Identification of ORM1, vWF, SPARC, and PPBP as immune-related proteins involved in immune thrombocytopenia by quantitative LC-MS/MS.

BACKGROUND: Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by loss of immune tolerance to platelet autoantigens leading to excessive destruction and insufficient production of platelets. METHOD: Quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to detect the differentially expressed proteins in bone marrow samples from active ITP patients and normal controls. RESULT: Our bioinformatic analysis identified two upregulated proteins (ORM1 and vWF) and two downregulated proteins (PPBP and SPARC) related to immune function. The four proteins were all found to be related to the tumor necrosis factor (TNF) -α signalling pathway and involved in the pathogenesis of ITP in KEGG pathway analysis. CONCLUSION: Bioinformatics analysis identified differentially expressed proteins in bone marrow that are involved in the TNF-α signalling pathway and are related to the activation of immune function in ITP patients. These findings could provide new ideas for research on the loss of immune tolerance in ITP patients.

Abnormal expression of autophagy-related proteins in immune thrombocytopenia.

Autophagy is a highly conserved protein degradation pathway that is essential for affecting some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between autophagy-related proteins and immune responses in ITP remains unclear. Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Beclin-1, SQSTM1/p62 and LC3 were measured in the peripheral blood mononuclear cells (PBMCs) of 20 newly diagnosed patients with active ITP, 16 ITP patients in remission and 21 healthy volunteers. The stained Beclin-1 and SQSTM1/p62 proteins were also observed in the bone marrow of active ITP patients and normal controls by immunofluorescence. SQSTM1/p62 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Beclin-1 mRNA was increased significantly. During the remission stages, the levels of these autophagy-related proteins were comparable with those observed in healthy controls. Taken together, these results suggest that the aberrant expression of autophagy-related proteins might be involved in the pathogenesis of ITP. Further study of the autophagy pathway may provide a new strategy and direction for the treatment of ITP.

Quantitative LC-MS/MS uncovers the regulatory role of autophagy in immune thrombocytopenia.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune haemorrhagic disease whose pathogenesis is associated with bone marrow megakaryocyte maturation disorder and destruction of the haematopoietic stem cell microenvironment. METHODS: In this study, we report the qualitative and quantitative profiles of the ITP proteome. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to elucidate the protein profiles of clinical bone marrow mononuclear cell (BMMC) samples from ITP patients and healthy donors (controls). Gene Ontology (GO) and Kyoto Encyclopaedia Genes and Genome (KEGG) pathway analyses were performed to annotate the differentially expressed proteins. A protein-protein interaction (PPI) network was constructed with the BLAST online database. Target proteins associated with autophagy were quantitatively identified by parallel reaction monitoring (PRM) analysis. RESULTS: Our approaches showed that the differentially expressed autophagy-related proteins, namely, HSPA8, PARK7, YWHAH, ITGB3 and CSF1R, were changed the most. The protein expression of CSF1R in ITP patients was higher than that in controls, while other autophagy-related proteins were expressed at lower levels in ITP patients than in controls. CONCLUSION: Bioinformatics analysis indicated that disruption of the autophagy pathway is a potential pathological mechanism of ITP. These results can provide a new direction for exploring the molecular mechanism of ITP.

Significant reductions in apoptosis-related proteins (HSPA6, HSPA8, ITGB3, YWHAH, and PRDX6) are involved in immune thrombocytopenia.

To investigate differences in the expression of plasma proteins in immune thrombocytopenia (ITP) and normal control groups, bone marrow samples were collected from 20 active ITP patients and 20 healthy controls. Quantitative proteomics analysis based on mass spectrometry was used to measure the protein levels and understand the protein networks. We found differentially expressed proteins in ITP patients and healthy controls. Parallel reaction monitoring (PRM), a targeted proteome quantification technique, was used to quantitatively confirm the identified target proteins and verify the proteomics data. In this study, a total of 829 proteins were identified, and the fold-change cut-off was set at 1.5 (patients vs controls); a total of 26 proteins were upregulated, and 69 proteins were downregulated. The bioinformatics analysis indicated that some differentially expressed proteins were associated with apoptosis. KEGG enrichment analysis showed that the apoptosis-related proteins were closely related to the PI3K-Akt signalling pathway. PRM demonstrated that apoptosis-related proteins were significantly decreased in ITP patients, which further confirmed the important effect of apoptosis on ITP pathogenesis. We hypothesised that apoptosis may be closely related to ITP pathogenesis through the PI3K-Akt signalling pathway.

