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1.
Ann Hematol ; 99(2): 215-221, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900500

RESUMO

Many studies have confirmed that overexpressed WT1 exists in leukemic cells, especially in AML. However, the immunophenotypic features of this sort of leukemic cells remain to be unclarified. We retrospectively analyzed the immunophenotype of 283 newly diagnosed AML patients with intermediated and poor cytogenetic risk to evaluate the correlation between phenotype and WT1 overexpression. EVI1 transcripts, KMT2A-PTD, FLT3-ITD, and NPM1 mutations were simultaneously assessed. Our results revealed that overexpressed WT1 was significantly associated with the expression of CD117, CD13, and CD123. Besides, leukemic cells with WT1 overexpression also lacked lymphoid and myeloid differentiation-related markers. FAB subtype M2 patients had higher WT1 levels, compared with other FAB subtype. Multivariate analysis was proved that NPM1 mutation, M2 subtype, and the expression of CD123 were independently associated with WT1 overexpression. These indicated that AML with overexpressed WT1 was proliferated and blocked in the early stage of AML development. It presumably provided some clues to detect overexpressed WT1 cells via multiparameter flow cytometry. CD123-targeted drugs might become one of the alternative treatments for patients with WT1 overexpression.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/metabolismo , Proteínas WT1/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Feminino , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fatores de Risco , Proteínas WT1/genética
2.
Cancer Med ; 8(12): 5459-5467, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364309

RESUMO

Acute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease. Although the detection of minimal residual disease (MRD), which is indicated by RUNX1-RUNX1T1 transcript levels, plays a key role in directing treatment, risk stratification needs to be improved, and other markers need to be assessed. A total of 66 t(8;21) AML patients were tested for aldehyde dehydrogenase (ALDH) activity by flow cytometry at diagnosis, and 52 patients were followed up for a median of 20 (1-34) months. The median percentage of CD34+ALDH+, CD34+CD38-ALDH+, and CD34+CD38+ALDH+ cells among nucleated cells were 0.028%, 0.012%, and 0.0070%, respectively. The CD34+ALDH+-H, CD34+CD38-ALDH+-H, and CD34+CD38+ALDH+-H statuses (the percentage of cells that were higher than the individual cutoffs) were all significantly associated with a lower 2-year relapse-free survival (RFS) rate in both the whole cohort and adult patients (P = .015, .016, and .049; P = .014, .018, and .032). Patients with < 3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy (defined as MRD-H) had a significantly lower 2-year RFS rate than patients with ≥ 3-log reduction (MRD-L) (P = .017). The CD34+ALDH+ status at diagnosis was then combined with the MRD status. CD34+ALDH+-L/MRD-H patients had similar 2-year RFS rates to both CD34+ALDH+-L/MRD-L and CD34+ALDH+-H/MRD-L patients (P = .50 and 1.0); and CD34+ALDH+-H/MRD-H patients had significantly lower 2-year RFS rate compared with CD34+ALDH+-L and/or MRD-L patients (P < .0001). Multivariate analysis showed that CD34+ALDH+-H/MRD-H was an independent adverse prognostic factor for relapse. In conclusion, ALDH status at diagnosis may improve MRD-based risk stratification in t(8;21) AML, and concurrent high levels of CD34+ALDH+ at diagnosis and MRD predict relapse.

3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(1): 141-148, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30738461

RESUMO

OBJECTIVE: To study the value of flow cytometric scoring system in the diagnosis of myelodysplastic syndromes (MDS). METHODS: The phenotypes of erythroid and immature cells were analyzed retrospectively in 130 MDS patients, 19 healthy controls and 89 pathological controls, all of them were well clinically immunophenotyped. The 4-parameter scoring system reported in the literature was studied, including myeloblast-related cluster size, B-progenitor-related cluster size, lymphocyte to myeloblast CD45 ratio, and granulocyte to lymphocyte side scatter ratio. The two flow cytomatric parameters of the erythroid scoring system were analyzed, including CD36 coefficient of variation (CV) and CD71CV. According to our previous study, the percentage of CD117+CD105- myeloid progenitor cells and the proportion of CD105+ cells in CD117+ cells were selected to establish a two-parameter scoring system, and compared with the four-parameter scoring system and the erythroid scoring system. RESULTS: The sensitivity of the four-parameter scoring system and the erythroid scoring system for the diagnosis of low-risk MDS was 43.5% and 63.0%, and the specificity was 87.0% and 63.9%, respectively. After combining the two scoring systems, the sensitivity to diagnose low-risk MDS was 73.9% and the specificity was 62.0%. The sensitivity of the two-parameter scoring system for the diagnosis of low-risk MDS was 76.1% with a specificity of 81.5%. Combined with the four-parameter scoring system, the sensitivity was increased to 78.3%, but the specificity was reduced to 71.3%. After combining with the erythroid scoring system, the sensitivity reached 87.0%, but the specificity was reduced to 54.6%. CONCLUSION: Using the two-parameter scoring system alone can achieve great sensitivity and specificity in the diagnosis of low risk MDS.


