Relationship between TET2 Gene SNP rs3733609 C/T and JAK2V617F Allele Burden in Patients with Myeloproliferative Neoplasms / 中国实验血液学杂志

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.

Effect of Icaritin on Proliferation and Apoptosis of THP-1 Cell and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of icaritin on the proliferation and apoptosis of THP-1 cells and its mechanism.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Icaritin can inhibit proliferation and induce apoptosis of THP-1 in vitro, Icaritin may induce apoptosis in THP-1 cells through the mitochondrial pathway.</p>

Thalidomide Effects in Patients with Hereditary Hemorrhagic Telangiectasia During Therapeutic Treatment and in Fli-EGFP Transgenic Zebrafish Model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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-β3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment.</p><p><b>RESULTS</b>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-β3 mRNA expression of five patients was lower after thalidomide therapy. The plasma VEGF protein expression was down-regulated in HHT patients.</p><p><b>CONCLUSIONS</b>Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-β3 and VEGF in HHT patients. It also leads to vascular remodeling in the zebrafish model.</p>

Effect of glucocorticoid on dendritic cells in children with chronic immune thrombocytopenia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the change in dendritic cells (DCs) in children with chronic immune thrombocytopenia (cITP) and the effect of glucocorticoid on DCs in children with cITP.</p><p><b>METHODS</b>Fifteen children with cITP and 20 healthy controls were included in the study. Flow cytometry was used to measure the DC subsets count in the 15 children with cITP before and after glucocorticoid treatment as well as the corresponding values in the 20 healthy controls. The DCs derived from peripheral blood monocytes in children with cITP were cultured in vitro and collected, and their immunophenotypes were determined by flow cytometry.</p><p><b>RESULTS</b>Before glucocorticoid treatment, the children with cITP showed no notable change in the absolute count of myeloid DCs (mDCs) but showed decreased absolute count of plasmacytoid DCs (pDCs) and increased mDC/pDC ratio compared with the healthy controls (P<0.05). After glucocorticoid treatment, the children with cITP demonstrated increased absolute count of pDCs and decreased absolute count of mDCs and mDC/pDC ratio compared with before treatment (P<0.05). Before glucocorticoid treatment, the children with cITP had significantly higher positive rates of HLA-DR, CD80, CD83 and CD86 on peripheral blood DCs than the healthy controls (P<0.01). All the positive rates were significantly decreased after glucocorticoid treatment (P<0.01), so that there was no significant difference from the healthy controls (P>0.05).</p><p><b>CONCLUSIONS</b>Disproportion and functional disturbance of DC subsets is associated with the pathogenesis of cITP in children. Glucocorticoid can strengthen the immunosuppression of DCs in children with cITP, which may contribute to the effectiveness of glucocorticoid as a treatment.</p>

Screen of phosphopeptide specific for acute leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen phosphopeptide specific for acute leukemia.</p><p><b>METHODS</b>Mononuclear cells from bone marrow were collected from 16 newly diagnosed acute lymphoblastic leukemia (ALL) and 20 acute myeloid leukemia (AML) patients. Peptides were extracted and purified, analyzed by immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry (LC-MS).</p><p><b>RESULTS</b>(1) Non-receptor tyrosine kinase family members Fyn, Yes, Src widely expressed in acute leukemia; (2) Some phosphopeptides, including non-receptor tyrosine kinase family members Abl/iso1 and Abl, non-receptor Ser/Thr protein kinase family members Bcr, JNK2, JNK2 iso2, Adaptor/scaffold members Cas-L, Cbl, CrkL CENTD1 (Centaurin delta1) ZO2, transcriptor GFR-1 and phosphatase SHIP-2 were detected in Ph positive ALL, but not in other kinds of ALL. (3) Hck, Lyn and Fgr selectively expressed in AML (except AML-M(3)).</p><p><b>CONCLUSION</b>Some phosphopeptides were specific for ALL and AML, and may be useful for diagnosis and therapy of acute leukemia.</p>

Effect of BAFF/APRIL mRNA expression induced by glucocorticoid and bortezomib in multiple myeloma cells in vitro / 中国实验血液学杂志

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.

