for 1 or 2 nucleotide insertion. After retrieved, 4 missense mutations and 1 frameshift mutation were found for the first time. Among the 34 mutations, 2 mutations in signal peptide, 7 mutations in propeptide and gla domain, 7 mutations in epidermal growth factor-like domain, 3 mutations in activation domain, 15 mutations in serine protease or catalytic domain. It is concluded that gene analysis can directly explain molecular mechanism of hemophilia B and also provides the foundation for further studies to the function of coagulation factor IX. There is obvious heterogeneity in F9 gene mutation and missense mutation is still the main way of mutation, which are closely related to clinical features. DNA sequencing and linkage analysis are efficient methods for HB carriers and prenatal gene diagnosis.
Base Sequence ; DNA Mutational Analysis ; Factor IX ; genetics ; Hemophilia B ; diagnosis ; genetics ; Humans ; Male

OBJECTIVETo investigate the role of CD4+ CD25+ regulatory T (Treg) cells in patients with ITP.

METHODSFlow cytometry was used to examine the number of CD4+ CD25+, CD4+ CD25 high, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ T cells. The level of Foxp3 mRNA expression was analyzed by realtime quantitative reverse transcriptase polymerase chain reaction (RT-PCR) . CD4+ CD25 high T cells was cocultured with CD4+ CD25 - T cells from patients or controls for assessing the regulatory properties of CD4+ CD25 Treg cells.

RESULTSThe proportion of CD4+ CD25+ T cells in the peripheral blood of patients with ITP [(15.64 +/- 5.82) %] was significantly higher than that in normal control group [(9.30 +/- 3.95)%] (P <0.01). There was no significant difference in the percentages of CD4+ CD25 high T cells between ITP patients and controls [(1.53 +/- 0.66)% versus (1.36 +/- 0.55)% (P = 0.317)]. But the number of CD4 Foxp 3+ T cells and CD4 + CD25+ Foxp3+ T cells in patients were significantly lower than that in control group (P <0.01). The expression of Foxp3 mRNA reduced (P < 0.01) and the suppressive activity of CD4+ CD25 high T cells is lower than that of healthy controls (P <0.01).

CONCLUSIONIn patients with ITP, both the number and immuno-regulative function of CD4+ CD25+ Treg cells are reduced.

Adolescent ; Adult ; Aged ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; metabolism ; RNA, Messenger ; metabolism ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVEThis study was designed to evaluate the efficacy and toxicity of modified BFM-90 regimen originated from Germany authors in the treatment of Chinese childhood and adolescent lymphoblastic lymphoma.

METHODSThirty-six untreated lymphoblastic lymphoma patients aged from 3 to 18 years were included, with 1 patient in stage II , 9 in stage III and 26 in stage IV. Of these 36 patients, 28 (77.7%) were diagnosed as T cell phenotype, 26 (72. 2%) were found to have mediastinal mass, 21 (58. 3%) had bone marrow involvement. All patients received chemotherapy of modified BFM-90 regimen consisting of induction remission, central nerve system prophylaxis, re-induction remission and maintenance therapy. Total treatment duration was two years. The difference from standard BFM-90 is that we omitted cranial radiotherapy but gave regular high dose methotrexate (MTX) iv infusion and intrathecal MTX therapy during maintenance therapy period. Kaplan-Meier method was used to evaluate survival rate.

RESULTSOf 36 patients, 32 (88%) achieved complete remission (CR) , 1 (2. 7%) partial remission (PR) with an overall response rate of 90.7%. One patient had disease progression ( DP). Two patients received autologous stem cell transplantation at CR1, and two patients received radiotherapy to mediastinum. Totally, 5 patients relapsed, while 2 of them were still alive after salvage chemotherapy. The other 3 died of tumor progression. Two patients died during induction remission, 1 of fungal septicemia, the other of cerebral hemorrhage; one PR and one DP patient died of disease, therefore, totally 7 patients died at last. Median follow-up time was 28 months. Overall three-year survival rate was 78. 3%. The major toxicity was myelosuppression.

CONCLUSIONModified BFM-90 protocol can improve the efficacy and survival of Chinese childhood and adolescent lymphoblastic lymphoma with tolerable toxicity. However, this modified protocol should only be used in experienced cancer center or hematological unit.

