Research progress of metabonomics in systemic lupus erythematosus / 中国医师杂志

By means of non-targeted quantitative determination of various low-molecular-weight metabolites in biological samples,metabonomics enables a better understanding of the metabolic variation in organisms subject to internal and external stimuli.As a result,it is particularly suitable for characterizing the metabonomic alternation associated with diseases and exploring the disease-related early metabolic biomarker clusters,which may provide a novel approach to the early diagnosis of diseases and individual medication.Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which autoantibodies can cause tissue damage and potentially life-threatening mul-tiple organ failure.The complicated pathogenesis and varied clinical symptoms of SLE pose great challenges for the diagnosis and monitoring of this disease.Development of new biomarkers that can be used to reliably confirm SLE or monitor its progression is still one of the main goals and challenges in SLE research.Metabolomics are alternative biomarker discovery approaches.In order to explore the application of metabolomics in SLE,we summarize the current progress in the field of metabtomics in the studies of SLE.

Research progress of metabonomics in systemic lupus erythematosus / 中国医师杂志

By means of non-targeted quantitative determination of various low-molecular-weight metabolites in biological samples, metabonomics enables a better understanding of the metabolic variation in organisms subject to internal and external stimuli. As a result, it is particularly suitable for characterizing the metabonomic alternation associated with diseases and exploring the disease-related early metabolic biomarker clusters, which may provide a novel approach to the early diagnosis of diseases and individual medication. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which autoantibodies can cause tissue damage and potentially life-threatening mul-tiple organ failure. The complicated pathogenesis and varied clinical symptoms of SLE pose great challenges for the diagnosis and monitoring of this disease. Development of new biomarkers that can be used to reliably confirm SLE or monitor its progression is still one of the main goals and challenges in SLE research. Metabolomics are alternative biomarker discovery approaches. In order to explore the application of metabolomics in SLE, we summarize the current progress in the field of metabtomics in the studies of SLE.

A preliminary study on SLAMF6 expression in patients with severe aplastic anemia / 中华血液学杂志

Objective@#To explore the expression of SLAMF6 on CD8+ T cells in patients with severe aplastic anemia (SAA) and its correlation with disease immune status.@*Methods@#By flow cytometry (FCM), SLAMF6 expression level in peripheral blood CD8+ T cells was detected in 21 patients with SAA and 15 normal controls respectively from February 2017 to April 2018. The correlation between SLAMF6 expression level and hematopoietic functions, including HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, hyperplasia degree (percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes in bone marrow) and perforin, granzyme B, IFN-γ expression level in CD8+ T cells were evaluated. To further confirm the effect of SLAMF6 on CD8+ T cells, anti-SLAMF6 Ab was used to block SLAMF6 pathway (IgG as control), and FCM was used to detect the perforin, granzyme B, and IFN-γ production of CD8+ T cells.@*Results@#The expression of SLAMF6 on CD8+ T cells in untreated SAA patients[(56.29±12.97)%]was significantly lower than that of normal controls[(80.96±7.36)%](t=-7.672, P<0.001). The expression of SLAMF6 on CD8+ T cells in SAA patients were positively correlated with the HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, percentage of granulocytes, erythrocytes in bone marrow (all P<0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow, and the expression of perforin, granzyme B, and IFN-γ of CD8+ T cells (all P<0.05). After blocking SLAMF6 pathway by anti-SLAMF6 Ab, the expression levels of perforin, granzyme B and IFN-γ in SAA patients were significantly higher than those in the untreated group, and the differences were statistically significant (all P<0.05).@*Conclusions@#SLAMF6 is significantly down-regulated on CD8+ T cells in SAA patients, which may act as a negative immunoregulatory molecule participating in the mechanism of SAA by affecting the functional molecules secretion on CD8+ T cells.

