Jianzhi Sun

Profile of Th17 cytokines (IL-17, TGF-β, IL-6) and Th1 cytokine (IFN-γ) in patients with immune thrombocytopenic purpura

The data on polarization of the immune system towards T helper 1 (Th1) or T helper 2 (Th2) cells in immune thrombocytopenic purpura (ITP) are limited and contradictory. Th17 characterized by the production of Interleukin 17 (IL-17) has been shown to play a crucial part in the induction of autoimmune diseases. To further investigate the role of Th cytokines in the pathogenesis of ITP, we measured the plasma concentration of three Th17-associated cytokines [IL-17, transforming growth factor-ß (TGF-ß), IL-6] and Th1 cytokine interferon-γ (IFN-γ) in ITP patients, and evaluated their clinical relevance. The concentration of IL-17, TGF-ß, IL-6, and IFN-γ in plasma specimens from 29 adults with chronic ITP and 38 controls was analyzed by enzyme-linked immunosorbent assay method. No significant differences of Th17 cytokines (IL-17, TGF-ß, and IL-6) and Th1 cytokine (IFN-γ) concentration were observed between patients with active ITP and the control group. And the IFN-γ/IL-17 ratio representing Th1/Th17 cytokine profile was not significantly different between ITP patients and control, either. However, significantly positive correlation between IL-6 and IFN-γ in ITP patients was observed (r = 0.48, P = 0.01). Among the ITP patients, Plasma IL-17 levels in male were marginally higher than those in female, while similar for TGF-ß, IL-6 or IFN-γ. There was a significantly positive correlation between age and IL-6 concentration in ITP patients (r = 0.56, P = 0.0002), while no statistical significance between age and the other three cytokines. No significant correlation between cytokine concentrations and platelets or megakaryocytes number was found in ITP patients. In summary, ITP may not be associated with changes of plasma Th17 and Th1 cytokine concentrations relative to control population.

De novo induction of platelet-specific CD4+CD25+ regulatory T cells from CD4+CD25- cells in patients with idiopathic thrombocytopenic purpura

CD4(+)CD25(+) regulatory T cells (Treg) play the critical role in maintenance of peripheral immune tolerance. However, the numbers of naturally occurring Treg (nTreg) that can be isolated from periphery are far too small to be clinically effective. The isolation and expansion of nTreg for treatment of autoimmune diseases encounter great difficulties. Whether autoantigen-specific Treg could be converted from CD4(+)CD25(-) T cells in patients with autoimmune diseases has not been reported. Here, we demonstrated that platelet glycoprotein (GP)-specific induced Treg (GP-iTreg) could be generated de novo from nonregulatory CD4(+)CD25(-)CD45RA(+) cells in patients with idiopathic thrombocytopenic purpura and induced both antigen-specific and linked suppression. GP-iTreg mediated regulatory effects via modulating the T cell-stimulatory capacity of dendritic cells. By investigating the gene expression profile of iTreg-modulated dendritic cells, we provided a genome-wide assessment of the changes induced by antigen-specific iTreg and identified that the Toll-like receptor, Notch and transforming growth factor-beta signaling pathways were related to the GP-specific tolerance, with the Toll-like receptor pathway being dominant. The findings in patients with idiopathic thrombocytopenic purpura will facilitate our understanding of the mechanisms of induction and maintenance of autoantigen-specific tolerance and highlight the considerable potential of antigen-specific iTreg for targeted immunotherapy in human auto-immune diseases.

High-dose dexamethasone shifts the balance of stimulatory and inhibitory Fcγ receptors on monocytes in patients with primary immune thrombocytopenia

The human Fcγ receptor (FcγR) system is composed of 2 opposing families, the activating FcγRs (FcγRI, FcγRIIa, and FcγRIII) and the inhibitory FcγR (FcγRIIb). The disturbed balance of the activating and inhibitory FcγRs has been implicated in the pathogenesis of many autoimmune diseases. In this study, the expression of FcγRs on monocytes was determined in 23 patients with primary immune thrombocytopenia (ITP) before and after high-dose dexamethasone (HD-DXM) treatment. The FcγRI expression was significantly higher in ITP patients and decreased after HD-DXM treatment. The ratio of FcγRIIa/IIb mRNA expression on monocytes was significantly higher in untreated patients than in healthy controls. After HD-DXM therapy, the ratio decreased and the increased expression of FcγRIIb mRNA and protein coincided with a remarkable decrease in the expression of FcγRIIa, FcγRI, and monocyte phagocytic capacity. There was no significant difference in FcγRIII expression on monocytes between patients and controls. In vitro cell-culture experiments showed that DXM could induce FcγRIIa and FcγRIIb expression in monocytes from ITP patients, with FcγRIIb at higher amplitudes. These findings suggested that the disturbed FcγR balance might play a role in the pathogenesis of ITP, and that HD-DXM therapy could shift monocyte FcγR balance toward the inhibitory FcγRIIb in patients with ITP.