Assuntos
Síndromes Mielodisplásicas , Endoglina , Citometria de Fluxo , Humanos , Imunofenotipagem , Síndromes Mielodisplásicas/diagnóstico , Proteínas Proto-Oncogênicas c-kit , Estudos Retrospectivos
4.
Am J Hematol ; 94(5): 512-521, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30680765

RESUMO

This study evaluated the effects of pretransplantation minimal residual disease (pre-MRD) on outcomes of patients with acute lymphoblastic leukemia (ALL) who underwent unmanipulated haploidentical stem cell transplantation (haplo-SCT). A retrospective study including 543 patients with ALL was performed. MRD was determined using multiparametric flow cytometry. Both in the entire cohort of patients and in subgroup cases with T-ALL or B-ALL, patients with positive pre-MRD had a higher incidence of relapse (CIR) than those with negative pre-MRD in MSDT settings (P < 0.01 for all). Landmark analysis at 6 months showed that MRD positivity was significantly and independently associated with inferior rates of relapse (HR, 1.908; P = 0.007), leukemia-free survival (LFS) (HR, 1.559; P = 0.038), and OS (HR, 1.545; P = 0.049). The levels of pre-MRD according to a logarithmic scale were also associated with leukemia relapse, LFS, and OS, except that cases with MRD <0.01% experienced comparable CIR and LFS to those with negative pre-MRD. A risk score for CIR was developed using the variables pre-MRD, disease status, and immunophenotype of ALL. The CIR was 14%, 26%, and 59% for subjects with scores of 0, 1, and 2-3, respectively (P < 0.001). Three-year LFS was 75%, 64%, and 42%, respectively (P < 0.001). Multivariate analysis confirmed the association of the risk score with CIR and LFS. The results indicate that positive pre-MRD, except for low level one (MRD < 0.01%), is associated with poor outcomes in patients with ALL who underwent unmanipulated haplo-SCT.


Assuntos
Citometria de Fluxo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transplante de Células-Tronco , Adolescente , Adulto , Aloenxertos , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Cuidados Pré-Operatórios , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo
5.
Onco Targets Ther ; 11: 5121-5132, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30197523

RESUMO

Background: Daunorubicin is a traditional chemotherapeutic agent that plays a pivotal role in leukemia therapy. However, the dose-related toxicity remains a considerable challenge. The apoptosis-regulating gene, PDCD5, is downregulated in various tumors, including leukemias, and may provide a potential target for the diagnosis and treatment of leukemia. The purpose of this study was to construct a triple-regulated oncolytic adenovirus carrying a PDCD5 gene expression cassette (SG611-PDCD5) and explore the combined antitumor efficacy of SG611-PDCD5 in combination with low dose daunorubicin on leukemic cells. Materials and methods: A variety of leukemic cell lines, including K562, MEG-01, KG-1a, HL-60, SUP-B15, and BV-173, were cultured according to the providers' instructions. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and PCR. The tumor-selective replication of the constructed conditionally replicating SG611-PDCD5 and its antitumor efficacy in combination with daunorubicin were characterized in leukemic cell lines in vitro and in a nude mouse xenograft model. Cell viability was detected using cell-counting kit-8. Apoptosis was detected in whole living cells using flow cytometry and in paraffin-embedded tumor tissues using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The triple-regulated CRAd carrying SG611-PDCD5 and nude mouse xenograft models of K562 cells were successfully constructed. In vitro treatment with SG611-PDCD5 in combination with low-dose daunorubicin elicited more potent anti-proliferative and proapoptotic effects in leukemic cells in a dose-dependent manner. The Chou-Talalay analysis revealed synergistic anti-proliferative effects in all of the above cell lines. In the nude mice xenograft model, the tumor size in the control, daunorubicin, SG611-PDCD5, and combined treatment groups on day 10 were 170.1±47.8, 111.9±81.1, 60.7±12.3, and 33.2±17.5 mm3, respectively (all P<0.05). The results of the TUNEL assay showed significantly more apoptotic cells in the SG611-PDCD5 plus daunorubicin group than in the SG611-PDCD5 or daunorubicin groups alone (25±0.82, 12.5±2.27, and 7.8±2.67 apoptotic cells/field, respectively) (P<0.05). Conclusion: The findings suggest that combined treatment with SG611-PDCD5 and daunorubicin may be a promising strategy for enhancing chemosensitivity and thus lowering the dose-related toxicity of daunorubicin in leukemia therapy.