Relationship between anemia and clinical features and prognosis of advanced lung cancer patients / 中华肿瘤杂志

Objective@#To investigate the relationship of anemia and clinicopathological features and prognosis of patients with advanced lung cancer.@*Methods@#The clinical data of 741 patients with stage Ⅲ~Ⅳ lung cancer were collected and analyzed retrospectively. We analyzed the incidence and type of anemia during the natural course of lung cancer patients, and its relationship with the gender, age, duration of disease, clinical stage, prognostic nutritional index (PNI) of these patients. Kaplan-Meier method and multivariate Cox regression model were used to analyze the effect of anemia on prognosis.@*Results@#Among 741 cases of lung cancer patients, 407 (54.9%) cases were accompanied with anemia, whose hemoglobin (Hb) was (89.39±15.76) g/L, including 214 cases of mild anemia, 173 cases of moderate anemia and 20 cases of severe anemia. The most common type of anemia is anemia of chronic disease (ACD), the incidence rate of which was 79.6% (324/407), followed by the iron deficiency anemia (IDA), the incidence rate of which was 4.2% (17/407). The incidence of anemia was marginally related to the gender and age (P>0.05), but significantly related to the duration of disease, clinical stage and PNI (all P<0.05). The degree of anemia was marginally related to the gender and age (P>0.05), but significantly related to the duration of disease, clinical stage and PNI (all P<0.05). The median survival time of the patients with anemia was 10.5 months (95% CI: 10.1~10.9 ), significantly shorter than 13.0 months (95% CI: 12.2~13.8) of patients without anemia (P<0.001). The median survival time of mild anemia patients was 11.0 months (95% CI: 10.7~11.3), significantly longer than 9.6 months (95% CI: 9.1~10.1) of moderate and severe anemia patients (P=0.048). The results of Cox regression survival analysis showed that the incidence and degree of anemia were independent factors of prognosis of patients with lung cancer (P<0.05).@*Conclusions@#During the natural course of advanced lung cancer, the incidence of anemia is high, especially ACD. The incidence and degree of anemia are substantially correlated with the duration of disease, clinical stage and PNI. The incidence and degree of anemia are independent prognostic factors of patients with lung cancer.

The clinical significance of CD4+CD69+T lymphocytes in patients with autoimmune hemolytic anemia/Evans syndrome / 中华内科杂志

Objective To investigate the significant of peripheral CD4+CD69+T lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome (ES).Methods In this study peripheral blood samples from 32 patients with AIHA/ES (15 hemolytic episode patients,17 remission patients) and 13 healthy controls were collected.Patients with AIHA/ES were recruited in Tianjin Medical University General Hospital from October 2015 to May 2016.The percentages of CD69+ T lymphocytes were analyzed by flow cytometry.The expression of CD69 mRNA in CD4+T lymphocytes which was sorted from peripheral blood by magnetic activated cell sorting (MACS) was detected using real-time PCR.Soluable CD69 was measured by ELISA.Results In hemolytic episode patients,the ratio of CD3+CD69+/CD3+T lymphocytes [(3.08 ± 1.48)％] was significantly higher than that in healthy controls [(1.28 ± 0.83)％,P＜0.01] and in remission group[(1.96± 1.33)％,P＜0.05].The absolute count of CD3+CD69+T lymphocytes in hemolytic episode group [(2.94± 1.81)× 107/L] was higher than that in healthy controls [(1.48± 1.42)× 107/L,P＜0.05].The ratio of CD3+CD4+CD69+/CD3+CD4+T cells in hemolytic episode group [(2.16± 1.56)％] was significantly higher than that in remission group [(1.16±0.62)％,P＜0.05] and healthy controls[(0.94±0.78)％,P＜0.05].The quantity of CD3+CD4+CD69+T lymphocytes in hemolytic episode group[(1.04±0.98)× 107/L] was higher than in healthy controls [(0.44± 0.38) × 107 / L,P＜0.05].The ratio of CD3+CD8+CD69+/CD3+ CD8+T lymphocyte in hemolytic episode group [(4.87±2.56)％] was significantly higher than that in healthy controls[(1.83± 1.27)％,P＜0.01].The quantity of CD3+CDs+CD69+T lymphocytes in three groups did not show significant difference.The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin (Hb) (P＜0.01),positively correlated with the percentage of reticulocytes (Ret％)(P=0.01)total bilirubin(TBil),indirect bilirubin(IBil) (P＜0.01) and not correlated with absolute reticulocytes count,lactic dehydrogenase (LDH),complement 3(C3),complement 4 (C4).The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in remission group was negatively correlated with Hb (P＜0.05).In hemolytic episode patients CD69 mRNA (32.26±35.11) was significantly higher than that in remission group(6.05±5.87)(P＜0.05)and healthy controls (1.76± 1.85)(P＜0.01).CD69 mRNA in remission group was significantly higher than healthy controls (P＜0.05).Serum CD69 in hemolytic episode patients [(494.21 ± 16.06) ng/L] was significantly higher than that in healthy controls [(441.39± 104.6) ng/L,P＜0.05].Conclusion Our findings suggest that the proportion of CD4+CD69+ T lymphocytes increase in AIHA/ES patients,which is correlated with the severity of disease.