High-Dose Dexamethasone Inhibits BAFF Expression in Patients with Immune Thrombocytopenia

IntroductionB-cell activating factor belonging to the TNF family (BAFF) is elevated in several autoimmune diseases including immune thrombocytopenia (ITP). High-dose dexamethasone (HD-DXM) has shown its clinical efficacy in ITP patients. Materials and MethodsThe plasma BAFF concentration and BAFF mRNA were measured in ITP patients before and after oral administration of 40 mg/day DXM for four consecutive days by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR. Moreover, we evaluated the effects of DXM on BAFF expression and proliferation of lymphocytes by ELISA, real-time quantitative PCR and cell proliferation respectively in in vitro experiment.ResultsBoth plasma BAFF concentration and BAFF mRNA were significantly increased in active ITP patients at pretherapy when compared with controls (P < 0.001). After 4-day treatment with HD-DXM, the BAFF and BAFF mRNA were decreased, and lower than that for controls. In in vitro assays, we found DXM-inhibited BAFF, IFN-γ expression, and the proliferation of lymphocytes in a dose-dependent manner.ConclusionThese results suggest that BAFF expression is increased in ITP patients with active disease, and DXM is an effective inhibitor of BAFF production. As immunosuppressant, DXM may play its role in ITP treatment partly through regulating BAFF expression.

Aberrant expression of Notch signaling molecules in patients with immune thrombocytopenic purpura

To investigate the role of Notch signaling pathway in immune thrombocytopenic purpura (ITP), we measured the expression of 11 Notch pathway molecules in ITP patients and evaluated their clinical relevance. Real-time reverse transcriptase polymerase chain reaction results showed there was aberrant expression of some Notch molecules in ITP. Notch1 and Notch3 expression elevated, while Notch2 decreased statistically in ITP patients. As for Notch ligands, only DLL1 was found downregulated in ITP. The expression of Notch target gene, Hes1, was also upregulated. In accordance with the mRNA level, Notch1 and Hes1 protein expression was also found elevated by Western blot. Immunocytochemistry showed that Notch1 expressed highly in the cytomembrane, cytoplasm, and part of cellular nucleus for ITP while weak in cytomembrane for controls, and Hes1 of ITP was found expressed higher in cellular nucleus than that of controls. Our findings suggest that the aberrant expression profile of Notch pathway may be involved in ITP, and blockage of Notch1 pathway is likely a promising therapeutic concept.

Decreased indoleamine 2,3-dioxygenase expression in dendritic cells and role of indoleamine 2,3-dioxygenase-expressing dendritic cells in immune thrombocytopenia

Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) can induce or maintain peripheral immune tolerance. Impaired IDO-mediated tryptophan catabolism has been observed in autoimmune diseases. In order to investigate the effects of IDO-mediated tryptophan catabolism and IDO-expressing DCs in immune thrombocytopenia, the concentrations of kynurenine were detected by high-pressure liquid chromatography. The expressions of IDO were analyzed by flow cytometry and western blot analysis. The effects of IDO+ DCs stimulated with CTLA-4-Ig on T cells proliferation and activation, lymphocyte apoptosis, and Tregs were measured by flow cytometry. We found that the expression of IDO in DCs of immune thrombocytopenia (ITP) patients was significantly decreased. CTLA-4-Ig significantly increased the expression of functional IDO in DCs of ITP patients. IDO+ DCs stimulated with CTLA-4-Ig suppressed T cells proliferation and activation, promoted lymphocyte apoptosis, and increased the percentage of Tregs. These results suggest that decreased IDO expression in DCs may play a critical role in ITP. CTLA-4-Ig successfully corrected the disorder of IDO expression in ITP. IDO+ DCs stimulated with CTLA-4-Ig inhibited immune responses by an IDO-dependent mechanism. Increasing the expression and activity of IDO in DCs might be a promising therapeutic approach for ITP.

Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method

Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99–100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90–98% cells were strongly positive. Four patients, including three patients treated with interferon-α and hydroxyurea and one patient treated with imatinib mesylate, had 26–82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/104 cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

Imbalance between CD205 and CD80/CD86 in dendritic cells in patients with immune thrombocytopenia.

INTRODUCTION CD205(DEC-205), a tolerance-associated receptor, is a member of the macrophage mannose receptor family of C-type lectin receptors. Antigen uptake via CD205 induces regulatory T cells, thereby regulating peripheral immune tolerance. However, the contribution of CD205 to autoimmune diseases has not been elucidated. Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by overdestruction of platelets. A previous study by the present authors found that CD205 expression in dendritic cells (DCs) was upregulated during induction of immune tolerance in patients with ITP. METHODS CD205 expression in monocyte-derived DCs and spleens from patients with ITP was analysed prior to and after high-dose dexamethasone (HD-DXM) treatment. Expression of CD80, CD86 and HLA-DR was also analysed in order to identify and define the maturation status of the DCs more precisely. RESULTS In patients with ITP, CD205 expression was found to be significantly decreased in DCs, and rare or absent in the border region of the spleen. However, the expression of CD80 and CD86 was increased in both monocyte-derived DCs and spleens in patients with ITP compared with controls. HD-DXM treatment may upregulate CD205 expression and downregulate CD80/CD86 expression, then rebalance the expression of CD205 and CD80/CD86 in DCs in patients with ITP. CONCLUSION Imbalance between CD205 and CD80/CD86 may contribute to the development of ITP. Therapies that aim to restore the balance between CD205 and CD80/CD86 may help to re-establish tolerance in patients with ITP.