Megakaryocytic dysfunction in immune thrombocytopenia is linked to autophagy.

Immune thrombocytopenic purpura (ITP) is a multifactorial autoimmune disease characterized by both increased platelet destruction and/or reduced platelet production. Even though they are detected in ≤ 50% of ITP patients, auto-antibodies play a pivotal role in the pathogenesis of ITP. Recent experimental and clinical observations have revealed abnormal autophagy in ITP patients. Autophagy is a catabolic process responsible for the elimination and recycling of cytoplasmic constituents, such as organelles and macromolecules, in eukaryotic cells. Additionally, it triggers cell death or promotes cell survival following various forms of stress, and maintains the microenvironment and stemness of haematopoietic stem cells. The role of autophagy in megakaryopoiesis, thrombopoiesis, and platelet function is slowly being uncovered. The abnormal autophagy in ITP patients may be caused by deletion of autophagy-related genes such as ATG7 and abnormal signalling due to overexpression of mTOR. These changes are thought to affect markers of haematopoietic stem cells, such as CD41 and CD61, and differentiation of megakaryocytes, ultimately decreasing the function and quantity of platelets and leading to the onset of ITP. This review highlights recent evidence on the essential role played by autophagy in megakaryopoiesis, megakaryocyte differentiation, thrombopoiesis, and platelet production. It also discusses the potential of targeting the autophagy pathway as a novel therapeutic approach against ITP.

Reduced tumour necrosis factor receptor superfamily 13C inversely correlated with tumour necrosis factor superfamily 13B in patients with immune thrombocytopenia.

To investigate the expression of tumour necrosis factor superfamily 13B (TNFSF13B) receptors in immune thrombocytopenia (ITP) and their correlation with disease activity, we investigated the protein and mRNA levels of TNFSF13B, tumour necrosis factor receptor superfamily 13C (TNFRSF13C), TNFRSF13B and TNFRSF17 by flow cytometry, enzyme-linked immunosorbent assay and real time quantitative polymerase chain reaction. All CD19(+) B lymphocytes expressed TNFRSF13C by flow cytometry, but the mean fluorescence intensity (MFI) was decreased in patients with active disease compared to patients in remission and healthy controls, while no significant difference of TNFRSF13C mRNA was found between ITP patients and controls. The mRNA and plasma TNFSF13B were elevated in active ITP patients, and TNFRSF13C MFI level was inversely correlated with plasma TNFSF13B in active patients. In vitro assays showed that TNFRSF13C MFI was decreased after long exposure to TNFSF13B. No significant difference for TNFRSF13B or TNFRSF17 was found between ITP patients and controls. In conclusion, TNFRSF13C expression is reduced on CD19(+) cells in active ITP patients. This down-regulation occurs through a post-transcriptional mechanism and could be a consequence of chronic increase of TNFSF13B.

Altered expression of T cell Immunoglobulin-Mucin (Tim) molecules in peripheral blood mononuclear cells in aplastic anemia.

To evaluate the balance between T-cell immunoglobulin and mucin domain (Tim) molecules(Tim)-1 and Tim-3 in patients with aplastic anemia (AA), plasma IFN-γ and IL-4 levels were measured in patients with active AA (n = 41), AA in remission (n = 29) and in healthy subjects (n = 40) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IFN-γ, IL-4, Tim-1 and Tim-3 were studied in all subjects. The results showed that the expression of Tim-3 in newly diagnosed patients was significantly deceased, compared with the controls. Meanwhile, Tim-1 mRNA expression in the active AA group was not significantly reduced, which resulted in a declined ratio of Tim-3/Tim-1 in patients with active disease. During the remission stages, the levels of these transcription factors were comparable with those observed in the healthy controls. These findings are the first data on the expression of the Tim-1 and Tim-3 molecules in AA. The reduced levels of Tim-3/Tim-1 in PBMCs during the active stages of disease suggest that they may play a possible role in the pathogenesis and course of AA.

Imbalanced expression of T-bet and T cell immunoglobulin mucin-3 in patients with aplastic anaemia.