Characteristic and function of peripheral blood mononuclear cells-induced macrophages in patients with myelodysplastic syndrome / 中华血液学杂志

Objective@#To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression.@*Methods@#A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry.@*Results@#The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t=3.529, P=0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t=4.551, P<0.001]. The SIRPα level of macrophages from MDS patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t=2.102, P=0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t=-6.253, P=0.008; (23.69±3.22) % vs (42.75±2.13) %, t=-6.982, P=0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t=3.464, P=0.001) by fluorescence microscopy.@*Conclusion@#The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

Study on autophagy in nucleated red blood cells in patients with myelodysplastic syndromes / 中华血液学杂志

Objective@#To investigate the change of autophagy level of bone marrow nucleated red blood cell (RBC) in patients with myelodysplastic syndromes (MDS) .@*Methods@#Fifty-four MDS patients and thirty-three controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM) . The level of autophagy-associated protein LC3B in GlycoA+ nucleated RBC was measured by flow cytometry. The expressions of ULK1 and mTOR mRNA in GlycoA+ nucleated RBC were measured by real-time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA+ nucleated RBC was detected by Western blot.@*Results@#Autophagosomes or autolysosomes were scarcely observed by TEM in MDS patients. The expression of LC3B in GlycoA+ nucleated RBC in high-risk MDS patients (0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.001) , and lower than that in low-risk MDS patients (0.40±0.16, P=0.001) . The expression of AMPK [0.26 (0.60) ] in GlycoA+ nucleated RBC in high-risk MDS patients was significantly lower than that in controls [1.00 (2.07) , P<0.017) . The expression of ULK1 mRNA in GlycoA+ nucleated RBC in high-risk MDS patients [0.27 (3.31) ] was significantly lower than that in controls [1.07 (4.41) , P<0.017]. The level of mTOR mRNA in GlycoA+ nucleated RBC in high-risk MDS patients [1.82 (3.74) ] was significantly higher than that in controls [1.26 (1.38) , P<0.017]. The level of LC3B in GlycoA+ nucleated RBC was negatively correlated with the HGB (r=0.529, P=0.009) in high-risk MDS patients. The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was 9.42±4.42.@*Conclusion@#Autophagy is impaired in nucleated RBC of MDS patients.

Research on the negative immune regulation of NK cells in patients with primary immune thrombocytopenia / 中华血液学杂志

Objective@#To investigate the levels of NK cells and their relevant cytokines (IL-10, TGF-β and IFN-γ) in patients with primary immune thrombocytopenia (ITP) .@*Methods@#All samples were obtained from 42 patients (22 newly diagnosed and 20 in remission) and 20 healthy volunteers. The levels of IL-10 and IFN-γ in blood serum were detected by enzyme-linked immunosorbent assay (ELISA) . The percentage of CD3- CD56+ NK cell, CD3- CD56bright CD16- NK cell, CD3- CD56dim CD16+ NK cell in peripheral blood lymphocyte were detected by flow cytometry. The NK cells were isolated by immunomagnetic microbeads. The mRNA expression levels of IL-10, TGF-β, and IFN-γ in NK cells were detected by real-time fluorescent quantitative PCR. Correlation between the above measured results was analyzed.@*Results@#① The blood serum level of IFN-γ in newly diagnosed ITP patients [ (653.0±221.6) ng/L] was higher than that in remission ITP patients [ (484.4±219.5) ng/L] and healthy control [ (390.9±253.5) ng/L] (P=0.022, P=0.001) . The blood serum level of IL-10 in newly diagnosed ITP patients was lower than that in healthy control [ (52.09±26.66) ng/L vs (79.44±38.43) ng/L, P=0.007]. ②The percentage of NK cell in newly diagnosed and remission ITP patients [ (9.53±3.93) %, (9.03±3.78) %] were significantly lower than that in healthy control [ (13.72±7.42) %] (P=0.013, P=0.007) . The ratio of CD3- CD56bright CD16- NK cell/total NK cells in newly diagnosed ITP patients was higher than that in healthy control [ (6.85±4.43) % vs (4.05±2.81) %, P=0.032]. The ratio of CD3-CD56dim CD16- NK cell/total NK cells in newly diagnosed ITP patients was lower than that in healthy control [ (93.14±4.43) % vs (95.94±2.81) %, P=0.032]. ③ There was no significant difference in the mRNA expression level of IFN-γ in NK cells of ITP patients and healthy control (all P>0.05) . The mRNA expression levels of IL-10 and TGF-β in NK cells in newly diagnosed ITP patients were significantly higher than that in healthy control (1.82±1.32 vs 1.02±1.03, P=0.023; 2.80±2.31 vs 1.46±1.37, P=0.028) . The ratio of CD3-CD56bright CD16- NK cell/total NK cells was positively correlated with the mRNA expression levels of IL-10, TGF-β in NK cells (r=0.424, P=0.001; r=0.432, P<0.001) .@*Conclusion@#NK cells may compensate for the deficiency of the number by enhancing the secretion of negative regulation cytokines, acting as "protective" roles in the disease.

