Donglei Zhang

Analysis of calreticulin mutations in Chinese patients with essential thrombocythemia: clinical implications in diagnosis, prognosis and treatment.

Analysis of calreticulin mutations in Chinese patients with essential thrombocythemia: clinical implications in diagnosis, prognosis and treatment

Immunosuppressive function of mesenchymal stem cells from human umbilical cord matrix in immune thrombocytopenia patients

Human umbilical cord matrix/Whartons jelly (hUC)-derived mesenchymal stem cells (MSC) have been shown to have marked therapeutic effects in a number of inflammatory diseases and autoimmune diseases in humans based on their potential for immunosuppression and their low immunogenicity. Currently, no data are available on the effectiveness of UC-MSC transplantation in immune thrombocytopenia (ITP) patients. It was the objective of this study to assess the effect of allogeneic UC-MSCs on ITP patients in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BM-MNCs) from ITP patients and healthy controls were co-cultured with UC-MSCs for three days and seven days, respectively. Flow cytometry and ELISA were applied to assess the various parameters. In PBMCs from ITP patients, the proliferation of autoreactive T, B lymphocytes and destruction of autologous platelets were dramatically suppressed by UC-MSCs. UC-MSCs not only suppressed co-stimulatory molecules CD80, CD40L and FasL expression but also in shifting Th1/Th2/Treg cytokines profile in ITP patients. UC-MSCs obviously reversed the dysfunctions of megakaryocytes by promoting platelet production and decreasing the number of living megakaryocytes as well as early apoptosis. In addition, the level of thrombopoietin was increased significantly. Our clinical study showed that UC-MSCs play a role in alleviating refractory ITP by increasing platelet numbers. These findings suggested that UC-MSCs transplantation might be a potential therapy for ITP.

Bmi-1 Regulates Autoreactive CD4+ T Cell Survival in Immune Thrombocytopenia Patients

Autoreactive T cells in immune thrombocytopenia (ITP) patients undergo a rapid clonal expansion and are resistant to apoptosis to maintain continuous effect in thrombocytopenia. As Bmi-1 is involved in memory CD4+ T cell survival and Th2 proliferation, we hypothesized that Bmi-1 may have a role in autoreactive CD4+ T cell clonal expansion and Th1/Th2 development in ITP patients. We found that CD4+ T cells from active ITP patients had a higher Bmi-1 expression in comparison with remission and healthy controls, and autoreactive CD4+ T cells had more capability to proliferate and resistance to apoptosis than that of healthy controls. We evaluated the part that Bmi-1 played in proliferation and Th1 bias condition of autoreactive CD4+ T cells in ITP. We used lentiviral transfer vectors containing Bmi-1 and shBmi-1 to infect CD4+ T cells from ITP patients and healthy controls during autologous platelets stimulation. Flow cytometry and ELISA were applied to detect various parameters. The results showed that suppression of Bmi-1 using short hairpin RNA inhibited the platelet-mediated proliferation and increased apoptosis of autoreactive CD4+ T cells from ITP patients. Increased Bmi-1 expression in CD4+ T cells from healthy controls promoted the proliferation and inhibited apoptosis of CD4+ T cells. Bmi-1 significantly promoted interleukin-4 secretion by CD4+ T cells. These findings suggest that Bmi-1 plays a part in autoreactive CD4+ T cell proliferative capability and apoptotic resistance in ITP patients.

Association of ABCB1 gene polymorphisms and haplotypes with therapeutic efficacy of glucocorticoids in Chinese patients with immune thrombocytopenia.

Resistance to glucocorticoids (GCs) remains a tricky problem complicating the therapy of ITP. Recently, ATP binding cassette gene B1 gene (ABCB1) was reported to be correlated with susceptibility and therapeutic efficacy of autoimmune diseases through P-glycoprotein (Pgp). We investigated three single nucleotide polymorphisms (SNPs) of ABCB1 and their haplotypes by PCR-RFLP (restriction fragment length polymorphism) method in 471 ITP patients and 383 healthy controls, patients were further assigned into GCs-responsive and -non-responsive group according to the therapeutic effects of GCs. We observed a remarkable difference in genotypes of G2677T/A between GCs-responsive and non-responsive group, but not between patients and controls. A frequently expression of T/A allele within G2677T/A was recorded in GCs-responsive group. Furthermore, we found that some haplotypes (CGC, CTC/CAC, CTT/CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) were presented significantly differences between non-responsive and responsive group. No difference of C1236T and C3435T polymorphisms was observed between ITP and controls, and between the GCs-responsive and -non-responsive group. Our findings suggest that ABCB1 polymorphisms, as well as haplotypes derived from C1235T, G2677T/A and C3435T, are associated with inter-individual differences of GCs treatment in ITP.

Reduced expression of MIR409-3p in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with many immune dysfunctions. MicroRNAs (miRNAs) are a class of non‐coding RNAs that post‐transcriptionally regulate gene expression by messenger RNA degradation or translational repression. Accumulating evidence has demonstrated that miRNAs play a vital role in the regulation of immunological functions and prevention of autoimmunity. However, whether miRNAs are involved in the pathogenesis of ITP is still unknown. To illustrate the role of miRNAs in ITP, the expression profile of miRNAs from peripheral blood mononuclear cells (PBMCs) in ITP patients was investigated by miRNA microarray, and further validated by TaqMan real‐time polymerase chain reaction. MIR409‐3p expression was decreased in PBMCs of active ITP patients, but this recovered after effective therapy. IFNG was identified and validated as one of the targeted genes of MIR409‐3p by bioinformatic prediction and reporter gene analysis. In addition, we found DGCR8 transcript was down‐regulated in ITP patients and positively correlated with MIR409‐3p. Thus, in ITP patients, decreased DGCR8 leads to down‐regulation of MIR409‐3p, which in turn results in up‐regulation of IFNG (IFN‐γ).