Changes of HLA-DR15 and immunoglobulin, T lymphocyte subsets in patients with aplastic anemia, myelodysplastic syndrome and their significance / 中国实验血液学杂志

The objective of this study was to detect the expression frequency of HLA-DR15 in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS), to investigate the relation of expression frequency with diseases and to analyze the relationship between immunoglobulin, T lymphocyte subsets and HLA-DR15. HLA-DR15 expression was detected by PCR-SSP; immunoglobulin was detected by immune turbidimetry; T cell subsets were detected by flow cytometry. The results showed that the expression rates of HLA-DR15 in AA and MDS as well as normal control groups were 78.6%, 63.2% and 24.6% respectively. The difference between AA, MDS and the normal control groups was statistically significant (p < 0.01). OR (odds ratios) values of AA and MDS groups were 11.262, 4.710 respectively. Compared with normal control group, expression rate of HLA-DR15 in hematologic malignancy group was not significantly different. The immunoglobulin level and abnormal T cell subsets in AA and MDS groups were statistically different between HLA-DR15 positive and negative groups (p > 0.05). It is concluded that the frequency of HLA-DR15 antigen in AA and MDS patients is significantly higher than that in normal control and hematologic malignancy group. OR value>1 showed a positive correlation between the diseases and HLA-DR15. HLA-DR15 is a susceptible gene in AA and MDS. The abnormalities of immunoglobulin level and ratios of T cell subsets in AA and MDS are common, but are not associated significantly with the expression of HLA-DR15.

Screening for drug resistance related microRNAs in K562 and K562/A02 cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between microRNA and drug resistance in leukemia treatment by screening and identifying the microRNAs which differentially express in K562 cell line and its adriamycin resistant cells-K562/A02 cell line.</p><p><b>METHODS</b>The drug resistance potency of K562/A02 cells was evaluated by MTT assay. P-gp expression of K562 and K562/A02 cells were detected by flow cytometry (FCM). The differentially expressed microRNAs in K562 and K562/A02 cells were analyzed by microarray technique and Real Time RT-PCR.</p><p><b>RESULTS</b>The resistance to adriamycin (ADM) of K562/A02 cells was 180 fold greater than that of K562 cells. P-gp expression rate of K562 and K562/A02 cells was 0.2% and 86%, respectively. Twenty-two microRNAs expressed differentially in K562 and K562/A02 cells (P < 0.01). As compared to K562 cells, expressions of miR-221, miR-155 and miR-451 were up-regulated by more than two fold, while expression of miR-98, miR-181a, let-7f, let-7g, miR-424 and miR-563 down-regulated by more than two fold in K562/A02 cells. The results of real time RT-PCR were consistent with that of microarray. Of note, differential expressions of miR-451, miR-155, miR-221, let-7f and miR-424 were remarkable.</p><p><b>CONCLUSION</b>K562/A02 cells show a different microRNA expression profile as compared to its parental K562 cells, suggesting microRNAs including miR-221, miR-155, miR-451, let-7f and miR-424 may be involved in the mechanism of drug resistance in leukemia. These differentially expressed microRNAs provide potential novel targets for overcoming drug-resistance.</p>

The frequency of JAK2 V617F mutation, expression level of phosphorylated JAK/STATs proteins and their clinical significance in myeloproliferative disorders patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features.</p><p><b>METHODS</b>The JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation.</p><p><b>RESULTS</b>1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group.</p><p><b>CONCLUSIONS</b>MPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.</p>

A non-familial May-Hegglin anomaly accompanying with MYH9 gene R1933X mutation and I1626V polymorphism / 中华血液学杂志

<p><b>OBJECTIVES</b>To identify the nonmuscle myosin heavy chain 9 (MYH9) gene mutation site in a May-Hegglin anomaly(MHA) patient, and to analyze the genotype of her relatives to exclude the inherit correlation between the proband and her family members.</p><p><b>METHODS</b>Inclusion bodies in neutrophils of the proband were examined by transmission electron microscope, and giant platelets by scanning electron microscope. The mutation "hot spot" on the MYH9 gene of the proband and her family members was amplified with polymerase chain reaction(PCR), and then sequenced in both directions to identify the mutant site.</p><p><b>RESULTS</b>(1) The proband manifested with the typical MHA triad of giant platelet, thrombocytopenia and Dohle-like inclusion bodies in neutrophil. However, all of the proband's family members had no such anomaly. (2) Transmission electron microscope and scanning electron microscope confirmed that giant platelets and neutrophils inclusion bodies existed in the proband peripheral blood cells. (3) There was a missense mutation 5797 C-->T in the exon 40 of MYH9 gene which led to Arg changing into termination codon (Arg1933 stop). The proband also had a heterozygous mutation 4876A-->G in exon 33. There was no abnormal finding in the sites mentioned above in her mother, while her father carried the homozygous 4876A-->G mutation.</p><p><b>CONCLUSIONS</b>This MHA case is a sporadic one, in whose family a mode for autosomal dominant inheritance can not be established. The 5797C-->T substitution in MYH9 gene is a pathogenic mutation, however, 4876A-->G is simply a polymorphism.</p>

Molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin / 中南大学学报(医学版)

OBJECTIVE@#To determine the molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin.@*METHODS@#Effects of ADR and puerarin on NF-kappaB activity of K562,K562/AO2 were tested by immunofluorescence. The expression of survivin of K562,K562/AO2 was examined by immunocytochemistry. The p-gp expression was detected by flow cytometry.@*RESULTS@#The NF-kappaB activity of K562 was significantly higher than that of K562/AO2. The NF-kappaB activity of K562 treated by ADR was significantly higher than untreated. The NF-kappaB activity of K562 which was pretreated by puerarin and then treated by ADR was much lower than that treated by ADR alone. The NF-kappaB activity of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin.The p-gp and survivin expression of K562/AO2 was significantly higher than K562. The p-gp and survivin expression of K562 treated by ADR was higher than that untreated by ADR. But the p-gp and survivin expression of K562 which was pretreated by puerarin and then treated by ADR was much lower than that not pretreated by puerarin.The p-gp and survivin expression of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin. The expression was negatively correlated to the duration of intervention. The inhibition effect demonstrated time dependence.@*CONCLUSION@#The activation of NF-kappaB can increase the expression of p-gp and survivin, which may be part of the molecular mechanism of multi-drug resistance of K562. Puerarin can prevent and stop the multi-drug resistance in K562 and reverse the multi-drug resistance of K562/AO2 to ADR by inhibiting the activity of NF-kappaB and the expression of p-gp and survivin.

Expression characteristics of heat shock protein 70 and pim-1 gene in bone marrow mononuclear cells from leukemia patients and their clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression characteristics of HSP70 protein/mRNA, pim-1 mRNA in bone marrow mononuclear cells from leukemia patients, and to clarify whether these changes are related to leukemia type, tumor burden of leukemia, therapeutic reaction and prognosis. HSP70 mRNA and pim-1 mRNA in BMMNCs were detected with semi-quantitative RT-PCR in 40 leukemia patients and 10 controls. HSP70 protein in BMMNCs was assayed with Western blot in 34 leukemia patients and 10 controls. Relation of HSP70 and pim-1 expression with leukemia classification, the degree of tumor burden and therapeutic reaction were analyzed. The results showed that the BMMNCs from both leukemia patients and controls expressed HSP70 protein/mRNA. The mean ODR value of HSP70 mRNA in BMMNCs from leukemia patients was significantly higher than that of the controls; the mean ODR value of HSP70 protein/mRNA in acute myeloid leukemia and chronic myeloid leukemia patients both were significantly higher than that of acute lymphocytic leukemia patients; the mean ODR value of HSP70 protein/mRNA in acute leukemia patients with high-degree tumor burden was higher than that of the patients with low-degree tumor burden; the mean ODR value of HSP70 protein/mRNA in the patients after chemotherapy was significantly higher than that of the patients before chemotherapy; the BMMNCs from both leukemia patients and controls expressed pim-1 mRNA. The mean ODR value of pim-1 mRNA in BMMNCs from leukemia patients was significantly higher than that of the controls; the mean ODR value of pim-1 mRNA in BMMNCs for Acute lymphocytic leukemia patients was significantly higher than that of the patients suffered from acute myeloid leukemia and chronic granulocytic leukemia; there was a positive relationship between pim-1 mRNA and HSP70 mRNA expressions in leukemia patients (p < 0.05). It is concluded that there are high expressions of HSP70 and pim-1 in leukemia and their positive correlation is shown. The over-expressions of HSP70 and pim-1 protein/mRNA are related to tumor burden in leukemia patients.