Immunophenotypes and clinical features of lymph ocytes in labial gland of patients with primary Sj(o)gren's syndrome / 中华风湿病学杂志

Objective To investigate the consistency of the lymphocyte immunophenotype between labial gland and peripheral blood of patients with primary Sj(o)gren's syndrome (pSS).Methods Seveny-one pSS patients (referred as pSS group) from rheumatology department and 35 subjects patients with maxillofacial trauma (referred as control group) from department of head and neck surgery of general hospital at tianjin medical university (Tianjin,China) during August 2013 to April 2016 were included into this study.Based on the ratio of CD20 to CD3 in labial gland from pSS patients,they were divided into CD20 high proportion group and CD20 low proportion group.Lymphocyte immunophenotypes in labial glands,course of disease,erythrocyte sedimentation rate (ESR),C-reactive protein (CRP) level,immuno-globulin (Ig) and complement levels were compared among groups.Results ① The levels of serum IgG,IgA,IgM and CRP,and ESR were higher,but C4 level was lower in pSS patients than in the control group [IgG:(22 990±8 060) mg/L vs (1 1560±1 290)mg/L,t=l 1.645,P＜0.01;IgA:(3 710±1 400) mg/L vs (2 390±800) mg/L,t=6.138,P＜0.01;IgM:(1 580±1 300)mg/L vs (920±390) mg/L,t=3.893,P＜0.01;ESR:(37±14) mm/1 h vs (14±4) mm/1 h,t=12.723,P＜0.01;CRP:(5.9±8.7) mg/L vs (2.5±1.2) mg/L,t=3.199,P＜0.01;C4:(180±60) mg/L vs (250±40) mg/L,t=-6.850,P＜0.01].② The levels of IgG,IgA,IgM and C3c in labial gland were higher (IgG:Z=-6.264,P＜0.01;IgA:Z=-1.997,P＜0.05;IgM:Z=-5.459,P＜0.01;C3c:Z=-8.533,P＜0.01),but Clq was lower in pSS group than in control group (Z=-4.363,P＜0.01).③ CD20 was detected in labial gland samples of all pSS patients,in which CD3 was positive in 66 patients,and negative in 5.④ Serum IgG level,ESR were higher in CD20 high proportion group than in low proportion group [IgG:(24 970±7 510) mg/L vs (18 860±7 740) mg/L,t=3.176,P＜0.01;ESR:(40±13) mn/1 h vs (32±15) mm/1 h,t=2.148,P＜0.05].⑤ The levels of IgG and Clq in labial gland were higher in CD20 high proportion group than in low proportion group [IgG:Z=-5.387,P＜0.01;C1q:Z=-4.724,P＜0.01].Conclusion pSS patients have a higher proportion of CD20 in infiltrating lymphocytes of the labial gland,accompanied with the changes of immunoglobulins and complements in both labia gland and peripheral blood.