6.
Leuk Res ; 72: 12-19, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30059823

RESUMO

A seven-color panel was used to detect minimal residual disease (MRD) in T cell acute lymphoblastic leukemia (T-ALL) via flow cytometry (FCM). Its availability and clinical significance were studied in T-ALL patients with newly diagnosed (n = 64), relapsed (n = 48) and morphologically complete remission (n = 103). The following four features were used to identify immature cCD3+ T cells: CD34+, TdT+, but mCD3-/dim+, and CD45dim+. Among these features, either TdT or CD34 expression was the most useful and were found in 93.8% of patients at diagnosis and 86.7% of patients who relapsed. Although some of the immature markers had disappeared in 23 of 59 cases after therapy, only one case presented with a false negative MRD. Of the 74 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), MRD-positive patients showed a higher relapse rate, a higher cumulative incidence of relapse at 4 years and a shorter median relapse-free survival than MRD-negative patients at post-HSCT(72.7% vs 17.3%, P = 0.000; 100% vs 19.9%, P < 0.0001; and 16 months vs undefined, P < 0.0001). We demonstrated that this panel could be applied to>97% of T-ALL patients to detect MRD and predict relapse after allo-HSCT even in the absence of the initial immunophenotype.


Assuntos
Antígenos CD34/sangue , DNA Nucleotidilexotransferase/sangue , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Proteínas de Neoplasias/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adolescente , Adulto , Idoso , Aloenxertos , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Indução de Remissão , Taxa de Sobrevida
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 25(5): 1289-1294, 2017 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-29070097

RESUMO

OBJECTIVE: To preliminarily identify the existence of CD34- leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test. METHODS: Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. Lin-CD34+ CD38-(abbreviation, CD34+CD38-), Lin-CD34+CD38+ (abbreviation, CD34+CD38+) and Lin-CD34-CD38-CD45dimSSClow(abbreviation, CD34-"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in G0 phase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR. RESULTS: The 3 tested samples displayed the same tendency in ratio of the cells in G0 phase: CD34-"LSC">CD34+ CD38->CD34+CD38+. The paired t-test of 53 patients showed that frequency of ALDHbr cells of both CD34+CD38- and CD34-"LSC" cell fractions was significantly higher than that of CD34+CD38+ (P<0.01), furthermore, the ALDHbr cell frequency was significantly higher in CD34-"LSC" than that in CD34+ CD38- (P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34-"LSC" than that in other 2 cell fractions. CONCLUSION: CD34- LSC may exist in t(8;21) AML, and may be more primitive than CD34+ LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.


Assuntos
Antígenos CD34 , Leucemia Mieloide Aguda/imunologia , ADP-Ribosil Ciclase 1 , Animais , Medula Óssea , Citometria de Fluxo , Células-Tronco Hematopoéticas , Humanos , Camundongos , Células-Tronco Neoplásicas , Células-Tronco , Transplante Heterólogo
9.
Stem Cells Dev ; 26(22): 1648-1661, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28946811

RESUMO

Immune thrombocytopenia (ITP) is an autoimmune disease in which dendritic cells (DCs) play a crucial role in the breakdown of self-tolerance. Studies have identified the function of mesenchymal stem cells (MSCs) in promoting the development of regulatory DCs (regDCs). Our previous work revealed that MSCs in ITP exerted senescence, apoptosis, and impaired immunosuppressive effects on T and B cells. However, it is unclear whether the effects of MSCs on regDC induction are altered in ITP. Our data demonstrated that MSCs in ITP were impaired in inhibiting CD1a+ DC and CD14+ DC differentiation from CD34+ hematopoietic progenitor cells (CD34+ HPCs). DCs differentiated with MSCs in ITP exhibited an increased expression of costimulatory molecules CD80/CD86 and secretion of proinflammatory interleukin-12 (IL-12). Accordingly, the tolerogenic characteristics were deficient in DCs induced by MSCs in ITP. DCs differentiated with MSCs in ITP exhibited an impaired ability to inhibit CD3+ T cell proliferation, to suppress T helper (Th)1 cell differentiation, and to induce anergic and regulatory T cells (Tregs). The expression of Notch signaling components was measured in MSCs in ITP. Reduced expression of the ligand Jagged-1, the receptor Notch-1 intracellular domain (NICD-1), and the target gene Hes-1 was identified in MSCs in ITP. The addition of biologically active Jagged-1 to CD34+ HPCs was observed to promote regDC differentiation. When cultured on Jagged-1-coated plates, MSCs in ITP showed an enhancement of the Notch-1 pathway activation, Jagged-1 expression, and the function in inducing regDCs. Pretreatment with all-trans retinoic acid (ATRA) was found to partially restore the capacity of MSCs in both ITP patients and healthy controls in inducing CD34+-derived regDCs. Our data elucidated that MSCs in ITP were impaired in inducing CD34+-regDCs, associated with the Notch-1/Jagged-1 signaling pathway. ATRA could partially correct the impairment of MSCs, suggesting that ATRA could serve as a potential therapeutic alternative for ITP.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteína Jagged-1/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores Notch/metabolismo , Tretinoína/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Trombocitopenia/metabolismo
10.
DNA Cell Biol ; 36(12): 1099-1107, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28953414

