zhen Li

Cell-mediated lysis of autologous platelets in chronic idiopathic thrombocytopenic purpura.

Abstract:  Objectives: Investigate the contribution and mechanism of cell‐mediated cytotoxicity to the pathogenesis of idiopathic thrombocytopenic purpura (ITP). Methods: We observed the cytotoxic effect of cytotoxic T‐lymphocyte (CTL) (CD8+) and natural killer cells (CD3−CD16+CD56+) toward chronic ITP patients autologous platelets, and investigated the expression of Fas ligand (FasL), tumor necrosis factor (TNF)‐α and TNF‐related apoptosis inducing ligand, as well as perforin and granzyme B mRNA in CD8+ cells using flow cytometry and reverse transcriptase‐polymerase chain reaction. Results: We found that platelet lysis was seen only using purified CD8+ T cells as effector cells; expression of FasL and TNF‐α in CD8+ T cells in ITP group was elevated. Moreover, the mRNA levels of granzyme B and perforin in CD8+ cells of ITP patients were increased. Conclusions: Our findings suggest that CTLs are activated in chronic ITP and might be involved in the pathogenesis of this disorder. Apoptosis and perforin/granzyme‐mediated cytotoxicity constitute an important pathway through which CTLs destruct autologous platelets. CTLs might be a reasonable target for a therapeutic strategy.

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.

Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 in T-ALL cell lines

Gain-of-function mutations in Notch-1 are common in T-cell lymphoblastic leukemia (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSIs). However, GSIs seem to be active in only a small fraction of T-ALL cell lines with constitutive Notch-1 activity and the downstream response of Notch signaling is only partially understood. To further investigate the molecular mechanisms underlying proliferation suppression and apoptosis and explore effective downstream target genes, we used RNA interference (RNAi) technology to down-regulate the expression of Notch-1 in GSIs-resistant T-ALL cell lines. Results showed that down-regulation of Notch-1 by transfection of a small interfering RNA (siRNA) could cause SupT1 cells proliferation inhibition by inducing G(0)/G(1) cell cycle arrest and apoptosis. The proliferation inhibitory and apoptotic effects resulting from down-regulation of Notch-1 may be mediated through regulating the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 and the activity of Akt signaling. In addition, our results demonstrated that down-regulation of Notch-1 signaling could sensitize SupT1 cells to adriamycin. Taken together, cell cycle regulatory proteins and Akt signaling may be attractive targets in T-ALL.

CD8+ T cells suppress autologous megakaryocyte apoptosis in idiopathic thrombocytopenic purpura.

To investigate the effect and mechanism of the CD8+ T cells in bone marrow on autologous megakaryocytopoiesis in idiopathic thrombocytopenic purpura (ITP) patients, we prepared bone marrow mononuclear cells (MNCs) from 15 chronic ITP patients and 13 controls. MNCs were cultured in vitro directly (MNC group) or after depleting CD8+ T cells (CD8+ T‐dep group) or adding purified autologous CD8+ T cells to CD8+ T‐dep MNCs (Coculture group) or adding dexamethasone to the coculture (DEX group) all in semi‐solid and liquid culture systems. The quantity and quality of megakaryocytes were measured. The megakaryocyte count was increased in the presence of autologous CD8+ T cells of patients with chronic ITP, while platelet production was reduced. In addition, lower percentages of polyploidy and apoptotic megakaryocytes, and higher levels of soluble Fas (sFas) in supernatant were observed. Dexamethasone successfully corrected this effect of CD8+ T cells on autologous megakaryocytopoiesis. These studies provide evidence that activated CD8+ T cells in bone marrow of patients with chronic ITP might suppress megakaryocyte apoptosis, leading to impaired platelet production. Megakaryocyte apoptosis would be a novel target for the management of ITP.

Interleukin 27 inhibits cytotoxic T-lymphocyte-mediated platelet destruction in primary immune thrombocytopenia

Cytotoxic T-lymphocyte (CTL)-mediated platelet destruction and aberrant cytokine profiles play important roles in the pathogenesis of primary immune thrombocytopenia (ITP). Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the effect of IL-27 on CTL activity in ITP has not been reported. In the present study, platelets from ITP patients were cultured with autologous CTLs in the presence of IL-27. We found that IL-27 could inhibit CTL-mediated platelet destruction. In these IL-27-treated CTLs, granzyme B and T-bet expression decreased significantly, whereas granzyme A, perforin, and eomesodermin were not affected. To further investigate the role of granzyme B in CTL-mediated platelet destruction, granzyme B inhibitor was added and platelet apoptosis was significantly inhibited. These results suggest that IL-27 negatively regulates CTL cytotoxicity toward platelets in ITP by decreasing granzyme B expression, which is associated with reduced T-bet expression. IL-27 may have a therapeutic role in treating ITP patients.

Down-regulation of Notch-1 increases co-cultured Jurkat cell sensitivity to chemotherapy

The bone marrow (BM) microenvironment plays a critical role in malignant cell growth, patient survival and response to chemotherapy in hematologic malignancies. However, the molecular mechanisms of BM stromal cells (BMSCs)-mediated survival of tumor cell remain unclear. In this study, to further evaluate the role of Notch-1 in vivo microenvironment, we investigated the influence of inhibiting Notch-1 pathway by Notch-1 siRNA on Jurkat cells in the co-culture system. We found that Notch-1 signaling in Jurkat cells was further activated by interaction with BMSCs, which inhibited drug-induced apoptosis in Jurkat cells. Notch-1 siRNA down-regulated Notch-1 and restored drug-induced apoptosis in co-cultured Jurkat cells. The possible mechanism of restoration of sensitivity to chemotherapy could be associated with repressed Akt signaling. The results indicated that Notch-1 may be a potential mechanism of BMSCs involvement in the protection of hematopoietic malignant cells from drug-induced apoptosis.

Polarization of Natural Killer T Cells towards an NKT2 Subpopulation Occurs after Stimulation with α-Galactosylceramide and rhG-CSF in Aplastic Anemia

Natural killer T (NKT) cells play an important role in the regulation of immune responses in a broad range of diseases, including autoimmune disorders, infectious diseases and cancer. So far, few studies have evaluated the roles of NKT cells in the pathogenesis of aplastic anemia (AA), an autoimmune disease. In this study, we investigated the quantitative and qualitative changes in NKT cells in bone marrow (BM) mononuclear cells of AA patients in response to in vitro stimulation with α-galactosylceramide. Compared to healthy controls, BM from AA patients had reduced fraction of NKT cells, which possessed a decreased potential to expand in vitro in response to α-galactosylceramide stimulation, producing more IFNγ+ NKT1 cells. In the presence of rhG-CSF, the expansion capacity of NKT cells stimulated by α-galactosylceramide was significantly reduced in both AA and control groups, with the majority of the activated NKT cells expressing intracellular IL-4, and the fractions of IFNγ+ NKT cells were significantly reduced. In summary, our results indicate that polarization of NKT cells towards the NKT2 subpopulation occurs after co-stimulation with α-galactosylceramide and rhG-CSF in AA.

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.