Yanhui Yang

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.

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).

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.

The polymorphisms of T cell-specific TBX21 gene may contribute to the susceptibility of chronic immune thrombocytopenia in Chinese population

Chronic primary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by both reduced platelet counts and suppression of megakaryocyte and platelet development. T cell-specific T-box transcription factor gene (TBX21) plays a critical role in the development and maintenance of T helper 1 (Th1) cells. Recently, several studies have confirmed that the T-1554C and T-1993C polymorphisms of this gene can influence its expression level and are associated with autoimmune diseases. Therefore, we speculated that TBX21 polymorphisms might be associated with the susceptibility of chronic ITP in Chinese population. We investigated the distributions of TBX21 (T-1514C and T-1993C) polymorphisms in 275 patients with chronic ITP and 261 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. We observed significant overrepresentation of T allele and T/T genotype at T-1993C (but not T-1514C) in patients compared with controls. Stratified analysis by gender and age of disease onset revealed comparable observations in both female and childhood ITP cohorts. In conclusion, the T-1993C polymorphisms of TBX21 gene may be associated with the susceptibility of chronic primary ITP in Chinese population.

Acquired hemophilia a: retrospective analysis of 49 cases from a single Chinese hemophilia center.

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by the autoantibody directed against factor VIII in patients without previous history of a bleeding disorder. We retrospectively analyzed the characteristics and outcomes of 49 patients with AHA diagnosed in our center from February 1994 to October 2012. Twenty-four patients with acute bleeding episodes were treated with prothrombin complex concentrate (PCC) at a relative low dose of 30 to -50 U/kg/d and achieved good outcomes without any adverse reaction. Corticosteroids alone or in combination with cyclophosphamide were used as the first-line therapy to eradicate the inhibitors. In 39 evaluable patients, 35 (89.7%) achieved complete remission (CR). This study demonstrates that when bypassing agents such as recombinant activated factor VII and activated PCCs are not affordable or available, low dose PCC is effective and safe to control acute bleeding in patients with AHA. First-line therapy achieved good outcomes with a CR rate of 89.7%.

Lack of association between NR3C1 polymorphism and glucocorticoid resistance in Chinese patients with immune thrombocytopenia

Abstract Resistance to glucocorticoids (GCs) is a tricky problem in therapy for immune thrombocytopenia (ITP). As GCs exert their effects through glucocorticoid receptor (GR), being a GR gene, NR3C1 is thought to connect with individual differences in GC responsiveness during GCs treatments. We analyzed the frequency of three novel single nucleotide polymorphisms (SNPs) of NR3C1 in ITP patients and evaluated the role of these genetic variants in GCs therapy. Four hundred and seventy-three patients with ITP and 160 healthy controls were recruited. Patients were allocated into GCs-responsive (n = 358) and -non-responsive group (n = 115). All subjects of the three groups were genotyped by the PCR-RFLP (restriction fragment length polymorphism) method for the BclI, N363S and ER22/23EK polymorphisms. Assess the statistical differences of genotypes between ITP and controls, and those between GCs- responsive and non-responsive groups. In healthy controls, BclI-GG/GC/CC occurred with 0.581/0.35/0.069 frequency. In ITP patients, BclI-GG/GC/CC was found with 0.617/0.353/0.03 frequency. There was no statistically differences between ITP and controls (p = 0.070). In GCs-responsive and -non-responsive group, BclI-GG, GC, CC occurred with frequencies of 0.628/0.352/0.02 and 0.583/0.357/0.061, respectively. No correlations in the variants of BclI was found between the GCs-responsive and -non-responsive group (p = 0.086). Neither N363S nor ER22/23EK polymorphism was observed in all 636 participants. The BclI polymorphism is not related to the response of GCs in patients with ITP. Furthermore, we did not observe N363S and ER22/23EK polymorphism in Chinese Han population.

Absence of Association of Interleukin-18 Gene Polymorphisms with Primary Immune Thrombocytopenia in a Chinese Han Population

Interleukin-18 (IL-18) is an inflammatory cytokine that plays an important role in autoimmune disease by inducing interferon-γ secretion. Considering the abnormal serum concentration of IL-18 in primary immune thrombocytopenia (ITP) patients and the regulated effect of IL-18 gene polymorphisms on its production, the aim of this study was to investigate a possible association between the IL-18 promoter polymorphisms (-137 G/C and -607 C/A sites) and genetic susceptibility to ITP in a Chinese Han population. A total of 181 ITP patients and 163 healthy controls were included in this study; IL-18 gene promoter polymorphisms were analyzed by a polymerase chain reaction with sequence-specific primers. No significant differences in genotype (-607: χ(2)=0.307, p=0.858; -137: χ(2)=0.378, p=0.828) and allele frequencies (-607: χ(2)=0.004, p=0.949; -137: χ(2)=0.307, p=0.858) were found between total ITP patients and normal controls. We further analyzed the association of IL-18 polymorphisms with clinical parameters of ITP patients, including first onset age and clinical therapy response to glucocorticoids, and no difference was revealed. In conclusion, IL-18 promoter polymorphisms may not be associated with genetic susceptibility to ITP in a Chinese Han population.

Abnormal Distribution and Function of Monocyte Subsets in Patients With Primary Immune Thrombocytopenia.

Human monocytes are heterogeneous and play an important role in autoimmune diseases. However, the distribution and function of monocyte subsets remain unclear in primary immune thrombocytopenia (ITP). In this study, we determined the frequencies of monocyte subsets in 71 untreated patients with active ITP and 49 healthy controls by flow cytometry. Compared with controls, the frequency of nonclassical monocytes was significantly increased in patients with active ITP but decreased after complete remission. The intermediate subset was also increased in patients with active ITP and produced the highest levels of tumor necrosis factor α and interleukin 1β. Both the nonclassical and intermediate subsets were negatively correlated with the platelet counts. We further determined the correlation between monocyte subsets and the proliferation of platelet-autoreactive T cells. The purified monocyte subsets were cocultured with CD4+ T cells and autologous platelets. The nonclassical subset showed the highest capability of promoting platelet reactive T-cell proliferation and significantly promoted the secretion of interferon γ among the 3 subsets. In conclusion, the nonclassical and intermediate monocyte subsets are both expanded and play different roles in the pathogenesis of ITP.