iTRAQ-based quantitative proteomics analysis of immune thrombocytopenia patients before and after Qishunbaolier treatment.

RATIONALE: Treatment of immune thrombocytopenia (ITP) usually involves long-term use of immunosuppressive corticosteroids and splenectomy. However, these treatments often have side effects in patients. The Mongolian medicine Qishunbaolier (QSBLE) has a high curative effect, reduces the chances of relapse, and has no obvious side effects. This study was designed to identify potential therapeutic targets of QSBLE for treating ITP. METHODS: To reveal differences in protein expression between ITP patients (ITPs) before and after QSBLE treatment, comparative proteomics studies were performed using isobaric tags for relative and absolute quantification (iTRAQ). The analysis used nanospray liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) in positive ion electrospray ionization mode. Key proteins relevant to ITP were revealed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and other bioinformatics tools. Real-time polymerase chain reaction (RT-PCR) analysis was carried out for confirmation of differentially expressed proteins. RESULTS: A total of 982 differentially expressed proteins were identified in ITPs compared with the controls. Compared with the pre-QSBLE treatment group, 61 differentially expressed proteins were identified in the post-QSBLE treatment group, with 48 proteins being significantly upregulated and 13 downregulated. Twenty-nine pathways were significantly enriched. Q6N030 and other proteins were the key players in the protein-pathway network. Twenty proteins that may play important roles in the treatment of ITP were further filtered. RT-PCR and Western blot analyses further confirmed that MIF, PGK1 and IGHM were upregulated in ITPs after QSBLE treatment, in accordance with the proteomics data. CONCLUSIONS: It is believed that the identified proteins and the results of bioinformatics analysis will provide a potential therapeutic target site for QSBLE for ITP therapy and biomarkers.

The abnormal function of CD39+ regulatory T cells could be corrected by high-dose dexamethasone in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disease, characterized with decreased platelet and increased risk of bleeding. Recent studies have shown the reduction and dysfunction of regulatory T (Treg) cells in ITP patients. CD39 is highly expressed on the surface of Treg cells. It degrades ATP to AMP and CD73 dephosphorylates AMP into adenosine. Then adenosine binds with adenosine receptor and suppresses immune response by activating Treg cells and inhibiting the release of inflammatory cytokines from effector T (Teff) cells. Adenosine receptor has several subtypes and adenosine A2A receptor (A2AR) plays a crucial role especially within lymphocytes. The CD39+ Treg cells and the expression of A2AR showed abnormality in some autoimmune disease. But knowledge of CD39+ Treg cells and A2AR which are crucial in the adenosine immunosuppressive pathway is still limited in ITP. Thirty-one adult patients with newly diagnosed ITP were enrolled in this study. CD39 and A2AR expression was measured by flow cytometry and RT-PCR. The function of CD39 was reflected by the change of ATP concentration detected by CellTiter-Glo Luminescent Cell Viability Assay. CD39 expression within CD4+CD25+ Treg cells in ITP patients was decreased compared to normal controls. After high-dose dexamethasone therapy, response (R) group showed increased CD39 expression within Treg cells while non-response (NR) group did not show any difference in contrast to those before treatment. The expression of A2AR in CD4+CD25- Teff and CD4+CD25+ Treg cells was both lower in ITP patients than that of normal controls. After therapy, CD4+CD25- Teff cells had higher A2AR expression while CD4+CD25+ Treg cells did not show any difference in comparison to that before treatment. The enzymatic activity of CD39 was damaged in ITP patients and improved after high-dose dexamethasone therapy. In ITP, there was not only numerical decrease but also impaired enzymatic activity in CD39+ Treg cells. After high-dose dexamethasone treatment, these two defects could be reversed. Our results also suggested that ITP patients had reduced A2AR expression in both CD4+CD25+ Treg cells and CD4+CD25- Teff cells. CD4+CD25- Teff cells had increased A2AR expression after treatment.

Effect of glucocorticoid treatment on BAFF and APRIL expression in patients with immune thrombocytopenia (ITP).

Immune thrombocytopenic purpura (ITP) is an idiopathic bleeding disorder. B cell activating factor (BAFF) and 'A proliferation-inducing ligand' (APRIL) have regulatory effects on B and T cells and may represent relevant factors in the pathogenesis of ITP. Serum levels and gene expression were investigated in ITP patients. Both BAFF and APRIL serum levels were significantly elevated in active ITP. However, gene expression analysis revealed both factors to have a tendency toward downregulation. Glucocorticoid treatment significantly reduced BAFF but not APRIL serum levels, which may be mediated by differences in transcription factor binding sites. The glucocorticoid receptor binding site is present in the BAFF promotor region, but not in the APRIL promotor region. Prednisolone in combination with vitamin D3 may be effective in reducing APRIL serum levels. In conclusion, glucocorticoid treatment exerts different regulatory effects on both BAFF and APRIL, whereas antioxidant supplementation may also be beneficial in reducing serum levels.

Effect of Yiqi Tongyang Decoction () on blood T cell subsets in patients with chronic immune thrombocytopenia.