RESUMO

Multiple myeloma (MM) patients commonly present abnormal expression of cancer-testis antigens, which may serve as immunotherapeutic targets and prognostic factors. We previously reported that preferentially expressed antigen of melanoma (PRAME) overexpression in bone marrow mononuclear cells is related to progression in MM patients treated with non-bortezomib-containing regimens. The mechanism underlying variations in PRAME expression remains unknown. To investigate the impact of gene copy number variation (CNV) on PRAME expression, plasma cells were sorted from 50 newly diagnosed patients and 8 healthy volunteers to measure PRAME transcript levels and gene copy numbers by real-time quantitative polymerase chain reaction. A total of 14 (28.0%), 7 (14.0%), and 29 (58.0%) patients exhibited overexpression, expression within the normal range, and low expression, respectively. PRAME overexpression was significantly related to a lower 1-year progression-free survival rate compared with PRAME low expression (20.0% vs. 88.9%, p = 0.043). The mean PRAME gene copy number relative to albumin (ALB) in normal samples was ∼1.0, whereas 4.0%, 24.0%, 70.0%, and 2.0% of patients had PRAME gene relative copy numbers of approximately 0, 0.5, 1.0, and 2.0, respectively. Patients with PRAME gene deletion (relative copy number of 0 or 0.5) had significantly higher frequency of PRAME nonoverexpression and lambda light chain expression than those with no deletion (p = 0.011 and 0.003). Thus, PRAME gene CNV occurs in MM. Gene deletion may be one mechanism leading to PRAME nonoverexpression and related to immunoglobulin lambda light chain locus rearrangement. PRAME overexpression in plasma cells might be an adverse prognostic factor for progression in MM.


Assuntos
Antígenos de Neoplasias/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Adulto , Idoso , Células da Medula Óssea/imunologia , Estudos de Casos e Controles , Feminino , Deleção de Genes , Expressão Gênica , Humanos , Cadeias lambda de Imunoglobulina/metabolismo , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real
11.
Leuk Res ; 43: 33-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26930456

RESUMO

To explore new risk predictors for a high risk of relapse in Philadelphia chromosome negative (Ph-) B cell acute lymphoblastic leukaemia (B-ALL) patients, 196 paediatric Ph- B-ALL patients (≤ 18 years) were retrospectively analysed. We mainly focus on investigating the prognostic value of CD38 and CD58 expression in leukemic blasts in these patients by four colour flow cytometry. The CD38+ CD58- group (n=16) had a higher relapse rate, a shorter 3-year event-free survival (EFS) and overall survival (OS) than the CD38+ CD58+ group (n=157; 31.3% vs 10.2%, P=0.04; 52.4% vs 92.3%, P<0.01; 32.5% vs 91.0%, P=0.01); CD38+ CD58- was an independent adverse prognostic predictor for relapse (hazard ratio [HR], 0.203; 95%CI, 0.063-0.656; P=0.01), 3-year EFS (HR, 0.091; 95%CI, 0.023-0.355; P<0.01) and OS (HR, 0.102; 95%CI, 0.026-0.3971; P<0.01) in this cohort, as determined by Cox multivariate analysis. We identified, for the first time, a higher risk population of paediatric Ph- B-ALL patients with CD38+ CD58- who had a higher relapse risk and a shorter survival. Our results may allow better risk stratification and individualized treatment.


Assuntos
ADP-Ribosil Ciclase 1/sangue , Antígenos CD58 , Proteínas de Neoplasias/sangue , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Taxa de Sobrevida
12.
Thromb Res ; 139: 1-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26916289

RESUMO

OBJECTIVES: The aim of this study was to investigate the role of prostacyclin (PGI2) in prolonged isolated thrombocytopenia (PT) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the effect of PGI2 on the activation and aggregation of platelets in PT. METHODS: We enrolled 37 patients with PT and 36 controls following allo-HSCT in this study. Platelet aggregation and activation and PGI2 levels were measured. Endothelial progenitor cells (EPCs) from either PT or control patients were cultured ex vivo with serum from either PT or control patients. PGI2 secretions were then measured. PGI2 was added to the platelets ex vivo, and platelet aggregation and activation and PI3K/Akt phosphorylation were analyzed. RESULTS: A higher PGI2 level was observed in the PT patients. The activation and aggregation of platelets were significantly lower in the PT patients. EPCs from PT patients cultured in PT serum secreted higher levels of PGI2, and PGI2 inhibited platelet activation and aggregation in a concentration-dependent manner ex vivo. PI3K/Akt phosphorylation of platelets was regulated by PGI2 after allo-HSCT. Disease status, serum PGI2 level and platelet aggregation were independent risk factors in patients with PT after allo-HSCT. CONCLUSIONS: Higher PGI2 levels and lower platelet activation and aggregation occurred simultaneously in PT patients. PGI2 inhibited platelet activation and aggregation, probably by regulating the phosphorylation of PI3K/Akt.