In vitro expansion of the adult human bone marrow mesenchymal stem cells for clinic application in HSCT / 中国实验血液学杂志

This study was aimed to investigate the efficiency of 4 different culture media for in vitro culture and expanding adult human bone marrow mesenchymal stem cells (ahBM-MSCs) so as to establish a protocol of culturing and expanding hBM-MSCs and provide exprimental basis for hematopoietic blood stem cell transplantation combined with BM-MSCs. BM-MSCs were obtained from 16 fresh adult human bone marrow aspirate by gradient centrifugation with Ficoll Paque, then cultured in DMEM/F12 with 10% umbilical cord blood serum, 10% fetal calf serum (FCS), human blood serum, and MesenCult culture medium. The surface antigens of BM-MSCs were detected by flow cytometry. BM-MSCs were differentiated into osteoblasts and adipocytes under culture in the conditioned medium special for osteogenesis, and adipogenesis and the differentiated MSCs were identified by morphological observation, immunophenotype and immunohistochemical staining. The results showed that BM-MSCs could be isolated from adult human bone marrow and cultured by all culture media. The effect of umbilical cord blood serum on BM-MSC proliferation and their purity were similar to that of MesenCult culture medium, but better than that of FCS and human blood serum. The positive rate of CD29, CD73, CD105 on BM-MSCs cultured in umbilical cord serum and MesenCult medium was higher than that in FCS and adult human serum (p < 0.05), and the positive rate of CD31 was lower than that in FCS and adult human serum (p < 0.05). The positive rate of BM-MSCs differentiated into osteoblasts and adipocytes under culture in the conditioned medium for osteogenesis and adipogenesis with umbilical cord blood serum and MesenCult culture medium was also higher than that in FCS and adult human serum (p < 0.05). It is concluded that BM-MSCs can be obtained by all the four methods. DMEM/F12 with 10% umbilical cord blood serum and MesenCult culture medium are better than the others for the purification and differentiation potency of BM-MSCs in vitro. The medium with umbilical cord serum is valuable for clinical application in HSCT.

Clinical analysis of histocytic necrotizing lymphadenitis / 中南大学学报(医学版)

OBJECTIVE@#To understand the clinical features and histopathology of histocytic necrotizing lymphadenitis (HNL) so as to better recognize the disease.@*METHODS@#The clinical features, histopathology, and diagnosis of 10 patients admitted to our hospital were retrospectively analyzed.@*RESULTS@#The clinical features of these 10 cases included: young females were the majority; lymphadenopathy and fever were the most common clinical manifestations; some cases were accompanied by connective tissue diseases. Histopathologic examination showed distinctive necrosis and around the necrotic foci, variable proliferations of histocytes but generally without infiltration of neutrophils.@*CONCLUSION@#HNL has some typical histopathological alterations and relatively fine prognosis,but it tends to be misdiagnosed as lymphoma or lymphoid tuberculosis and may be accompanied by other diseases.

Specific cell immune response mediated by dendritic cells in multiple myeloma patients in vitro / 中南大学学报(医学版)

OBJECTIVE@#To explore KM3 multiple myeloma (MM) cell line specific cytotoxic T lymphocyte (CTL) response in vitro mediated by autologous dendritic cells (DCs) aroused by KM3 cells' lysates and acid-eluted peptides.@*METHODS@#Monocytes were isolated from the peripheral blood of healthy individuals and MM patients, and were cultured in serum medium with IL-4, GM-CSF, and TNF-alpha to generate DCs. These DCs were pulsed by KM3 cells' lysates or the acid-eluted peptides, then incubated with autologous T lymphocytes for 3 days to induce KM3 cell antigen specific CTL. MTT assay was performed to examine the specific KM3 cells' lysing ability of CTL.@*RESULTS@#DCs were generated in peripheral blood monocytes cultured in the serum medium containing GM-CSF, IL-4, and TNF-alpha. Autologous T lymphocytes induced by IL-2 and DCs pulsed with KM3 cells' lysates or the acid-eluted peptides showed strong killing effect on KM3 cells.@*CONCLUSION@#The DCs pulsed by KM3 cells' lysates or the acid-eluted peptides incubated with autologous T lymphocytes can induce KM3 cell antigen specific CTL.