Hypomethylation and overexpression of CD70 in CD4+ T cells of the patients with immuno-related pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of CD70 and the methylation level of CD70 promoter in immuno-related pancytopenia（IRP）patients, and explore the role of CD70 in the pathogenesis of IRP.</p><p><b>METHODS</b>Thirty- five IRP patients and fifteen healthy donors were enrolled in this study. Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation, and CD4(+) T cells were isolated by immunomagnetic beads. The mRNA level and the percentage of CD70 of CD4(+) T cells were measured by real- time quantitative polymerase chain reaction（RT- PCR）and flow cytometry respectively. Methylation- Specific PCR（MSP）was performed to determine the methylation level of CD70 promoter.</p><p><b>RESULTS</b>The percentage of CD70 expression on CD4(+) T cells of untreated IRP patients［（7.46±1.51）%］was significantly higher than that of recovered ones［（5.95±1.34）%］and normal controls［（1.83 ± 0.60）%］, and that of recovered IRP patients was significantly higher than of normal controls（P<0.05）. The relative expressions of CD70 mRNA in CD4(+) T cells were 2.314（0.200-6.084）, 1.021（0.135-3.434）, 0.353（0.008-2.258）in three groups respectively. The differences among untreated IRP patients, recovered IRP patients and normal controls were significant（P<0.05）. The CD70 promoter methylation level in CD4(+) T cells of all IRP patients was significantly lower than that of normal controls （P<0.05）. The expression of CD70 positively correlated to the ratio of CD5(+) B cells（r=0.533, P<0.01）.</p><p><b>CONCLUSION</b>The overexpression of CD70 may lead to immunologic disarrangement of patients, which may play important role in the pathogenesis of IRP.</p>

Autophagy level of bone marrow mononuclear cells in patients with myelodysplastic syndromes / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）.</p><p><b>METHODS</b>Thirty- eight patients with MDS and 26 megaloblastic anemia patients were enrolled in this study. The autophagic vacuoles were observed by transmission electron microscopy （TEM） and the quantity of autophagic vacuoles was detected by monodansylcadaverine （MDC） staining. The LC3 protein positive cells were counted by immunofluorescence assays. The expression of Beclin 1, LC3A, mTOR mRNA were measured by real time PCR. The expression of Beclin 1 proteins were detected by Western blotting.</p><p><b>RESULTS</b>The autophgic vacuoles of double membrane that surrounds lysosomes appeared in MDS patients. The percentage of MDC positive cells was significantly higher in MDS patients［（9.75±2.63）%］than that of controls［（2.90± 0.89）%, P＜0.05）. The percentage of LC3 protein cells was also increased in MDS patients（6.13±1.03）% vs（1.5±0.58）%, P＜0.05）. The expression of Beclin 1 and LC3A mRNA in low-risk and intermediate-1 MDS were higher compared with controls （3.61 ± 3.02 vs 1.55 ± 1.03 and 6.56 ± 3.97 vs 1.21 ± 0.95 respectively, both P＜0.05）. The expression of mTOR mRNA was down- regulated in low- risk and intermediate-1 MDS compared with controls（0.39±0.37 vs 1.50±1.03, P＜0.05）. There were no significant difference in expression of Beclin 1, LC3 and mTOR mRNA among intermediate-2 and high-risk MDS and controls. Beclin 1 protein expression was higher in low- risk and intermediate- 1 MDS patients（1.257 ± 0.197）than that of controls（0.528±0.086）and inermediate-2 and high-risk MDS patients（0.622±0.118）.</p><p><b>CONCLUSION</b>The autophagy levels were increased in low- risk and intermediate- 1 MDS, while not enhanced in intermediate-2 MDS. Autophagy might be considered as a cell protective mechanism in MDS. The relatively defective autophagy in intermediate- 2 and high- risk MDS might contribute to disease's progression.</p>