Assuntos
Epoprostenol/sangue , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Fosfatidilinositol 3-Quinases/metabolismo , Ativação Plaquetária , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombocitopenia/sangue , Trombocitopenia/etiologia , Adulto , Plaquetas/metabolismo , Plaquetas/patologia , Epoprostenol/metabolismo , Feminino , Humanos , Masculino , Agregação Plaquetária , Transdução de Sinais/efeitos dos fármacos , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Transplante Homólogo , Adulto Jovem
13.
J Transl Med ; 13: 145, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25948100

RESUMO

BACKGROUND: Regulatory T cells (Treg) in allografts are important for the prevention of graft-versus-host disease (GVHD) post-transplantation. The aim of this study was to compare the contents of Tregs and effector T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts (G-BM) and peripheral blood grafts (G-PB). METHOD: G-BM and G-PB were obtained from 20 allogeneic donors. T-cell subgroups, including conventional T cells and different types of Treg cells, as well as the percentage of Ki67 expression on CD4(+)CD25(high)Foxp3(+) Treg cells, were analyzed using flow cytometry. The levels of interferon-γ (IFN-γ) and interleukin-17 (IL-17) secreted by T cells stimulated with PMA and ionomycin were also determined by flow cytometry. RESULTS: The percentage of CD4(+)CD25(high)CD127(-/dim)CD62L(+) Treg cells was significantly higher in the G-BM group, with higher proportions of CD45RA(+) naïve Treg cells and higher expression of CD69 on Treg cells in G-BM (P < 0.05). The percentage of Ki67 expression in CD4(+)CD25(high)Foxp3(+) Treg cells in G-BM was significantly higher than that on G-PB. The suppressive functions of Treg cells in inhibiting T-cell activation were comparable between G-BM and G-PB. The proportions of CD4(+)CD25(-)CD69(+) Treg subsets as well as Th1 cells in G-BM were also significantly higher than those in G-PB (P < 0.001). The proportions of conventional T cells and Th17 effector cells were comparable in G-BM compared with those in G-PB. Thus, the ratio of conventional T cells and CD4(+)CD25(high)CD127(-/dim) regulatory T cells were lower in G-BM than that in G-PB (P = 0.014). CONCLUSION: In addition to the much higher T-cell counts in G-PB grafts that may contribute to more severe GVHD, the higher frequency of Treg cells and lower ratio of conventional T cells to Treg cells in G-BM compared with G-PB grafts might reduce GVHD post-transplantation in G-BM compared with G-PB transplantation.


Assuntos
Células da Medula Óssea/citologia , Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco de Sangue Periférico , Linfócitos T Reguladores/citologia , Adolescente , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Feminino , Citometria de Fluxo , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-17/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ionomicina/química , Antígeno Ki-67/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Pessoa de Meia-Idade , Acetato de Tetradecanoilforbol , Transplante Homólogo , Adulto Jovem
14.
Biol Blood Marrow Transplant ; 20(8): 1190-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24792870

RESUMO

Prolonged isolated thrombocytopenia (PT) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, it remains unclear whether abnormalities of the bone marrow (BM) microenvironment are involved in the pathogenesis of PT. This prospective, nested case-control study included 20 patients with PT, 40 matched patients with good graft function (GGF) after allo-HSCT, and 16 healthy donors (HDs). Cellular elements of the BM microenvironment, including BM endothelial cells (BMECs), perivascular cells, and endosteal cells, were analyzed via flow cytometry and via hematoxylin-eosin and immunohistochemical staining in situ. Moreover, stromal-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) were measured in the plasma of BM via an enzyme-linked immunosorbent assay. No significant differences in endosteal cells (15 per high-power field [hpf] versus 16 per hpf versus 20 per hpf, P > .05) were demonstrated among the patients with PT, GGF, and the HDs. The PT patients exhibited remarkable decreases in cellular elements of the vascular microenvironment, including BMECs (.01% versus .18% versus .20%, P < .0001) and perivascular cells (.01% versus .12% versus .13%, P < .0001), compared with the GGF allo-HSCT recipients and the HDs, respectively. Moreover, significantly lower levels of SDF-1 (3163 pg/mL versus 3928 pg/mL, P = .0002) and VEGF (56 pg/mL versus 123 pg/mL, P < .0001) were found in the BM plasma of the PT patients compared with the BM of the GGF patients. A multivariate analysis revealed that BMECs (odds ratio [OR] = 171.57, P = .002) and cytomegalovirus infection after HSCT (OR = 4.35, P = .009) were independent risk factors for PT. Our data suggested that an impaired BM vascular microenvironment and megakaryocyte-active factors may contribute to the occurrence of PT after HSCT.