Identification of IgG subclass and FVIII binding epitope of an acquired FVIII inhibitor in a bullous pemphigoid patient / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the clinical and laboratory diagnosis of a bullous pemphigoid patient with acquired hemophilia A (AH-A). To identify FVIII binding epitope and IgG subclass of the FVIII inhibitor, and explore the molecular mechanism for AH-A pathogenesis.</p><p><b>METHODS</b>Plasma FVIII activity( FVIII: C) was determined by one-stage assay, the titre of FYIII inhibitor by Bethesda Unit (BU). IgG purification of patient plasma or normal pooled plasma was finished by protein A-agarose column chromatography. Activated partial thromboplastin time (APTT) was assayed for uncovering FVIII inhibitor effect on FVIII in vivo. Combined Western blot analysis by anti-IgG1, IgG2, IgG3 and IgG4 antibodies was used to determine the relative concentration of patient' s IgG subclass. IgG subclass concentrations were quantified by nephelometric method. Solid-phase binding assay of FVIII and FVIII inhibitor, combined with Western blot was used to recognize the binding epitope at which the FVIII inhibitor bound to FVIII.</p><p><b>RESULTS</b>(1) Plasma APTT value of patient was prolonged evidently and could not be corrected by normal pooled plasma. Patient's FVIII: C was < 1.5%. The titre of FVIII inhibitor in patient plasma was 147.8 BU. (2) The purified patient IgG was able to inhibit FVIII: C of normal pooled plasma significantly with a dose dependent manner, and the patient plasma could prolong rabbit plasma APTT markedly with a time dependent manner. (3) The FVIII inhibitor was predominantly then of IgG4 subtype with a minority IgG1, and the concentration of IgG4 and IgG1 in the patient was higher than that in normal. The FVIII inhibitor reacted with FVIII 44 x 10(3) fragment epitope.</p><p><b>CONCLUSIONS</b>The inhibiting effect of FVIII inhibitors on FVIII: C in the bullous pemphigoid patient with AH-A is determined and the IgG subclass of the FVIII inhibitor is identified. A binding epitope for the FVIII inhibitor is a FVIII 44 x 10(3) fragment. The results provides evidence for understanding the pathogenesis of AH-A.</p>

Analysis of angiogenesis related proteins and its implication in type-2 hereditary hemorrhagic telangiectasia / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the level of transforming growth factor-beta1 (TGF-beta1), TGF-beta2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor-alpha (PDGFRalpha) in plasma and peripheral blood leukocytes in a hereditary hemorrhagic telangiectasia type 2 (HHT-2) family, and explore the implication of angiogenesis related proteins in HHT-2 pathogenesis.</p><p><b>METHODS</b>The diagnosis of the HHT-2 patient was based on clinical features and further confirmed by determining a C1231T mutation of activin receptor-like kinase 1 (ALK1) gene. Five other new members in this family were evaluated with ALK1 gene screening and clinical manifestation. Plasma level of TGF-beta1, TGF-beta2 or VEGF was measured by ELISA, and the expression of PDGFRalpha,TGF-beta1, and VEGF in peripheral blood leukocytes by flow cytometry combined with direct or indirect immunofluorescence.</p><p><b>RESULTS</b>No C1231T mutation was detected in exon 8 of ALK1 gene in the 5 new members. Plasma TGF-beta1 and TGF-beta2 concentration in 3 affected HHT case was (16 954 +/- 3 709) ng/L and (11 548 +/- 2 611) ng/L, respectively, compared with that of normal control, the difference was not significant (P > 0.05). VEGF concentration in the 3 HHT patients, 6 unaffected family members and 6 normal controls was (179.2 +/- 22.0) microg/L, (149.8 +/- 22.7) microg/L and (132.9 +/- 21.0) microg/ L, respectively. Plasma VEGF level in HHT patients was significantly higher than that in normal subjects (P < 0.025). Peripheral leukocyte PDGFRalpha and VEGF in HHT patients and unaffected family members were markedly higher than that of normal control (P < 0.05 and P < 0.02), while TGF-beta1 distribution was similar in HHT patients and normal subjects.</p><p><b>CONCLUSION</b>Compared with normal controls there is no difference in plasma TGF-beta1 concentration on peripheral leukocytes of HHT patients. Plasma VEGF concentration or leukocytes VEGF expression in HHT is significantly higher than that of normal subjects. Leukocytes PDGFRalpha expression in HHT is significantly higher than that of normal control. These changes may be associated with a compensable mechanism in HHT.</p>

Hematostatic function in myeloproliferative diseases / 中国实验血液学杂志

This study was purposed to investigate the change of early hemostatic function in patients with myeloproliferative diseases (MPD) and to explore its significance in combination with clinical data. The platelet aggregative function was measured by using ristomycin, adenosine diphosphate, collagen and adrenine as inductors, the plasma von Willebrand factor-associated antigen (vWF: Ag) level was measured by enzyme-linked immunosorbent assay (ELISA), the plasma von Wellebrand factor-ristomycin cofactor (vWF: Rco) activity was measured in 6 patients with obviously low ristomycin induced platelet aggregation (RIPA). The results showed that the platelet aggregative function obviously decreased in 35 patients, there were distinct differences in maximal platelet aggregative rate between patients and normal controls induced by 4 inductors respectively (P < 0.001, P < 0.001, P = 0.002, P < 0.001). There was no obvious difference between patients with MPD and healthy controls in plasma vWF: Ag level (P > 0.50). Plasma vWF: Rco activity in all 6 patients with MPD chosen was in the normal range, except one patient with essential thrombocytosis (ET) whose plasma vWF: Rco activity was much lower than normal. No correlation was found between platelet count and plasma vWF: Ag level in the patients (r = -0.180). No correlation was found between platelet count and maximal platelet aggregative rate induced by 4 inductors respectively in patients. It is concluded that the occurrence of abnormal platelet aggregative function is high in patients with MPD. The RIPA and vWF: Rco activity decrease in one patient with ET. However, the shortage of vWF polymer existed in his plasma have needs for further research. No correlation was observed between hemostasis and clinical manifestations. However, because of the high occurrence of platelet dysfunction in MPD patients, the clinical application of anti-platelet drugs should be considered carefully.