BACKGROUND: Activated T helper (Th)-1 CD4+ cells and their mediators are essential for pathogenesis processes in aplastic anaemia (AA). Recently, T-cell immunoglobulin and mucin domain 3 (Tim-3) molecules, a Th1-specific type 1 membrane protein, have been suggested to be important regulators of both Th1 proliferation and the development of tolerance. Moreover, T-box expressed in T cells (T-bet) is a major T cell transcription factor that regulates the expression of Th1 cytokine genes and plays a crucial role in T cell differentiation. The function of Tim-3 and its association with T-bet in the pathophysiology of AA remain unclear. DESIGN AND METHODS: Plasma IL-18, IFN-γ and IL-4 levels were measured in patients with newly diagnosed AA (n = 29), AA in remission (n = 22) and healthy subjects (n = 30) via enzyme-linked immunosorbent assay (ELISA). CD4+ Tim-3+ cells were evaluated via flow cytometry and expressed as a percentage of the total number of CD4+ cells. Using real-time quantitative polymerase chain reaction (RT-PCR) and mRNA expression analysis the expression levels of Tim-3, IL-18, IFN-γ and T-box (T-bet) were examined in all subjects. RESULTS: Tim-3 was expressed on CD4+T cells. The percentages of Tim-3 cells identified in newly diagnosed patients were significantly deceased compared with the controls. Meanwhile T-bet, IL-18 and IFN-γ levels were significantly elevated in patients, which resulted in an increased ratio of T-bet/Tim-3 expression levels in patients with active disease. During the remission stages, the levels of these cytokines were comparable with those observed in the healthy controls. CONCLUSIONS: These results suggest that the imbalanced expression of Tim-3 and T-bet may play a role in the pathogenesis and course of AA, and the downregulation of T-bet/Tim-3 may represent a reasonable therapeutic strategy for AA treatment.

Gene mutations in the PI3K/Akt signaling pathway were related to immune thrombocytopenia pathogenesis.

BACKGROUND: Immune thrombocytopenic (ITP) is an autoimmune bleeding disease with genetic susceptibility. Twenty newly diagnosed active primary ITP patients who had not been treated with glucocorticosteroids, immune globulin or immunosuppressants prior to sampling were enrolled in this study. Bone marrow blood mononuclear cells were used for whole exome sequencing to further elucidation the variant genes of ITP. METHODS: High-molecular-weight genomic DNA was extracted from freshly frozen bone marrow blood mononuclear cells from 20 active ITP patients. Next, the samples were subjected to molecular genetic analysis by whole-exome sequencing, and the results were confirmed by Sanger sequencing. The signaling pathways and cellular processes associated with the mutated genes were identified with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. RESULTS: The results showed that there were 3998 missense mutations involving 2269 genes in more than 10 individuals. Unique genetic variants including phosphatase and tensin homolog, insulin receptor, and coagulation factor C homology were the most associated with the pathogenesis of ITP. Functional analysis revealed these mutation genes mainly affect Phosphatidylinositol 3 kinase/serine/threonine kinase B signaling pathways (signal transduction) and platelet activation (immune system). CONCLUSION: Our finding further demonstrates the functional connections between these variant genes and ITP. Although the substantial mechanism and the impact of genetic variation are required further investigation, the application of next generation sequencing in ITP in this paper is a valuable method to reveal the genetic susceptibility.

High-throughput DNA methylation analysis in ITP confirms NOTCH1 hypermethylation through the Th1 and Th2 cell differentiation pathways.

BACKGROUND: Immune thrombocytopenia (ITP) is a prevalent autoimmune disease with a complex aetiology where DNA methylation changes are becoming triggers. METHOD: To investigate novel abnormally methylated genes in the pathogenesis of ITP, we performed a high-throughput methylation analysis on 21 ITP patients and 9 normal control samples. We analysed the extent of key methylated genes and their downstream cytokines through Luminex assay or qRT-PCR. Then, bone marrow mononuclear cells were extracted from ITP patients, and decitabine (demethylation drug) was added to the culture medium of cultured cells. qRT-PCR and ELISA were used to detect whether decitabine could effectively affect target genes and related cytokines. RESULTS: Through the STRING and Metascape databases, hypermethylated NOTCH1 can be identified and can influence ITP by regulating many downstream cytokines through Th1 and Th2 cell differentiation pathways. Compared with those in the normal control group, the expression levels of NOTCH1 and its downstream Th2 cytokines (IL-4, IL-10, and GATA3) were significantly decreased and those of Th1 cytokines (IFN-γ, IL-12, and TNF-α) were significantly increased in the ITP group. Decitabine exerts its demethylation effect, so the expression of NOTCH1 and its related cytokines in the ITP group treated with 100 nM decitabine were significantly reversed. CONCLUSIONS: Our results suggest that the pathogenesis of ITP may exert its influence on epigenetics through alteration of DNA methylation at regulatory regions of the target NOTCH1 gene in the Th1 and Th2 cell differentiation pathways. At the same time, decitabine may achieve a therapeutic effect on ITP by demethylation.

