Increased exhausted CD8+ T cells with programmed death-1, T-cell immunoglobulin and mucin-domain-containing-3 phenotype in patients with multiple myeloma.

AIM: The immunosuppressive microenvironment plays a crucial role in T-cell immunodeficiency in multiple myeloma (MM). Overexpression of T-cell immunosuppressive receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain-containing-3 (Tim-3), may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow (BM) microenvironment may contribute to such immunosuppression. The purpose of this study was to analyze the distribution of PD-1+ and/or Tim-3+ T cells in different T-cell subset in patients with MM. METHODS: The expression of PD-1 and Tim-3 with exhausted (CD244+ and CD57+ ) CD3+ , CD4+ and CD8+ T cells between BM and peripheral blood (PB) from 10 patients with untreated MM was detected by multicolor flow cytometry assay. RESULTS: A significant increase in both PD-1+ CD57+ and Tim-3+ CD57+ CD3+ T cells and PD-1+ Tim-3+ CD3+ T cells was detected in PB from patients with MM compared with 10 healthy individuals (HIs), and the alteration was mostly in the CD8+ T-cell subset. Significant higher percentage of PD-1+ CD3+ T cells was found in BM compared with PB from patients with MM. The level of PD-1+ Tim-3+ CD3+ , CD4+ , and CD8+ T cells was high in BM group compared with PB. Moreover, PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells, particularly PD-1+ CD244+ and PD-1+ CD57+ CD8+ T cells were significantly higher in BM than in PB. In addition, limited dynamic detection data from three MM cases who achieved complete remission after treatment showed that the numbers of either PD-1+ or PD-1+ Tim-3+ T cells in different T-cell subsets were decreased in both BM and PB. CONCLUSION: We characterized the distribution of PD-1 and TIM-3 concurrent with exhausted CD3+ , CD4+ and CD8+ T cells between BM and PB from patients with MM. Higher numbers of PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells in BM from patients with MM may contribute to mediate the BM immunosuppressive microenvironment. Although heterogeneous alterations in Tim-3+ T cells may represent a complex immunosuppressive pattern in MM. Overall, higher levels of PD-1+ CD244+ or PD-1/Tim-3+ CD57+ CD8+ T cells may be a major reason for lower T-cell activation and T-cell immunodeficiency in MM.

Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia.

OBJECTIVE: To investigate the association between the T cell inhibitory receptor programmed death 1 (PD-1) and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia (AML) and AML in complete remission (CR). METHODS: Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3+, CD4+ and CD8+ T cells from peripheral blood samples from 20 newly diagnosed, untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry. Twenty-three healthy individuals served as control. RESULTS: A significantly higher percentage of PD-1+ cells were found for CD3+ T cells in the de novo AML group compared with healthy controls. In addition, an increased level of PD-1+CD8+ T cells, but not PD-1+CD4+, was found for CD3+ T cells in the de novo AML and AML-CR samples. A higher percentage of CD244+CD4+, CD244+CD8+, CD57+CD4+ and CD57+CD8+ T cells was found in CD3+ T cells in samples from those with de novo AML compared with those from healthy controls. Strong increased PD-1+CD244+ and PD-1+CD57+ co-expression was found for CD4+ and CD8+ T cells in the de novo AML group compared with healthy controls. CONCLUSIONS: We characterized the major T cell defects, including co-expression of PD-1 and CD244, CD57-exhausted T cells in patients with de novo AML, and found a particular influence on CD8+ T cells, suggesting a poor anti-leukemia immune response in these patients.

Overexpression of MALT1-A20-NF-κB in adult B-cell acute lymphoblastic leukemia.

