Kanger Zhu

Incidence, risks, and outcome of idiopathic pneumonia syndrome early after allogeneic hematopoietic stem cell transplantation

We retrospectively analyzed 23 cases with early‐onset idiopathic pneumonia syndrome (IPS) of 192 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) from April 1997 to October 2007. Risk factors for IPS development were evaluated using Cox proportional hazards model, including age, gender, underlying disease, disease status at transplant, transplant type, conditioning regimens, donor type, acute graft‐vs.‐host disease (GVHD), severity of acute GVHD (aGVHD), human leukocyte antigen (HLA) disparity, and organ involvement of aGVHD. Factors that were significant at the 0.1 level on univariate analysis were evaluated by multivariate analysis. Twenty‐three of 192 patients developed IPS (12.0%). Median time to IPS onset after allogeneic HSCT was 76 d (range 32–120 d); median time to death after the diagnosis of IPS was 9 d (range 3–92 d); 20 patients with IPS died because of the rapid progression of respiratory failure (87.0%). Nineteen patients with IPS developed aGVHD (82.6%), with grade III–IV aGVHD in 11 patients (47.8%) and aGVHD of gut in 16 patients (69.6%). The following six factors were associated with an increased risk of IPS by univariate analysis: not in remission, unrelated donor, HLA disparity, occurrence of aGVHD, grade III–IV aGVHD and aGVHD of gut. These risk factors were entered into a multivariate analysis model. Only unrelated donor, grade III–IV aGVHD and aGVHD of gut are identified as being significantly associated with the occurrence of IPS, and among them, aGVHD of gut was associated with the largest risk of IPS, suggesting that the lung may be a target organ of aGVHD.

Clinical features and risk factors of pure red cell aplasia following major ABO-incompatible allogeneic hematopoietic stem cell transplantation

Abstract The objective of this paper was to study the incidence, risk factors, clinical outcome, management and prevention of pure red cell aplasia (PRCA) following major ABO-incompatible allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively analyzed 11 cases of PRCA from a series of 42 patients undergoing major ABO-incompatible allo-HSCT from April 1997 to December 2005. Eleven out of the 42 patients developed PRCA (26.1%). All the 11 cases of PRCA were in blood group O recipients of grafts from blood group A donor (n = 9) or blood group B donor (n = 2). The following factors were associated with an increased risk of PRCA: (1) blood group O recipient; (2) blood group A donor; and (3) blood group O/A in recipient/donor pair. Only blood group a/A in recipient/donor pair was identified as being significantly associated with the occurrence of PRCA by multivariate analysis. Six patients who received donor-type plasma exchange did not develop PRCA and among them 5 cases were the blood group O recipients. Eight patients obtained spontaneous remission and in the remaining 3 patients 2 with long-lasting PRCA were successfully treated with plasma exchange with donor-type plasma replacement and the other one who was also complicated by EBV-associated lymphoproliferative disorder (EBV-PTLD) responded rapidly to anti-CD20 monoclonal antibody and achieved complete resolution of clinical finding and symptom of both EBV-PTLD and PRCA. We conclude that blood group A/O in donor/recipient pair is identified as being significantly associated with the occurrence of PRCA by multivariate analysis. Donor-type plasma exchange and anti-CD20 monoclonal antibody is an effective approach for the treatment of PRCA. PRCA could be prevented by plasma exchange prior to transplantation.

Proliferation inhibition and apoptosis induction of imatinib-resistant chronic myeloid leukemia cells via PPP2R5C down-regulation

Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. The aim of this study was to evaluate proliferation inhibition and apoptosis induction by down-regulating PPP2R5C gene expression in the imatinib-sensitive and imatinib-resistant CML cell lines K562, K562R (imatinib resistant without an Abl gene mutation), 32D-Bcr-Abl WT (imatinib-sensitive murine CML cell line with a wild type Abl gene) and 32D-Bcr-Abl T315I (imatinib resistant with a T315I Abl gene mutation) and primary cells from CML patients by RNA interference. PPP2R5C siRNAs numbered 799 and 991 were obtained by chemosynthesis. Non-silencing siRNA scrambled control (SC)-treated, mock-transfected, and untreated cells were used as controls. The PPP2R5C mRNA and protein expression levels in treated CML cells were analyzed by quantitative real-time PCR and Western blotting, and in vitro cell proliferation was assayed with the cell counting kit-8 method. The morphology and percentage of apoptosis were revealed by Hoechst 33258 staining and flow cytometry (FCM). The results demonstrated that both siRNAs had the best silencing results after nucleofection in all four cell lines and primary cells. A reduction in PPP2R5C mRNA and protein levels was observed in the treated cells. The proliferation rate of the PPP2R5C-siRNA-treated CML cell lines was significantly decreased at 72 h, and apoptosis was significantly increased. Significantly higher proliferation inhibition and apoptosis induction were found in K562R cells treated with PPP2R5C-siRNA799 than K562 cells. In conclusion, the suppression of PPP2R5C by RNA interference could inhibit proliferation and effectively induce apoptosis in CML cells that were either imatinib sensitive or resistant. Down-regulating PPP2R5C gene expression might be considered as a new therapeutic target strategy for CML, particularly for imatinib-resistant CML.