The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia.

Elevated level of B-cell activating factor (BAFF) has been implicated in the pathogenesis of some autoimmune diseases. Blockade of receptor and ligand binding by decoy receptor has demonstrated a clinical benefit in both oncologic and immunologic diseases. In this report, we have detected plasma BAFF and BAFF mRNA expression in immune thrombocytopenia (ITP) patients by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). The effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of interferon gamma (IFNgamma), and interleukin-4 (IL-4) were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. In in vitro assays, rhBAFF promoted the survival of CD19(+) and CD8(+) cells, and increased the apoptosis of platelets and the secretion of IFN-gamma. BR3-Fc successfully corrected the effects of rhBAFF on lymphocytes, platelets, and cytokines. These findings suggest that BAFF may play a pathogenic role in ITP by promoting the survival of CD19(+) and CD8(+) cells, and increasing the apoptosis of platelets and the secretion of IFN-gamma. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

[Role of IL-18/IL-18BP balance in spleen of patients with primary immune thrombocytopenia].

OBJECTIVE: To investigate the role of IL-18 and IL-18BP balance in the spleens of patients with primary immune thrombocytopenia (ITP). METHODS: A total of 12 active ITP patients and 10 normal controls were recruited. Their expressions of IL-18 and IL-18BP were measured by immunohistochemistry (IHC) and immunofluorescence. The mRNA expressions of IL-18, IL-18BP, IFN-γ and IL-4 were studied by reverse transcription-polymerase chain reaction (RT-PCR) in all subjects. RESULTS: The positive rate of IL-18 was higher in the ITP patients than that of the normal control group (100% vs 90.0%). And the positive rate of IL-18BP was higher in the ITP patients than that of the normal control spleen (83.3% vs 60.0%). The relative amount of mRNA gene expression of IL-18 was increased 6.0-fold in active patients compared to controls (P<0.05). But the elevated mRNA content of IL-18BP failed to compensate for the sharply elevated content of IL-18. As compared with the normal control group, the expression level of IFN-γ mRNA expression was significantly increased 10.2-fold in ITP patients (P<0.05). The decrease observed in IL-4 was 41.7% in active patients compared to controls (P<0.05). CONCLUSION: The IL-18/IL-18BP imbalance plays an important role in pathogenesis of ITP and its correction may become a potential therapeutic target for ITP.

[Expression of interleukin-18 and interleukin-18 receptor alpha chain of the peripheral white blood cells in immune thrombocytopenia].

OBJECTIVE: To detect the expression of interleukin (IL)-18 of the peripheral blood cells and IL-18 receptor alpha chain (IL-18Ralpha) on the surface of CD(3)(+) cells in patients newly diagnosed as immune thrombocytopenia (ITP) before medication and to explore the roles of IL-18 and IL-18Ralpha in the development of ITP. METHODS: Eighteen out-patients or inpatients with acute ITP accepting treatment in Qilu Hospital were enrolled in this study and 15 matching healthy subjects were taken as control. Plasma IL-18 level was detected with enzyme linked immunosorbent assay (ELISA), the expression of IL-18Ralpha on CD(3)(+) lymphocytes and total lymphocytes were measured with flow cytometry;T-bet and GATA-3 mRNA were measured with reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The expression of IL-18 in acute ITP plasma was (468.57 + or - 141.62) ng/L and IL-18Ralpha on the surface of CD(3)(+) cells and lymphocytes were (8.50 + or - 3.16)% and (9.16 + or - 2.98)% respectively. The levels of IL-18 and IL-18Ralpha were increased in active ITP patients as compared with those in the controls (P < 0.05). The levels of IL-18 mRNA (0.12 + or - 0.02) and T-bet mRNA (0.07 + or - 0.02) were significantly increased in patients with active ITP as compared with those in the controls (P < 0.05), while GATA-3 mRNA (0.0039 + or - 0.0014) were significantly decreased in patients with active ITP (P < 0.05). The balance between T-bet and GATA-3 was significantly disturbed in ITP. CONCLUSIONS: Through the variation of the levels of gene and protein, our study showed that IL-18 and IL-18Ralpha might upregulate the expression of Th1-cytokines in ITP patients. It is also suggested that IL-18 has potential association with the development of ITP. Especially, it may provide a new treatment method for ITP by regulating the ratio of T-bet and GATA-3 and resuming the balance of Th1/Th2.