Study on C5b-9 deposited on the membrane of platelets and its dysfunction in patients with paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the expression levels of terminal complement complex (C5b-9) and CD62p on platelets and the soluble C5b-9 (sC5b-9) level in serum in patients with PNH or PNH-aplastic anemia (AA).</p><p><b>METHODS</b>Serum levels of sC5b-9, complement C3 and C4 were detected by using ELISA in 25 patients with PNH/PNH-AA. The quantities of C5b-9 and CD62p on the membrane of platelets were detected by flow cytometry.</p><p><b>RESULTS</b>①In PNH/PNH-AA group, the serum sC5b-9 level [390.27(265.73-676.87) μg/L] was lower than that in control group [540.39(344.20-1 576.78) μg/L] (P<0.01). ②The platelet PNH clone (CD59⁻CD61⁺/CD61⁺) size [50.58(23.29-81.60)%] was bigger in the PNH/PNH-AA group than that [23.57(15.58-29.02)%] in control group (P<0.01). The percentages of C5b-9 deposition (C5b-9⁺CD61⁺/CD61⁺) were higher on the PNH clone platelets (CD59⁻CD61⁺) in the PNH/PNH-AA group [(17.53 ± 6.27)%] than those on the normal platelets (CD59⁺CD61⁺) in PNH patients 11.33±5.03）%］ and control [(10.88±3.58)%] group (P<0.01). ③ The expression of CD62p (CD62p⁺CD61⁺/CD61⁺) on PNH clone platelets in PNH patients [(61.98 ± 11.71)%] was higher than that on the normal platelets in PNH patients [(43.76±11.30)%] and control group [(38.23±18.07)%] (P<0.01). In addition, the expression of CD62p on normal platelets was higher in PNH patients than control (P<0.05). ④The deposition of C5b-9 positively correlated with the expression of CD62p on the platelets (r=0.559, P=0.002).</p><p><b>CONCLUSION</b>Deficiency of CD59 antigen on platelets in PNH patients may lead to the deposition of C5b-9 on its membrane and its dysfunction, which may contribute to thrombosis events in PNH.</p>

An investigation of problem-based learning in American medical education / 中华医学教育探索杂志

[Abstrct] Based on field studying at University of California, Los Angeles (UCLA),discussing with the teacher and student representatives and reviewing related literatures, we found that the suc-cessful experience of PBL at UCLA were small group teaching, the use of different sources of tutor, serious case preparation, and good hardware support. The major difficulties of PBL at UCLA were high teaching costs, unstable teacher group and high hardware requirements. Because of the cultural differ-ences between East and West, Chinese students are not good at active learning, so we recommended gradually carrying out PBL teaching in our country, making more PBL skills training to teachers and students, strengthening the hardware construction, seeking school administrative support, and giving timely repair according to the feedback of scientific research.

Research progress on treatment of myelodysplastic syndromes with demethylating drugs / 中国肿瘤临床

Abnormal epigenetics play important roles in the pathogenesis of myelodysplastic syndromes (MDS). DNA hypermeth-ylation is the most common epigenetic abnormality in MDS. Demethylating DNA hypermethylation may improve the quality of life of MDS patients and prolong their overall survival. Azacitidine and decitabine are the demethylating drugs approved for MDS treatment. These drugs showed clinical effects on all subgroups of MDS patients.

Abnormal WT1 gene expression in paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria (PNH).</p><p><b>METHODS</b>The expression of WT1 mRNA in CD59⁻ and CD59⁺ bone marrow mononuclear cells (BMMNC) were measured by semi-quantitative reverse transcription PCR. After WT1 gene silence by RNA interference (RNAi) technology, biological characteristics of BMMNC were investigated by flow cytometry.</p><p><b>RESULTS</b>The relative expression of WT1 mRNA in PNH CD59⁻ BMMNC (1.06 ± 0.12) was significantly higher than that in PNH CD59⁺ BMMNC (0.90 ± 0.12) and normal BMMNC (0.86 ± 0.05, P<0.05), but there was no significant difference between PNH CD59⁺ BMMNC and normal BMMNC (P>0.05). WT1 mRNA expression in PNH was positively correlated with the proportion of CD59⁻ cells (r²=0.490, P=0.016), but had no relationship with the proportion of CD59⁺ cells. After WT1 gene silence by siRNA in PNH CD59⁻ BMMNC, WT1 mRNA expression was decreased. The proportions of G0/G1 phase in PNH CD59⁻ cell blank control group and siRNA-scr transfected group were (92.73 ± 3.71)% and (93.06 ± 4.14)%, and the proportions of S phase were (6.99 ± 3.61)% and (6.73 ± 4.08)%, respectively. The proportions of G0/G1 and S phase in siRNA-WT1 transfected group was (94.46 ± 3.71)% and (5.40 ± 3.55)%, respectively. There were significant differences in the proportions of G0/G1 phase and S phase among the controls, siRNA-WT1 transfected group and siRNA-scr transfected group (P<0.05). The rate of apoptosis in siRNA-WT1 transfected group [(35.91 ± 22.36)%] was significantly higher than those in controls [(26.12 ± 17.10)%] and siRNA-scr transfected group [(27.39 ± 18.99)%] (P<0.05).</p><p><b>CONCLUSION</b>siRNA-WT1 could effectively suppress the WT1 gene expression of CD59⁻ clone in PNH patients, inhibit its proliferation, and promote its apoptosis. WT1 gene expression might contribute to PNH clone proliferation.</p>