GATA-1, -2 and -3 genes expression in bone marrow microenviroment with chronic aplastic anemia

Abstract Both bone marrow stromal cells (BMSCs) and transcription factors (GATA-1, GATA-2 and GATA-3) are important in the normal hematopoiesis and the pathogenesis of hematopoietic disease. The purpose of this study was to investigate the expression of GATA-1, GATA-2 and GATA-3 genes in the bone marrow (BM) microenvironment from patients with chronic aplastic anemia (cAA) and normal individuals. Mononuclear cells (MNCs) were isolated from BM of patients with cAA (8 cases) and normal controls (9 cases). Adherent cells (i.e. BMSCs) were collected after long-term culture in vitro. The semi-quantitative expression levels of GATA genes were analyzed by using RT-PCR-enzyme linked immunosorbent assay (RT-PCR-ELISA). The BMSCs with cAA grew slowly compared with the normal BMSCs. In BMSCs, only the expression ratio of GATA-3 gene from cAA group (50.0%) was significant lower than the normal controls (P < 0.05), the expression ratios of other GATA genes from cAA group were similar to the normal controls. There was no difference in the expression level of GATA-1 gene in the BMSCs between normal controls and cAA group. The expression level of GATA-2 gene in BMSCs from cAA was significantly lower than that from normal controls (P < 0.05). The expression level of GATA-3 gene in BMSCs from cAA was significantly higher than that from normal controls (P < 0.05). The dominant expression of GATA-3 gene was found in the BMSCs from cAA and normal controls. GATA genes can be expressed in the BMSCs and may play a role in the regulation of hematopoiesis in normal individuals, as well as in patients with cAA. The change of expression levels of GATA genes may influence the hematopoiesis in BM microenvironment and relate to the pathogenesis and development of aplastic anemia.

Frequency analysis of TRBV subfamily sjTRECs to characterize T-cell reconstitution in acute leukemia patients after allogeneic hematopoietic stem cell transplantation

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) leads to a prolonged state of immunodeficiency and requires reconstitution of normal T-cell immunity. Signal joint T-cell receptor excision DNA circles (sjTRECs) are markers of developmental proximity to the thymus that have been used to evaluate thymic function related to T-cell immune reconstitution after HSCT. To assess the proliferative history in different T-cell receptor beta variable region (TRBV) subfamilies of T cells after HSCT, expansion of TRBV subfamily-naive T cells was determined by analysis of a series of TRBV-BD1 sjTRECs.MethodssjTRECs levels were detected by real-time quantitative polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMCs) from 43 Chinese acute leukemia patients who underwent allo-HSCT. Twenty-three TRBV-BD1 sjTRECs were amplified by semi-nested PCR. Sixteen age-matched healthy volunteers served as normal controls.ResultssjTRECs levels were low or undetectable in the first 6 weeks after allo-HSCT and increased after 8 weeks post HSCT; however, sjTRECs levels at week 20 post-HSCT were still less than normal controls. Frequencies of TRBV subfamily sjTRECs in PBMCs from recipients at week 8 post-HSCT (29.17 ± 20.97%) or at week 16 post-HSCT (38.33 ± 9.03%) were significantly lower than those in donors (47.92 ± 13.82%) or recipients at pre-HSCT (45.83 ± 14.03%). However, frequencies of TRBV subfamily sjTRECs in recipients at week 30 post-HSCT (42.71 ± 21.62%) were similar to those in donors and recipients at pre-HSCT. sjTRECs levels in donors had a positive linear correlation with sjTRECs levels in recipients within 8-12 weeks post-HSCT. Patients with acute graft-versus-host disease (GVHD) or chronic GVHD had profoundly reduced TRECs levels during the first year post-HSCT. Frequencies of BV22-BD1 sjTRECs and BV23-BD1 sjTRECs in patients with GVHD were significantly lower than those in recipients at pre-HSCT, and the frequencies of BV22-BD1 sjTRECs in patients with GVHD were significantly lower than those in donors.ConclusionsReconstitution of thymic output function resulted in a period of immunodeficiency, with low or undetectable TRECs after transplantation, although fludarabine-based dose-reduced conditioning regimens were used. GVHD could affect reconstitution of thymic output function and reduce sjTRECs levels and frequencies of TRBV-BD1 sjTRECs. Low frequency of BV22-BD1 and BV23-BD1 sjTRECs might be associated with GVHD.