BACKGROUND: A20 is a dual inhibitor of NF-κB activation and apoptosis in the tumor necrosis factor receptor 1 signaling pathway, and both are related to tumorigenesis. A20 is frequently inactivated by deletions and/or mutations in several B and T cell lymphoma subtypes; however, knowledge of the role of A20 in B-cell acute lymphoblastic leukemia (B-ALL) remains limited. In this study, we characterized the A20 gene expression pattern, the expression level of its upstream regulating factor MALT1, and its downstream target NF-κB in adult B-ALL. METHODS: The expression level of MALT1, A20 and NF-κB1 was detected in peripheral blood mononuclear cells (PBMCs) from 20 patients with adult B-ALL (including 12 de novo B-ALL and 8 refractory/relapse B-ALL cases), and nine patients with B-ALL in complete remission (CR) using real-time PCR. Sixteen healthy individuals served as controls. RESULTS: Significant A20 overexpression was found in the B-ALL (median: 13.489) compared with B-ALL CR (median: 3.755) (P = 0.003) patients and healthy individuals (median: 8.748) (P = 0.002), while there was no significant difference in A20 expression between B-ALL CR patients and healthy individuals (P = 0.107). Interestingly, the A20 expression level in the B-ALL samples was relatively different with approximately 50% of the B-ALL cases showing a relatively high A20 expression level, while the remaining 50% cases demonstrated slight upregulation or a similar expression level as the healthy controls. However, there was no significant difference in the A20 expression level between de novo B-ALL (median 12.252) and refractory/relapse B-ALL patients (median 21.342) (P = 0.616). Similarly, a significantly higher expression level of NF-κB1 was found in the B-ALL (median 1.062) patients compared with healthy individuals (median 0.335) (P < 0.0001), while the NF-κB1 expression level was downregulated in the B-ALL CR group (median 0.339), which was significantly lower than that in those with B-ALL (P = 0.001). Moreover, the MALT1 expression level in B-ALL was upregulated (median 1.938) and significantly higher than that in healthy individuals (median 0.677) (P = 0.002) and B-ALL CR patients (median 0.153) (P = 0.008). The correlation of the expression levels of all three genes was lost in B-ALL. CONCLUSIONS: We found that MALT1-A20-NF-κB is overexpressed in adult B-ALL, which may be related to the pathogenesis of B-ALL, and this pathway may be considered a potentially attractive target for the development of B-ALL therapeutics.

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 QuantibodyArray 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.

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 Vß 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 Vß subfamily T cells expressed was increased; however, all of the TCR Vß 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 Vß repertoire in AML patients.

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.

[Overexpression of eIF4E gene in acute myeloid leukemia and its relation with disease progression].

The aim of this study was to detect the expression level of eIF4E gene in patients with non-treated, remission and non-remission/relapse acute myeloid leukemia (AML), and other non-malignant haematologic diseases so as to analyze and reveal the relationship of eIF4E gene expression with AML progression. SYBR Green I RT-PCR was used to assay the expression level of eIF4E mRNA extracted from bone marrow mononuclear cells in 30 patients with AML (6 in M2, 5 in M3, 8 in M4, 10 in M5, 1 in M6) and 20 patients with non-malignant hematologic diseases. The ß2-microglubin(ß2M) was used as internal reference and the formula 2(-ΔCt)×100% was applied to calculate the expression level of eIF4E gene. The results showed that the eIF4E expression level (7.098 ± 5.544)% in patients with non-treated and non-remitted/relapsed AML was significantly higher than that in patients with remission (0.964 ± 0.312)% (P < 0.01) and non-malignant hematologic diseases (0.248 ± 0.163)% (P < 0.01). There was no difference between latter two group patients, even though the expression level of eIF4E gene in patients with M4 and M5 was higher. As compared with non-malignant hematologic diseases, the expression level of eIF4E gene of patients with remission patients showed no significant difference. It is concluded that the over-expression of eIF4E gene has been found in patients with AML, and its level obviously decreases along with remission of disease, thus the eIF4E gene may be a surveillance parameter for disease progression.

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

BACKGROUND: Allogeneic 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. METHODS: sjTRECs 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. RESULTS: sjTRECs 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. CONCLUSIONS: Reconstitution 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.

INTRODUCTION: The 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. OBJECTIVE: To investigate the dynamic change of clonal proliferation of T cell receptor (TCR) Valpha and Vbeta subfamilies in one CML patient who developed Ph-negative acute lymphoblastic leukemia (ALL) after interferon and imatinib therapy. METHODS: The 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 Valpha and Vbeta repertoire were detected by RT-PCR. The PCR products were further analyzed by genescan to identify T cell clonality. RESULTS: The CML patient who achieved complete cytogenetic remission (CCR) after 5 years of IFN-alpha therapy suddenly developed Ph-negative ALL 6 months following switch to imatinib therapy. The expression pattern and clonality of TCR Valpha/Vbeta T cells changed in different disease stages. The restrictive expression of Valpha/Vbeta subfamilies could be found in all three stages, and partial subfamily of T cells showed clonal proliferation. Additionally, there have been obvious differences in Valpha/Vbeta subfamily of T cells between the stages of Ph-positive CML-CP and Ph-negative ALL. The Valpha10 and Vbeta3 T cells evolved from oligoclonality to polyclonality, the Vbeta13 T cells changed from bioclonality to polyclonality, when Ph-negative ALL developed. CONCLUSIONS: Restrictive usage and clonal proliferation of different Valpha/Vbeta 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 TRGV and TRDV repertoire distribution and clonality in patients with immune thrombocytopenic purpura.