Telomere length of peripheral lymphocytes in patients with immuno-related pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the changes of relative telomere length (RTL) of peripheral blood (PB) CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺T lymphocytes, CD19⁺B lymphocytes and bone marrow (BM) CD34⁺ cells and its association with disease severity in untreated patients with immuno-related pancytopenia (IRP).</p><p><b>METHODS</b>The PB CD3⁺ , CD3⁺ CD4⁺ , CD3⁺ CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and BM CD34⁺ cells were purified by magnetic activated cell sorting (MACS), and RTL were measured with flow-fluorescence in situ hybridization (FLOW-FISH).</p><p><b>RESULTS</b>The RTL of CD3⁺, CD3⁺CD4⁺ , and CD3⁺CD8⁺T lymphocytes in untreated IRP patients were (27.754 ± 16.323)%, (7.526 ± 3.745)% and (25.854 ± 14.789)%, respectivly, which were significantly shorter than those in healthy-controls (54.555 ± 19.782)%, (12.096 ± 2.805)%, and (38.367 ± 4.626)% (P<0.05). The RTL of CD19⁺ lymphocytes in untreated IRP patients was (22.136 ± 16.142)%, which was significantly shorter than that in healthy controls (42.846 ± 16.353)% (P<0.01). There was no significant difference of BM CD34⁺ cells RTL between the untreated IRP patients (22.528 ± 21.601)% and the healthy controls (23.936 ± 19.822)% (P>0.05). There were significantly positive correlations between the RTL of B lymphocytes and the count of white blood cell (r=0.706, P=0.015). There were negative correlations between RTL of B lymphocytes and the clinical symptoms (r=-0.613, P=0.045) and positive correlations with therapeutic effect (r=0.775, P=0.005).</p><p><b>CONCLUSION</b>The shorter RTL of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ lymphocytes, and the normal RTL of BM CD34⁺ cells in untreated IRP patients were identified, which might imply that IRP is a type of acquired autoimmune diseases.</p>

Effect of CCL3 on osteoblast in myeloma bone disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM).</p><p><b>METHODS</b>Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3.</p><p><b>RESULTS</b>Bone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.</p><p><b>CONCLUSION</b>Bone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.</p>

Memory B (CD5⁺ CD19⁺ CD27⁺) lymphocyte in patients with immune-related pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect memory B lymphocyte (Bm) in peripheral blood (PB) of immune-related pancytopenia (IRP).</p><p><b>METHODS</b>86 patients with IRP and 11 health volunteers were enrolled in this study. Bm (CD5⁺ CD19⁺ CD27⁺) and bone marrow mononucleated cell antibodies (BMMNC-Ab) were determined via fluorescence-activated cell sorting, and clinical outcomes of these patients were analyzed.</p><p><b>RESULTS</b>(1)43 initial patients achieved obvious remission in all 52 initial cases after conventional immunosuppression therapy. 16 relapsed patients with IRP received Rituximab (RTX) and 14 cases achieved obvious remission, among which 7 cases were refractory to conventional immunosuppression therapy, 5 cases exhibited obvious remission, and 2 cases did not respond. Other 18 relapsed cases received conventional immunosuppression therapy and 13 cases achieved obvious remission. (1)The level of Bm in PB in 52 initial patients with IRP was(1.81 ± 0.97)%, and no significant difference was observed between the initial patients and health volunteers (1.75 ± 0.55)% (P>0.05). The level of Bm in PB in 34 relapsed patients with IRP was obviously higher than that in the initial IRP patients and health volunteers (P<0.05). Significant difference was observed in the level of Bm in PB in 16 relapsed IRP patients between pre-therapy and post-therapy with RTX (P<0.05). No statistical difference was found between the remission and no-response groups in relapsed patients treated with RTX. RTX regimen produced more effective outcome than conventional immunosuppression therapy, which better eliminated Bm than the latter (P<0.05). Initial patients with IRP who relapsed within a two-year follow-up period had a lower level of Bm in PB compared with un-relapsed patients (P<0.05). Majority of BMMNC- Ab antibodies in relapsed patients were IgG (82.4%) and IgM (69.2%) autoantibodies in patients with initial IRP.</p><p><b>CONCLUSION</b>The level of Bm in PB was associated with relapsed patients with IRP. Bm did not respond to conventional immunosuppression therapy,but responded to RTX.</p>