Evolution of T-cell clonality in a patient with Ph-negative acute lymphocytic leukemia occurring after interferon and imatinib therapy for Ph-positive chronic myeloid leukemia

IntroductionThe development of Philadelphia chromosome (Ph) negative acute leukemia/myelodysplastic syndrome (MDS) in patients with Ph-positive chronic myeloid leukemia (CML) is very rare. The features of restrictive usage and absence of partial T cell clones have been found in patients with CML. However, the T-cell clonal evolution of Ph-negative malignancies during treatment for CML is still unknown.ObjectiveTo investigate the dynamic change of clonal proliferation of T cell receptor (TCR) Vα and Vβ subfamilies in one CML patient who developed Ph-negative acute lymphoblastic leukemia (ALL) after interferon and imatinib therapy.MethodsThe peripheral blood mononuclear cells (PBMC) samples were collected at the 3 time points (diagnosis of Ph-positive chronic phase (CP) CML, developing Ph-negative ALL and post inductive chemotherapy (CT) for Ph-negative ALL, respectively). The CDR3 size of TCR Vα and Vβ repertoire were detected by RT-PCR. The PCR products were further analyzed by genescan to identify T cell clonality.ResultsThe CML patient who achieved complete cytogenetic remission (CCR) after 5 years of IFN-α therapy suddenly developed Ph-negative ALL 6 months following switch to imatinib therapy. The expression pattern and clonality of TCR Vα/Vβ T cells changed in different disease stages. The restrictive expression of Vα/Vβ subfamilies could be found in all three stages, and partial subfamily of T cells showed clonal proliferation. Additionally, there have been obvious differences in Vα/Vβ subfamily of T cells between the stages of Ph-positive CML-CP and Ph-negative ALL. The Vα10 and Vβ3 T cells evolved from oligoclonality to polyclonality, the Vβ13 T cells changed from bioclonality to polyclonality, when Ph-negative ALL developed.ConclusionsRestrictive usage and clonal proliferation of different Vα/Vβ subfamily T cells between the stages of Ph-positive CP and Ph-negative ALL were detected in one patient. These changes may play a role in Ph- negative leukemogenesis.

The feature of clonal expansion of TCR Vβ repertoire, thymic recent output function and TCRζ chain expression in patients with immune thrombocytopenic purpura

In order to identify the feature of T cells immune status in idiopathic/immune thrombocytopenic purpura (ITP) patients, TCR Vβ repertoire, T‐cell receptor (TCR) excision DNA circles (TRECs) and TCRζ chain gene expression were examined. Reverse‐transcriptase polymerase chain reaction, genescan and real‐time PCR techniques were used to analyze. DNA and cDNA from peripheral blood mononuclear cells (PBMCs) of 18 ITP patients were investigated and 25 normal individuals served as control. The results showed that only 4–11 Vβ subfamilies could be detected in each ITP case. The most frequently expressed Vβ subfamilies were Vβ2 and Vβ3, while 11 Vβ subfamilies were absent. Clonal expansion of Vβ T cells were found in all patients. The most frequent clonal expansion T cell was Vβ21. The number of TRECs in ITP patients (2.60 ± 2.99 copies/1000 PBMCs) was not significant different, compared with control (3.76 ± 3.42 copies/1000 PBMCs). The expression level of TCRζ gene was significantly lower in ITP samples than those in control (P = 0.017). In summary, T cells in ITP patients were characterized by restricted expression of TCR Vβ subfamilies and clonal expansion predominant in Vβ21. The alteration of peripheral TCR Vβ repertoire pattern may not relate to the thymic, recent output function in ITP patients. Our report is the first description of decreased TCRζ mRNA level in the majority of ITP patients.

Enhancement of the TCRζ expression, polyclonal expansion, and activation of t cells from patients with acute myeloid leukemia after IL-2, IL-7, and IL-12 induction.