External validation and clinical evaluation of the International Prognostic Score of Thrombosis for Essential Thrombocythemia (IPSET-thrombosis) in a large cohort of Chinese patients

In patients with essential thrombocythemia (ET), vascular complications contribute to both morbidity and mortality. To better predict the occurrence of thrombotic events, an International Prognostic Score of thrombosis for ET (IPSET‐thrombosis) was recently developed. We hereby presented an external validation and analysis of this model in a large Cohort of Chinese Patients.

The defective bone marrow-derived mesenchymal stem cells in patients with chronic immune thrombocytopenia

Abstract Chronic immune thrombocytopenia (ITP) is characterized by autoimmune-mediated platelet destruction and impairment of thrombopoiesis. Mesenchymal stem cells (MSCs) are proposed to exhibit immune modulatory functions in self-tolerance maintenance. In this study, we aimed to characterize phenotypically and functionally bone marrow (BM)-derived MSCs from adult chronic ITP patients. Our results showed that BM-MSCs from patients with chronic ITP exhibited impaired proliferation, abnormal morphology and excessive apoptosis, and these defects could be ameliorated by modifying the culture environment. BM-MSCs from chronic ITP patients were shown to have similar immunophenotype and capacities to differentiate along adipogenic and osteogenic lineages as those from normal controls. However, the immune-inhibiting potential and the regulatory T cell-inducing ability of BM-MSCs from patients were defective compared to that of normal BM-MSCs. These findings suggest that the BM-MSCs were defective in chronic ITP patients. Whether the defective BM-MSCs are relevant to the pathogenesis of chronic ITP remains to be determined.

Decreased IL-35 levels in patients with immune thrombocytopenia

IL-35 is a novel heterodimeric anti-inflammatory cytokine consisting of Epstein-Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12. IL-35 has been shown to possess the potency of inhibiting the CD4+ effector T cells and alleviating autoimmune diseases. In the study we investigated the levels of IL-35 as well as its prospective role in immune thrombocytopenia (ITP).ELISA was adopted to measure plasma IL-35, TGF-β and IL-10 levels. The mRNA expression levels of P35 and EBI3 in peripheral blood mononuclear cells (PBMCs) were studied based on real-time quantitative PCR. The correlation between plasma cytokine levels and clinical parameters was analyzed. Significantly lower plasma IL-35 levels were found in active ITP patients compared with those in remission (p = 0.017) and the healthy controls (p < 0.001). In active ITP patients, the plasma IL-35 levels displayed a significantly positive correlation with platelet counts (r = 0.5335, p < 0.0008). Further, P35 mRNA expression levels were lower in patients with active ITP than patients in remission (p = 0.033) and normal controls (p = 0.016).Thus, for the first time, this research reported a dramatically decreased IL-35 levels in ITP patients, suggesting that IL-35 may be involved in the pathogenesis of ITP.

Increased expressions of DNA methyltransferases contribute to CD70 promoter hypomethylation and over expression of CD70 in ITP

CD70 (TNFSF7), as a methylation susceptive immune gene, was hypomethylated in some autoimmune diseases. To investigate the status of CD70 methylation and the expressions of genes that regulated methylation in immune thrombocytopenia (ITP) patients, the expressions of CD70 mRNA and protein in CD4(+) T cells from ITP and controls were measured respectively by real-time PCR and flow cytometry. Methylation specific high resolution melting (MS-HRM) technology was applied to detect CD70 promoter methylation indices. Transcription levels of DNA methyltransferases (DNMTs), methylated CpG binding protein 2 (MBD2) were measured. The results showed that CD70, DNMTs and MBD2 was over expressed and methylation indices of CD70 promoter in CD4(+) T cells from ITP patients were lower than that from healthy controls. The transcription levels of CD70 showed significantly inverse correlation with methylation indices in ITP patients but significantly positive correlation with that of DNMTs. We concluded that DNMTs functioned as demethylases as MBD2, while increased DNMTs and MBD2 may cause demethylation and over expression of CD70 in CD4(+) T cells, potentially contributing to the pathogenesis of ITP.

Interleukin 35 may contribute to the loss of immunological self-tolerance in patients with primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder. Interleukin‐35 (IL35) can suppress T cell proliferation and elicit the development of inducible regulatory T cells (Tregs). Previous studies have shown decreased plasma IL35 levels and dysfunctional T cells in patients with ITP. In this study, we determined whether decreased IL35 levels correlate with T cell dysfunction in ITP patients. Plasma IL35 levels were found to be lower in ITP patients than in healthy controls, were positively correlated with platelet levels and the percentage of peripheral circulating Tregs, and negatively correlated with the levels of T helper‐1 cells in ITP patients. We also evaluated the effects of IL35 on cytokines contributing to T cell proliferation. IL35 promoted the secretion of interleukin 10 (IL10) and transforming growth factor–β1 but reduced the levels of interferon‐γ and IL17A (also termed IL17). Moreover, IL35 inhibited the proliferation of CD4+ and CD8+ T cells but induced the differentiation and proliferation of Tregs in ITP. In summary, IL35 appears to contribute to the loss of immunological self‐tolerance in ITP patients by modulating T cells and immunoregulatory cytokines.