Immunomodulatory cytokine interleukin-35 and immune thrombocytopaenia.

Considerable attention has been paid to interleukin (IL)-35 because of its immunosuppressive effects in a variety of autoimmune diseases. IL-35, a recently identified cytokine of the IL-12 family, is a negative regulatory factor secreted by IL-35-inducible regulatory T cells (iTr35 cells) and the recently reported regulatory B cells (Breg cells). Four biological effects of IL-35 have been discovered in vitro and in vivo: (i) suppression of T cell proliferation; (ii) conversion of naive T cells into iTr35 cells; (iii) downregulation of type 17 helper T (Th17) cells; and (iv) conversion of Breg cells into a Breg subset that produces IL-35 and IL-10. IL-35 plays an important role in a variety of autoimmune diseases, such as rheumatoid arthritis, allergic asthma and systemic lupus erythematosus. Primary immune thrombocytopaenia (ITP), which is characterized by isolated thrombocytopaenia and mild mucocutaneous to life-threatening bleeding, is an autoimmune disease with complex dysregulation of the immune system. Both antibody-mediated and/or T cell-mediated platelet destruction are key processes. In addition, impairment of T cells and cytokine imbalances have now been recognized to be important. This review summarizes the immunomodulatory effects of IL-35 and its role in the pathogenesis of ITP as mediated by T and B cells.

DNA methylation plays an important role in immune thrombocytopenia.

DNA methylation is the covalent addition of a methyl group to a DNA base, typically the cytosine of cytosine-phosphate-guanosine (CpG) dinucleotides. It is catalysed by methyltransferase enzymes using an S-adenosyl methionine donor, which is a heritable, stable and reversible DNA modification. Aberrant DNA methylation can influence gene expression without changing nucleotide sequences, inducing occurrence and development in autoimmune diseases, such as systemic lupus erythematosus and immune thrombocytopenia. Immune thrombocytopenia is an autoimmune disease characterised by bleeding and thrombocytopenia of peripheral blood, a normal or increased number of megakaryocytes and a maturation disorder. Recently, it was proven that aberrant DNA methylation is associated with the aetiology of immune thrombocytopenia. The defective methylation induces overexpression of methylation-related genes, such as CD70 and FOXP3, which can take part in autoreactive immune responses, and ultimately accelerated the progression of immune thrombocytopenia. Targeting the DNA methylation can be used as a new treatment for immune thrombocytopenia. As a demethylated drug, decitabine promotes megakaryocyte maturation and platelet release under the action of tumour necrosis factor-related apoptosis inducing ligand (TRAIL) promoter. This review highlights recent evidence on the role of DNA methylation in immune thrombocytopenia by describing the relationship between DNA methylation and immune thrombocytopenia, and the DNA methylation-related genes. Identifying and regulating abnormal DNA methylation provides new ideas for the diagnosis and treatment of immune thrombocytopenia.

Research Progress on Artemisinin and Its Derivatives against Hematological Malignancies.

Although current therapeutic methods against hematological malignancies are effective in the early stage, they usually lose their effectiveness because of the development of drug resistances. Seeking new drugs with significant therapeutic effects is one of the current research hotspots. Artemisinin, an extract from the plant Artemisia annua Linne, and its derivatives have excellent antimalarial effects in clinical applications as well as excellent safety. Recent studies have documented that artemisinin and its derivatives (ARTs) also have significant effects against multiple types of tumours, including hematological malignancies. This review focuses on the latest research achievements of ARTs in the treatment of hematological malignancies as well as its mechanisms and future applications. The mechanisms of ARTs against different types of hematological malignancies mainly include cell cycle arrest, induction autophagy and apoptosis, inhibition of angiogenesis, production of reactive oxygen species, and induction of differentiation. Additionally, the review also summarizes the anticancer effects of ARTs in many drug-resistant hematological malignancies and its synergistic effects with other drugs.

Mucin mutations and aberrant expression are associated with the pathogenesis of immune thrombocytopenia.