Defective T cell receptor (TCR) signaling resulting in lower T cell function plays a crucial role in the pathogenesis of T cell immunodeficiency in leukemia. Previous studies have indicated that lower TCRζ levels are a common characteristic of patients with leukemia, and upregulating TCRζ could partially recover T cell function. In this study, we characterized the effect of the stimulating factor induction on the TCRζ, Zap-70, and FcɛRIγ levels, IFN-γ secretion, and the distribution and clonal expansion of TCR Vβ subfamilies in CD3+ T cells sorted from peripheral blood from acute myeloid leukemia (AML) patients. The induction included single stimulating factor or a combination with different cytokines (IL-2, IL-7, IL-2+IL-7, IL-7+IL-12, CD3, CD3+CD28 antibody, CD3+CD28 antibody+IL-2, and CD3+CD28 antibody+IL-7) at 72 h. The results showed that increased TCRζ and Zap-70 levels with deceased FcɛRIγ in T cells after induction, and different responses to cytokine in T cell from different cases may indicate th...Defective T cell receptor (TCR) signaling resulting in lower T cell function plays a crucial role in the pathogenesis of T cell immunodeficiency in leukemia. Previous studies have indicated that lower TCRζ levels are a common characteristic of patients with leukemia, and upregulating TCRζ could partially recover T cell function. In this study, we characterized the effect of the stimulating factor induction on the TCRζ, Zap-70, and FcɛRIγ levels, IFN-γ secretion, and the distribution and clonal expansion of TCR Vβ subfamilies in CD3(+) T cells sorted from peripheral blood from acute myeloid leukemia (AML) patients. The induction included single stimulating factor or a combination with different cytokines (IL-2, IL-7, IL-2+IL-7, IL-7+IL-12, CD3, CD3+CD28 antibody, CD3+CD28 antibody+IL-2, and CD3+CD28 antibody+IL-7) at 72 h. The results showed that increased TCRζ and Zap-70 levels with deceased FcɛRIγ in T cells after induction, and different responses to cytokine in T cell from different cases may indicate the heterogeneity of T cells and different immune statuses in different AML cases. Increased IFN-γ levels in T cells from AML patients were detected after induction in the IL-12+IL-7, CD3+CD28+IL-2, and CD3+CD28+IL-7 groups. Moreover, the number of TCR Vβ subfamily T cells expressed was increased; however, all of the TCR Vβ subfamily T cells in the AML patients could not be completely recovered after induction. In conclusion, the cytotoxicity and activation function of T cells could be enhanced after induction by different stimuli accompanied by an increase in TCRζ and Zap-70 and recovery of the TCR Vβ repertoire in AML patients.

The feature of TRGV and TRDV repertoire distribution and clonality in patients with immune thrombocytopenic purpura

Abstract Chronic idiopathic (immune) thrombocytopenic purpura (ITP) is an autoimmune disorder in which anti-platelet antibodies induce platelet destruction due to an imbalanced immune response. Recently, data indicated the γδ+T cells may play an important role in autoimmune disease. Our previous study has shown the restricted expression of TRBV subfamilies and the alteration of peripheral TRBV repertoire pattern in the majority of ITP patients. In the present study, we further analyze the feature of TRGV and TRDV repertoire distribution and clonality in patients with ITP. The CDR3 size of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 11 cases with ITP, using RT-PCR and GeneScan techniques. To determine the expression level of TRGV subfamily genes, quantitative analysis of TRGV I–III subfamilies was performed by real-time PCR. TRGV I–III subfamilies could be detected in the most samples from ITP as well as in healthy controls. However, clonal expansion of TRGV was identified in five cases with ITP, which displayed polyclonality in all of samples from healthy controls. The expression level of all TRGV I–III subfamilies in ITP was significantly lower than that from healthy controls (p=0·048, 0·001, 0·035, respectively). The expression pattern of TRGV I–III repertoire in ITP was TRGV I>TRGV III>TRGV II, in contrast, TRGV II>TRGV I>TRGV II was found in healthy controls. TRDV 1 and TRDV 2 could be detected in most samples from ITP as well as in healthy controls, whereas TRDV 3 could be detected in only two out of 11 cases with ITP, which could be found in 90% of healthy controls (p=0·02). Oligoclonally expanded TRDV 1 and TRDV 2 T cells could be identified in the half of the ITP samples, with similar results in healthy control. In conclusion, the alteration of peripheral TRGV and TRDV repertoire pattern might play a role in the pathogenesis of immune-mediated platelet destruction in some cases with ITP.

Upregulated TCRζ improves cytokine secretion in T cells from patients with AML

Previous studies indicated that upregulating TCRζ partially recovers T cell function in patients with leukemia. In this study, we characterized the cytokine profile of TCRζ-transfected T cells from acute myeloid leukemia (AML) patients by Quantibody®Array Glass Chip. Firstly, the significantly lower expression of TCRζ in CD3+/TCRζ+ cells from AML patients was found. Increased secretion of IL-2, IL-8, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, growth-regulated oncogene (GRO), MIP-1b, and regulated on activation, normal T cell expressed and secreted (RANTES) could be detected in T cells from AML patients after TCRζ upregulating. We concluded that upregulating TCRζ in T cells from AML can alter the secretion profile of cytokines and chemokine which are involved in T cell proliferation and activation.