Culture and regulation of osteoblasts in multiple myeloma patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological characteristics of osteoblasts cultured in vitro from bone marrow (BM) of multiple myeloma (MM) patients and to explore their generation and osteogenic potential. Effects of some factors such as bortezomib and MM patient serum on the osteoblasts were observed.</p><p><b>METHODS</b>Twenty MM patients and 10 healthy donors as controls were enrolled in this study. Osteoblasts from MM patients'BM were cultured in vitro. The generation and osteogenic potential of osteoblasts from MM patients and normal subjects were compared. The changes of their osteogenic potential and biological characteristics were observed. The antigens (CD34,CD138,CD45) on osteoblasts were catalyzed by flow cytometry. The levels of IL-7 were measured by ELISA. The BMP2 mRNAs were measured by RT-PCR.</p><p><b>RESULTS</b>Osteoblasts from MM patients'BM could be cultured in vitro. The quantity of osteoblasts from MM patients (6.3±1.5) was less than normal subjects (8.2±2.6) (P<0.05). The osteoblasts cultured with MM patient serum (7.4±1.1) were less than those without patient serum (8.2±2.6) (P<0.05). Bortezomib increased those from MM patients after 6 days culture (8.9±2.1 vs 6.3±1.5) (P<0.05). Von Kossa staining showed that there were more calcium depositions in MM osteoblasts with bortezomib (8.8±1.9) than those without bortezomib (6.1±1.6) (P<0.05), but less than those from normal controls (15.8±2.2) (P<0.05). CD138, CD45, CD34 were not detected on the cultured cells. The level of IL-7 in MM patients'serum (2.07±0.71) was higher than that in normal controls (1.62±0.15) (P<0.05). The expression of BMP2 mRNA was seen in the normal osteoblasts and MM patients'osteoblasts cultured with bortezomib, but not be seen in those without bortezomib.</p><p><b>CONCLUSION</b>Osteoblasts could be cultured in vitro from MM patients' BM. The proliferation and osteogenic potential of osteoblasts from MM patients were decreased. Bortezomib was a positive regulatory of osteoblasts and MM patient serum was a negative one. They both could affect the proliferation and osteogenic potential of osteoblasts.</p>

Quantity and apoptosis-related protein levels of CD4+, CD25+, and CD127low regulatory T cells in peripheral blood of multiple myeloma patients / 中国肿瘤临床

To investigate the role of CD4+, CD25+, and CD127low regulatory T cells (Tregs) in multiple myeloma (MM). Methods:Levels of CD4+T cells and Tregs, as well as expression of CTLA-4 and apoptosis-related proteins, such as CD95, bcl-2, and Caspase3 of Tregs in peripheral blood of 30 patients with newly diagnosed cases, 27 patients under of complete remission (CR) from multiple myeloma patients, and 25 healthy adults were analyzed by flow cytometry. Results:The percentage of CD4+T cells in the untreated group was significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was significantly higher than that of the CR group and control group (P<0.05), which in ISSⅢpatients of the untreated group was significantly higher than that in I/II(P<0.05). No significant difference of CD95 expression in Tregs was observed among the three groups. The expression of CTLA-4 in Tregs from the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls (P<0.05). The expression of bcl-2 in Tregs in the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls(P<0.05). The expression of Caspase3 in Tregs from the untreated group and CR group were all significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was positively correlated with the proportion of bone marrow plasma cells (P<0.05). The percentage of Tregs in CD4+T cells from 15 MM patients who received bortezamib and dexamethasone (VD) chemotherapy was negatively correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r=0.735, P<0.01). Conclusion:The level of Tregs in the peripheral blood of MM patients was positively correlated with tumor burden and progression of disease, but was negatively correlated with curative effect. The increased level of Tregs was associated with their strengthened anti-apoptosis function.