Assuntos
Células da Medula Óssea/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Trombocitopenia/etiologia , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo/efeitos adversos , Adolescente , Adulto , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/métodos , Adulto Jovem
15.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 22(1): 73-7, 2014 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-24598655

RESUMO

The purpose of this study was to establish a novel xenotransplant mouse model with human Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). The bone marrow mononuclear cells (BMMNC) were separated from newly diagnosed Ph(+)ALL patients, and injected into 2.1 Gy of (60)Co irradiated and anti-CD122-conditioned NOD/SCID mice through intra femoral injection. Human hematopoietic chimerism in bone marrow and spleen of the recipients was detected by flow cytometry. Morphological analysis of murine marrow cells were performed using May-Giemsa staining. BCR/ABL1 level was detected by RQ-PCR and FISH assays. Furthermore, leukemia infiltration in the organs was evaluated by hematoxylin-eosin staining, immunohistochemical staining with anti-human CD19 and anti-human CD34 antibodies. The results indicated that the unsorted BMMNC from Ph(+)ALL patients were able to repopulate human Ph(+)ALL in vivo. The percentages of human CD45(+)CD19(+) cells in bone marrow, and spleen of the recipient mice were 87.2% ± 10.1% and 79.9% ± 9.2%, respectively. Furthermore, the engrafted cells possessed same morphology, phenotypic and cytogenetic characteristics as cells from the original Ph(+)ALL patients. Compatible with the clinical features, transplanted Ph(+)ALL cells infiltrated into the brain, liver, and kidney of the recipients. It is concluded that the human-mouse xenotransplant established model using intra femoral injection of an anti-CD122-conditioned NOD/SCID repopulation may provide a promising system to study the biology of human Ph(+)ALL in vivo.


Assuntos
Modelos Animais de Doenças , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Adulto , Animais , Células da Medula Óssea/citologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Baço/citologia , Adulto Jovem
16.
Zhongguo Dang Dai Er Ke Za Zhi ; 16(3): 301-5, 2014 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-24661526

RESUMO

OBJECTIVE: To investigate the effects of 1,25-(OH)(2)D(3) on the airway remodeling and expression of high-mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) in the lungs among asthmatic mice. METHODS: Thirty female mice (BALB/c strain) were randomly divided into control, asthma and 1,25-(OH)(2)D(3) intervention groups. An asthmatic mouse model was established by intraperitoneal injection and aerosol inhalation of ovalbumin. The intervention group was given 1,25-(OH)(2)D(3) by intraperitoneal injection 0.5 hour before each aerosol inhalation, while the control group used normal saline instead. The hematoxylin-eosin staining was used to observe the mouse airway structural changes. The mRNA and protein expression of HMGB1 and TLR4 was measured by RT-PCR and immunohistochemistry, respectively. Pearson correlation analysis was performed. RESULTS: The asthma group had a significantly increased airway wall thickness compared with the control group (P<0.05); the intervention group had a significantly lower increase in airway wall thickness than the asthma group (P<0.05). The mRNA and protein expression of HMGB1 and TLR4 was significantly higher in the asthma group than in the control group (P<0.05); the mRNA and protein expression of HMGB1 and TLR4 in the intervention group was significantly lower than that in the asthma group, but still higher than that in the control group (P<0.05). A positive correlation was found between the protein expression of HMGB1 and TLR4 (P<0.01), and so was their mRNA expression (P<0.01). CONCLUSIONS: HMGB1 and TLR4 may be involved in asthmatic airway remodeling. 1,25-(OH)(2)D(3) can reduce the airway remodeling in asthmatic mice, which may be related to the downregulation of HMGB1 and TLR4 expression in the lungs of asthmatic mice.