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 gammadelta(+)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.

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.

[Construction and expression of idiotype TCR Valpha1-pIRES-TCR Vbeta8 eukaryotic vectors in vitro].

AIM: To construct the eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 of Jurkat cell line and investigate their expression in vitro after transferred into eukaryotic cells. METHODS: TCR Valpha and Vbeta subfamilies of Jurkat cells were analyzed by using RT-PCR and genescan technique. Then the monoclonal TCR Valpha1 and Vbeta8 genes of Jurkat cells were cloned into multiple clone site (MCS) A and MCS B of the eukaryotic vector pIRES respectively. Its sequences were identified by restriction enzyme cutting and sequence analysis. The expression of TCR mRNA and idiotypic protein in transferred A549 and Molt4 cells was tested by RT-PCR, indirect immunophenotyping fluorescein dyeing and flow cytometry, respectively. RESULTS: Two recombinavot eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 were developed successfully and the expression of both idiotypic TCR mRNA and protein was detected in transferred A549 and Molt4 cells. CONCLUSION: The successful construction of the two eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 will provide a basic method for further study of the specific TCR gene modified T cells.

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

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.

[Low dose heparin for the prevention of hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the safety and efficacy of low dose heparin for the prevention of hepatic veno-occlusive disease (VOD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) conditioned with busulfan and cyclophosphamide (BU-CY2) and the ideal time frame of the heparin administration. METHODS: From April 1997 to December 2005, 134 patients (75 male, 59 female; median age 25 years old) were transplanted in our bone marrow transplantation unit. All patients were conditioned with BU-CY2 or busulfan-based regimen and scheduled to receive a continuous injection of heparin at a dose of 100 IUxkg-1xd-1. The patients were divided into different groups: 87 patients received related donor transplantation and 47 patients unrelated donor transplantation; 40 patients had abnormal liver function prior to transplantation and the remaining 94 patients normal liver function; 68 patients received heparin from day -7 to day +21 post HSCT and the other 66 patients from day -7 to day +14. RESULTS: All 134 patients had sustained engraftment. The median time for neutrophils to reach 0.5 x 10(9)/L and for platelets to reach 20x10(9)/L were 12 days (range 9-28) and 20 days (range 6-65), respectively. The disease-free survival at day +100 for leukemia patients was 82.2% (97/118). None of 134 patients developed VOD (0%), including 10 patients with class 3 thalassemia major. The incidence and severity of clinical bleeding events in 15 patients monitored with prothrombin time and activated partial thromboplastin time is comparable with those not monitored. CONCLUSIONS: It is suggested that low dose heparin alone is effective and safe for the prophylaxis of VOD following allo-HSCT conditioned with BU-CY regimen. Shortening the duration of administering heparin from -7 d to +21 d to -7 d to +14 d does not affect the effectiveness of low dose heparin to prevent VOD.

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

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 O/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.

Treatment of Epstein-Barr virus--associated lymphoproliferative disorder (EBV-PTLD) and pure red cell aplasia (PRCA) with Rituximab following unrelated cord blood transplantation: a case report and literature review.

OBJECTIVE: We report the first case of EBV-PTLD and pure red cell aplasia (PRCA) after an unrelated cord blood transplant in China, who was successfully treated with Rituximab. METHODS: Case report and literature review. RESULTS: A 5-year-old girl with CML underwent a major ABO-incompatible HLA-identical unrelated cord blood transplantation (U-CBT) in January 2003. The post transplant course was complicated by PRCA. She presented on day +75 with fever, followed by rapid enlargement of tonsils, and a cluster of lymph nodes in the cervical and submandibular regions. A cervical lymph node biopsy revealed the histopathologic findings consistent with PTLD. The immunoblasts were shown to contain EBV viral genomic DNA by PCR, and immunocytochemistry study for the latent membrane protein 1 (LMP-1) and in situ hybridization for Epstein-Barr encoded RNAs1 (EBER1) were both positive. She responded rapidly to Rituximab and achieved complete resolution of clinical findings and symptoms of both EBV-PTLD and PRCA. CONCLUSION: EBV-PTLD may occur with increasing frequency due to the increasing numbers of transplant recipients, transplant physician should be aware of this life-threatening complication. Rituximab alone may be an effective therapeutic strategy for patients who develop EBV-PTLD and PRCA after U-CBT.