OBJECTIVE: To measure the proportions of blood T cell subsets, Th1, Th2, Th17, Th22, and Treg cells, and other parameters in patients with chronic immune thrombocytopenia (CITP) before and after treatment with Yiqi Tongyang Decoction (, YTD) to explore T cell status of patients with CITP, and to defifine the mechanism of action of YTD. METHODS: The changes in peripheral blood T lymphocyte subsets, and those of Th1, Th2, Th17, Th22, and Treg cells in 30 patients with CITP (22 females and 8 males) were analyzed using multiparametric flflow cytometry before and after treatment with YTD for 6 months, and 26 healthy volunteers (14 males and 12 females) acted as a control. T-box expressed in T-cells (T-bet) and GATA binding protein 3 (GATA-3) mRNA levels in patients and controls were analyzed using real-time reverse transcription-polymerase chain reaction. RESULTS: The proportions of Th1, Th17, Th22, Th1/Th2, and Th17/Treg cells increased in the peripheral blood of patients with CITP compared to those in controls before YTD therapy (P<0.05). Th1 cell numbers and the Th1/Th2 ratio fell in the treated patients with CITP to approximate the values of the control group (P>0.05). Th17 cell numbers and the Th17/Treg ratio also decreased in the treatment group (P<0.05), but not to the levels of the controls. The number of Treg cells in the peripheral blood of patients with CITP before treatment was lower than that in the control group (P<0.05), but increased after YTD treatment P<0.05), but not to the level of controls. T-bet and GATA-3 mRNA levels in peripheral blood were initially higher in patients before treatment than controls (P<0.05), but decreased after YTD therapy (P<0.05). CONCLUSIONS: Imbalances in T lymphocyte levels, particularly those of Th1/Th2 and Th17/Treg cells, play important roles in the pathogenesis of CITP. YTD effificiently regulated the dynamics of Th1/Th2 and Th17/Treg equilibria.

Profiling of miRNA expression in immune thrombocytopenia patients before and after Qishunbaolier (QSBLE) treatment.

Immune thrombocytopenia (ITP), also known as idiopathic thrombocytopenic purpura, is an autoimmune disease characterized by low platelet count and increased bleeding tendency. Currently, glucocorticoid and splenectomy are the main therapies for ITP but with obvious side effects including tendency of relapse and risk of internal bleeding. In this study, we report the Mongolian medicine Qishunbaolier (QSBLE) can significantly and efficiently increase platelet count with a low recurrent rate and unnoticeable side effect. We profiled the microRNA (miRNA) expression in the blood sample of ITP patients and identified 44 miRNAs that are differentially expressed in ITP patients before and after QSBLE treatment. Out of these 44 miRNAs, 25 are expressed in control subjects and are downregulated in ITP patients, whereas the treatment with QSBLE restores their expressions to the level of control subjects. This result suggests that abnormal expression of these 25 miRNAs might be connected to the pathogenesis of ITP. Interestingly, 14 of those 44 miNRAs are predicted to target at least once on 31 known IPT associated genes, indicating the possible mechanism of QSBLE on ITP therapy.

Bone mass and biochemical markers of bone turnover in children and adolescents with chronic immune thrombocytopenia: relation to corticosteroid therapy and vitamin D receptor gene polymorphisms.

Optional drug therapy in refractory chronic immune thrombocytopenia (ITP) includes standard oral, pulsed high-dose steroid therapy, intravenous gamma globulin, anti-D, and immunosuppressive therapy or thrombopoietin receptor agonists. This work aimed to study the bone mass in children and adolescents with chronic ITP in relation to biochemical markers of bone turnover, cumulative steroid therapy, and the possible modulating effect of vitamin D receptor (VDR) gene polymorphisms. Thirty-six children and adolescents with chronic ITP were recruited from the Hematology Clinic, Children's Hospital, Ain Shams University and the Hematology Clinic of the National Research Centre in Egypt and compared with 43 healthy age- and sex-matched controls. The total cumulative dose of steroids was calculated. Bone markers (serum osteocalcin (OC) and propeptide I precollagen (PICP) and urinary deoxypyridinoline (DPD) excretion), analysis of VDR gene distribution, and dual energy X-ray absorptiometry at lumbar and hip regions were performed for patients and controls. Compared to controls, chronic ITP patients had higher body mass index (BMI) and lower height for age standard deviation score (SDS). Chronic ITP patients had lower levels of OC and C-terminal propeptide of type I procollagen (PICP) and higher urinary DPD excretion, and bone mineral density (BMD) was significantly lower for both spine and hip z-score (<0.001). BMD was inversely correlated with urinary DPD excretion, age, BMI, and cumulative steroid dose. There was significant negative correlation between cumulative oral steroid dose and BMD (r = -0.4, P = 0.01 and r = -0.45, p = 0.001 for spine and hip z-scores, respectively), but the correlation was non-significant in relation to cumulative pulsed steroid therapy. FokI polymorphism was significantly related to BMD for both spine and hip z-score (p = 0.015 and p = 0.008, respectively), but there was no relation between BMD and Bsm1 polymorphism. FokI gene polymorphism may be one of the contributing factors in bone loss in patients on chronic steroid therapy. High cumulative doses of corticosteroids increased bone resorption in young chronic ITP patients. Longitudinal studies are needed to confirm the effect of different steroid protocols on bone turnover. Protocols of therapy of chronic ITP should restrict corticosteroid use in growing children and favor alternative less harmful therapies.