PURPOSE: Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease of unknown aetiology. In this study, we aimed to identify the mutations and aberrant expression of mucins associated with ITP pathogenesis. METHODS: First, we investigated the DNA mutation profile of bone marrow samples from patients with ITP (n = 20) by using next-generation sequencing (NGS). In addition, MUC3A, MUC5B and MUC6 were mutated in all patients with ITP. ELISA (enzyme-linked immunoassay) was used to measure MUC3A, MUC5B and MUC6 levels in the plasma of bone marrow fluid mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs). Real-time quantitative PCR was used to study the mRNA expression levels of MUC3A, MUC5B and MUC6 in BMMCs and PBMCs. RESULTS: The results indicated that there were 3998 missense mutations involving 2269 genes in more than 10 individuals. MUC3A levels were not significantly different among the three groups, whereas MUC5B and MUC6 expression were significantly down-regulated in patients with ITP compared with healthy controls. In addition, serum MUC5B and MUC6 levels were significantly higher in patients with ITP in clinical remission than in patients with active ITP. CONCLUSIONS: Taken together, these results suggest that genetic alterations and the aberrant serum expression of mucins might be involved in the pathogenesis of ITP.

Reduced IL-35 in patients with immune thrombocytopenia.

: The occurrence and development of primary immune thrombocytopenia is closely related to autoimmune imbalanced. Thus, we conducted the current study to investigate the modulation of IL-35, a newly identified immunological self-tolerance factor on immune thrombocytopenic purpura (ITP). We were enrolled peripheral blood in 21 adult healthy volunteers, 21 active primary ITP patients and 16 ITP patients in remission. In the same period, bone marrow plasma was drawn from active primary ITP patients and 16 bone marrow donors. Enzyme-linked immunoassay was used to measure IL-35 levels in bone marrow mononuclear cells and peripheral blood mononuclear cells. Real-time quantitative PCR was used to study the mRNA expression levels of p35, Epstein-Barr virus-induced gene 3 in bone marrow mononuclear cells and peripheral blood mononuclear cells. Compared with the normal group, IL-35 levels of in ITP patients were decreased significantly. IL-35 level in bone marrow plasma was decreased more significantly than that in peripheral blood plasma at the same stage. The results showed that plasma IL-35 levels were significantly decreased in patients with active ITP compared with those of control individuals, and IL-35 levels in bone marrow plasma were decreased more significantly compared with those at the same stage. The pathogenesis of ITP is associated with decreased IL-35 levels. Further studies are needed to expand sample content and explore more in-depth investigate a possible role of IL-35 in the pathogenesis and course of ITP.

Rapamycin induces megakaryocytic differentiation through increasing autophagy in Dami cells.

: Autophagy is a conserved cellular process that involves the degradation of cytoplasmic components in eukaryotic cells. However, the correlation between autophagy and megakaryocyte development is unclear. This study aims to explore the role of autophagy in megakaryocyte differentiation. To test our hypothesis, we used the Dami cell line in-vitro experiments. Rapamycin and Bafilomycin A1 were used to stimulate Dami cells. CD41 expression and apoptosis were analysed by flow cytometry. Autophagy-related proteins were detected by Western blotting. 12-O-Tetradecanoylphorbol 13-acetate-treated Dami cells can simulate endomitosis of megakaryocytes in vitro. Rapamycin-induced autophagic cell death was verified by LC3-II conversion upregulation. Meanwhile, Bafilomycin A1 blocked endomitosis and autophagy of Dami cells. Our results provide evidence that autophagy is involved in megakaryocyte endomitosis and platelet development. Rapamycin inhibited cell viability and induced multiple cellular events, including apoptosis, autophagic cell death, and megakaryocytic differentiation, in human Dami cells. Upregulated autophagy triggered by rapamycin can promote the differentiation of Dami cells, while endomitosis is accompanied by enhanced autophagy.

Interleukin 18 and interleukin 18 binding protein in patients with idiopathic thrombocytopenic purpura.

To evaluate the balance of interleukin IL18 and its endogenous antagonist IL18 binding protein (IL18BP) in patients with idiopathic thrombocytopenic purpura (ITP), plasma IL18, IL18BP, interferon gamma (IFNG) and IL4 levels, as well as platelet counts were measured in patients with active ITP (n = 23), ITP in remission (n = 21) and in healthy subjects (n = 24) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction, the mRNA expression of IL18, IL18BP, IFNG, IL4, T-box (TBX21) and GATA-binding protein 3(GATA3) were studied in all subjects. The results showed that IL18 and IFNG protein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but that IL18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL18/IL18BP in patients with active disease. During remission stages, the levels of these cytokines were comparable to those of healthy controls. The elevated levels of IL18/IL18BP in plasma during active stages of disease suggest a possible role in the pathogenesis and course of ITP.