Clinical significance of serum vascular endothelial growth factor and interleukin-6 in multiple myeloma / 中南大学学报(医学版)

OBJECTIVE@#To investigate the clinical significance of the changes of vascular endothelial growth factor ( VEGF ) and interleukin-6 ( IL-6 ) level in multiple myeloma (MM), solid tumor following bone metastasis.@*METHODS@#Thirty- seven MM patients, including 7 in Stage I , 8 in Stage II , 22 in Stage III, 8 solid tumor with bone metastasis patientsly, and 17 healthy controls were enrolled in this study. The serum VEGF and IL-6 levels were determined by ELISA.@*RESULTS@#Serum VEGF and IL-6 concentrations in patients with MM and solid tumor were significantly higher than those of the healthy controls (P <0.01 ), and the VEGF level was higher in MM than in solid tumor with bone metastasis. There was significant difference in VEGF and IL-6 levels in various clinical stages of MM. VEGF levels in Stage II were significantly higher than in Stage I (P < 0.05 ) and IL-6 levels in Stage II were significantly higher than in Stage I (P < 0.05). The levels of IL-6 showed great difference according to bone lesion scores (P < 0.05). There was a positive correlation between IL-6 and serum calcium or C-reactive protein( P <0.01) , and there was a positive correlation between VEGF and serum Cr or urinary Bene-Jones protein lambda (P < 0.01 ). The IL-6 levels had significant differences between patients with the normal serum CRP, serum calcium, and beta2-MG and patients with abnormal ones (P < 0.05). VEGF levels showed significant differences between the patients with normal serum Cr, serum calcium Bene-Jones protein lambda, and urinary Bene-Jones protein lambda and patients with abnormal ones (P < 0.05).@*CONCLUSION@#Serum VEGF and IL-6 levels are helpful to diagnose the clinical stages, and understand bone lesion and serevity of MM.

Expressive profile of retinoblastoma-associated protein 46 and its clinical significance in acute leukemias / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of retinoblastoma-associated protein 46 (RbAp46) or RbAp 46 mRNA in bone marrow mononuclear cells (BMMNC) of acute leukemia (AL) patients and determine whether the expression is related to the classification and prognosis of ALs.</p><p><b>METHODS</b>The expression of RbAp46 protein in BMMNC was detected by Western blot in 46 AL patients and the expression of RbAp46 mRNA in BMMNC by semi-quantitative RT-PCR in 22 AL patients. The indirect immunofluorescence staining technique was applied to the localization of RbAp46 protein in BMMNC both in leukemia patients and control subjects.</p><p><b>RESULTS</b>(1) Both RbAp46 protein and mRNA were expressed in AL BMMNC and no significant difference was found among different leukemia types. (2) The expression of RbAp46 protein was lower in AL patients with high-degree tumor burden than in those with low-degree tumor burden (mean A, 93.4 +/- 37.2 vs 127.2 +/- 15.8, P < 0. 05). (3) The expression of RbAp46 protein was lower in refractory leukemia than those in non-refractory leukemia (mean A, 87.1 +/- 33.8 vs 126.6 +/- 21.2, P < 0. 05). (4) The expression of RbAp46 mRNA was lower in AL patients with high-degree tumor burden than in those with low-degree tumor burden (mean A R, 0.19 +/- 0.08 vs 0.31 +/- 0.12, P < 0. 05). (5) RbAp46 protein was mainly localized in nucleus of BMMNC in both AL patients and control subjects.</p><p><b>CONCLUSION</b>Both RbAp46 protein and mRNA are expressed in AL patients BMMNC. The downregulation of RbAp46 expression is associated with high leukemic burden and refractory to treatment. RbAp46 gene might be a tumor suppressor gene for leukemia.</p>