Assuntos
Asma/tratamento farmacológico , Calcitriol/farmacologia , Proteína HMGB1/genética , Pulmão/metabolismo , Receptor 4 Toll-Like/genética , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Asma/metabolismo , Calcitriol/uso terapêutico , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise
17.
Beijing Da Xue Xue Bao Yi Xue Ban ; 46(1): 115-9, 2014 Feb 18.
Artigo em Chinês | MEDLINE | ID: mdl-24535362

RESUMO

OBJECTIVE: To explore the functional different natural killer (NK) cell subsets through the expression of killer immunoglobulin receptor (KIR) and CD57 on NK cells. METHODS: From May 2012 to June 2012, the peripheral blood samples of 10 related healthy donors for hematopoietic stem cells transplantation were collected to analyze KIR, CD57 expression and the intracellular cytokines of interferon-γ(IFN-γ), and the CD107a secreted by NK cells through 6-colour flow cytometer to compare the cytokine secretion and cytotoxic function among different NK subset. RESULTS: The expression of CD57 on NK cells were significantly higher than those of KIR on NK cells[(60.71% ± 5.71%) vs. (24.47% ± 3.95%), P < 0.001]. All the NK cells were separated into KIR+CD57-, KIR+CD57+, KIR-CD57+, KIR-CD57- cells based on the expressions of KIR and CD57. The proportions of KIR-CD57+ NK cells (43.03% ± 5.70%) and KIR-CD57-NK cells (32.45% ± 5.50%) among NK cells were comparable(P = 0.189), and were higher than those of KIR+CD57+ NK cells (17.67% ± 3.39%) and KIR+CD57- NK cells (6.69% ± 0.95%). Further functional experiments demonstrated that the cytotoxic function and IFN-γ cytokine secretion of CD57+ NK cells and KIR+ NK cells were comparable, which were significantly lower than those of CD56(bri) NK cells (P = 0.046 and 0.035, respectively), but were equal to those of CD56(dim) NK cells. The cytotoxic function and the IFN-γ secretion of KIR-CD57- NK cells (46.22% ± 9.24% and 23.41% ± 5.82%) were significantly higher than those of the other NK subsets including KIR+CD57- NK cells,KIR-CD57+ NK cells and KIR+CD57+ NK cells, which were similar to those of CD56(bri) NK cells. The cytotoxic function and IFN-γ secretion of KIR+CD57- NK cells were lower than those of KIR-CD57- NK cells, but were higher than those of CD57+ NK cells, whether KIR positive or negative. The cytotoxic function and IFN-γ secretion were similar between KIR+CD57+ and KIR-CD57+ cells. CONCLUSION: The expressions of KIR and CD57 are correlated with the function of NK cells. Therefore, CD57+ cells might be the end stage of NK cells, KIR-CD57- NK cells might be the early stage of NK cells, however, KIR+CD57- showed to be the intermediate stage of the NK cells.


Assuntos
Antígenos CD57/metabolismo , Células Matadoras Naturais/imunologia , Receptores KIR/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Interferon gama/metabolismo
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 21(6): 1385-9, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24370016

RESUMO

This study was purposed to compare the immunophenotypic and clinical characteristics of NPM1 mutated acute myeloid leukemia with a normal karyotype under the similar constituent ratio of FAB subtypes. Immunophenotyping and NPM1 gene mutation type-A,B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 77 AML patients with a normal karyotype (NK) and mutated NPM1 gene (NPM1m(+)AML) detected by immunophenotyping assay were included in this study. 55 cases without NPM1 mutation (NPM1m(-)AML) and with normal karyotype were served as negative control. The results showed that there was significant difference between NPM1m(+)AML and NPM1m(-)AML in terms of sex, white blood count, platelet counts, blast, WT1 expression level, FLT3-ITD mutation positive rate and response to treatment. The characteristic immunophenotype is lower expression of early differentiation-associated antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2) and higher expression of CD33 and CD123 (P < 0.05). When above features was further analyzed between the M1/2 and M4/5 subgroups in NPM1m(+)AML patients, the M1/2 cases retained a higher frequency in women and a higher WT1 expression level (P < 0.05) . Monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens CD7 were higher expressed and CD117 was lower expressed in M4/5 subgroup (P < 0.05). It is concluded that under condition of similar constituent ratio of M1/2 and M4/5 subtype and normal karyotype, NPM1m(+)AML patients have higher WT1 expression level and better response to treatment. The major immunophenotype features of NPM1m(+)AML patients are lower expression of early differentiation antigens and lymphoid lineage antigens and higher expression of CD33 and CD123. Monocytic differentiation-associated antigens only higher are expressed in M4/5 cases when compared with M1/2 cases within NPM1m(+) AML patients.


Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Cariótipo , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
19.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 21(6): 1585-90, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24370053

RESUMO

This study was aimed to distinguish abnormal cells and to diagnose hematologic diseases through recognizing antigen expression pattern and percentage of peripheral blood cells in normal elderly men. Antigen expression of blast cells, granulocytes, monocytes, lymphocytes, nucleated red blood cells and plasma cells was detected by seven-color flow cytometry in a total of 88 peripheral blood samples from normal elderly men, aged median 82 years old, from 70 to 98 years. Groups were divided according to age, region and underlying diseases, and the percentages of different subgroup cells were examined to confirm whether the differences were significant or not. The results showed that the median proportion of CD34(+) blast cells in peripheral blood from normal elderly men were 0.017% (0.015%-0.020%), with high expression of HLA-DR, CD33, CD13 and CD117, low expression of myeloid antigens, such as CD15, CD11b and CD16, while lymphoid antigens were seldom positive, including CD7, CD19 and CD56. Dim-expression of CD38 was found in peripheral blood blast cells, CD38(dim)+/- cell percentage in blast cells was 61.36% ± 18.26%. In the differentiation and development of granulocytes, CD16(-), CD13(+) CD16(+) (intermediate) and CD16(+) (strong) CD13(+) cells appeared in sequence from immature to mature granulocytes, whose median proportions in nuclear cells were 0.04%, 0.30% and 61.30%, respectively. The percentages of immature monocytes, such as CD64(+) CD14(-) and HLA-DR(+) CD11b(-) cells, were from 0.00% to 0.10% and from 0.07% to 0.68%, separately. No significant differences were found between different subgroups (P > 0.05). It is concluded that the immunophenotypic characteristics and referential percentages of CD34(+) blast cells, granulocytes and monocytes with different development stages in peripheral blood from normal elderly men are recognized, which can help to discriminate abnormal cells.


Assuntos
Células Sanguíneas/imunologia , Imunofenotipagem , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Citometria de Fluxo/métodos , Humanos , Contagem de Leucócitos , Masculino
20.
Zhonghua Xue Ye Xue Za Zhi ; 34(9): 745-50, 2013 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-24103870

RESUMO

OBJECTIVE: To compare the differences of the T helper cell reconstitution kinetics between HLA matched or HLA mismatched allo-HSCT through exploring the reconstitution kinetics of CD4+ CD25+Foxp3+ cells (CD4+ Treg), CD8+CD25+Foxp3+ cells (CD8+Treg), CD4+CD25-CD127+ conventional T cells (Tcon) and the secretion of IL-17a and IFN-γ in CD4+ T cells (Th17 and Th1 cells) or CD8+ T cells (Tc17 and Tc17 cells) post allogeneic hematopoietic stem cells transplantation (allo-HSCT). METHODS: From December 2011 to October 2012, the peripheral blood (PB) of 20 patients undergoing HLA matched (10 patients) or mismatched (10 patients) allo- HSCT without acute graft-versus-host disease (aGVHD) and of 10 related healthy donors were collected to analyze the expression of CD25+Foxp3+, IL-17a, IFN-γ and CD127 expression through 8-colour Flow cytometer. RESULTS: (1) The reconstitution kinetics of CD3+ T cells, CD4+ T cells, CD8+ T cells absolute numbers were comparable within 2 month post HLA matched and mismatched transplantation. (2)The absolute numbers of CD4+ Treg cells[+30 d, 8.46 (0.36-27.41) cells/µl 1.10 (0.04-8.03) cells/µl, P<0.05; +60 d, 8.50 (1.16-36.20) cells/µl vs 2.73 (0.34-6.84) cells/µl, P<0.05], Tcon cells[+30 d, 72.69 (3.85-211.73) cells/µl vs 13.41 (0.48-96.17) cells/µl, P<0.05; +60 d, 100.85 (16.28-267.20) cells/µl vs 47.75 (6.34-143.04) cells/µl, P<0.05], as well as Th17 cells[+30 d, 2.34 (0.02-6.87) cells/µl vs 0.20 (0.02-1.34) cells/µl, P<0.05; + 60 d, 1.90 (0.36- 7.82) cells/µl vs 0.46 (0.03-1.39) cells/µl, P<0.05]and Tc17 cells[+ 30 d, 1.08 (0.07-15.03) cells/µl vs 0.25 (0.01- 0.81) cells/µl, P<0.05;+60 d, 1.85 (0.63-26.57) cells/µl vs 0.46 (0.01-3.66) cells/µl, P<0.05]within 2 month post HLA matched HSCT were significantly higher than those post HLA- mismatched HSCT. However, the absolute numbers of Th1 cells or Tc1 cells within 2 month post HLA-matched or HLA-mismatched HSCT were comparable. (3) The ratio of Th1 and Th17 cells, or the ratio of Tc1 and Tc17 cells were significantly higher within 2 month post HLA-mismatched allo-HSCT compared to those post HLA-matched HSCT. CONCLUSION: The reconstitution kinetics of T helper cells subset were different at early stage post HLA-matched or HLA-mismatched allo-HSCT, which might be help to explain the different rate or the different involved organ of the acute graft-versus-host diseases (aGVHD) post HLA-matched or -mismatched allo-HSCT.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Linfócitos T Auxiliares-Indutores , Adulto , Feminino , Antígenos HLA , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores , Células Th1 , Células Th17 , Transplante Homólogo , Adulto Jovem
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