Adolescent ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Asian Continental Ancestry Group ; Asparaginase ; therapeutic use ; Child ; Child, Preschool ; China ; Cyclophosphamide ; therapeutic use ; Cytarabine ; therapeutic use ; Daunorubicin ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Kaplan-Meier Estimate ; Male ; Mercaptopurine ; therapeutic use ; Methotrexate ; therapeutic use ; Neoplasm Recurrence, Local ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; ethnology ; Prednisone ; therapeutic use ; Remission Induction ; Treatment Outcome ; Vincristine ; therapeutic use

Objective To study the effect of transforming growth factor-β (TGF-β) signaling pathway blockage on vasculogenic mimicry (VM) in gliomas and explore its possible mechanism.Methods Three-dimentional culture was performed on the glioma cell lines U251 and SHG44; the effects of U251 culture supematant and TGF-β on VM formation of SHG44 cells were observed; the capability of VM formation of U251 and SHG44 cells after being treated with 0 μg/mL (PBS group),15 μg/mL TGF-β neutralizing antibody (Ab15 group) and 30 μg/mL TGF-β neutralizing antibody (Ab30 group) was evaluated.ELISA was used to detect the concentrations of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the supematant of U251 cells from the blank group,PBS group,Ab 15 group and Ab30 group and the concentrations of VEGF and PDGF in the supernatant of SHG44 cells from the blank group,TGF-β treatment group,PBS group,Ab15 group and Ab30 group.Results VM was formed in the U251 cells while not in the SHG44 cells during the three-dimentional culture; SHG44 cells could only gather into colonies of different sizes.U251 culture supernatant could induce SHG44 cells to form VM,enjoying the most obvious effect at 24-48 h of culture; TGF-β could not induce SHG44 cells to form VM.The number of U251 cells annulation in PBS group,Ab15 group and Ab30 group decreased in sequence with significant difference (P＜0.05).The number of U251 cells armulation in SHG44 cells cultured in U251 culture supematant from the PBS group,Ab15 group and Ab30 group decreased in sequence after being added TGF-β antibody with significant difference (P＜0.05).As compared with that in the blank group and PBS group,significant decrease of VEGF and PDGF concentrations in the U251 cells from Ab15 group and Ab30 group was noted (P＜0.05); as compared with that in the blank group and TGF-β treatment group,significant increase of VEGF and PDGF concentrations in the SHG44 cells from PBS group,Ab15 group and Ab30 group was noted.Conclusion Blockage of TGF-β signaling pathways inhibits VM in glioma,and it maybe probably due to the decrease of VEGF and PDGF expressions..

BACKGROUNDA generally accepted guideline ("41 rules") published by the International Consensus Group for Hematology Review (ICGHR) can not be suitable for all the laboratories because the facility type, laboratory requirements, sample volume, review rate, turn around time, instrument model and characters etc. are quite different from each other, which may cause a higher workload for microscopy review or lead to false or misleading results. Therefore, we decided to develop the personalized review criteria for 4 series of hematology analyzers in the same hospital, and describe all the implement procedures in detail.

METHODSThe total 1770 blood samples were collected from Peking Union Medical College Hospital. Referring to the suggested criteria by international consensus group for hematology review ("41 rules"), the personalized review criteria for 4 series of hematology analyzers including Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i were established and validated by adjusting the rules in order to reduce the false positive rate and keep the false negative acceptable by clinical.

RESULTSUsing the "41 rules", high review rates of 37.94%, 35.56%, 33.44% and 37.94% were got respectively in Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i. Three false positive rules mainly were observed in all of 4 analyzers: white blood cell < 3 × 10(9)/L or >30 × 10(9)/L, platelet < 100 × 10(9)/L or > 1000 × 10(9)/L and immature granulocyte. Specialized rules were observed in different series of analyzers, atypical/variant lymphs flag were found mainly in Sysmex XE-2100, Aniso-RBC were found mainly in Sysmex XT-1800i, flag of "immature granulocyte" mainly in Sysmex XS-800i, Micro-RBC, Macro-RBC and Aniso-RBC mainly in Siemens Advia 2120. Rules of immature granulocyte, blast, and NRBC flag would be mainly triggered by hematology malignant tumor. We could not delete these rules due to the risk of false negative of serious disease, other rules were deleted or revised. After continually optimizing to the rules, we finalized the criteria suitable for Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i in our laboratory. The false negative rates were 2.94%, 2.86%, 3.10% and 2.78%, the review rates were 31.07%, 30.00%, 30.01% and 30.09%, and there was no hematology malignant tumor missed. Validated by 547 samples, the false negative rates of our optimized rules were 0.37%, 0.55%, 0.55%, and 0.91% respectively.

CONCLUSIONThe criteria can be based on the criteria established by International Consensus Group for Hematology Review but must be optimized according to the different requirements.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; China ; Female ; Hematologic Tests ; standards ; Hospitals ; standards ; Humans ; Male ; Middle Aged ; Young Adult