Low-dose decitabine modulates myeloid-derived suppressor cell fitness via LKB1 in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic fitness of MDSCs is fundamental for its suppressive activity toward effector T cells. Our previous studies showed that the number and inhibitory function of MDSCs were impaired in patients with immune thrombocytopenia (ITP) compared with healthy controls. In this study, we analyzed the effects of decitabine on MDSCs from patients with ITP, both in vitro and in vivo. We found that low-dose decitabine promoted the generation of MDSCs and enhanced their aerobic metabolism and immunosuppressive functions. Lower expression of liver kinase 1 (LKB1) was found in MDSCs from patients with ITP, which was corrected by decitabine therapy. LKB1 short hairpin RNA (shRNA) transfection effectively blocked the function of MDSCs and almost offset the enhanced effect of decitabine on impaired MDSCs. Subsequently, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP in murine models. Passive transfer of decitabine-modulated MDSCs significantly raised platelet counts compared with that of phosphate buffered saline-modulated MDSCs. However, when LKB1 shRNA-transfected MDSCs were transferred into SCID mice, the therapeutic effect of decitabine in alleviating thrombocytopenia was quenched. In conclusion, our study suggests that the impaired aerobic metabolism of MDSCs is involved in the pathogenesis of ITP, and the modulatory effect of decitabine on MDSC metabolism contributes to the improvement of its immunosuppressive function. This provides a possible mechanism for sustained remission elicited by low-dose decitabine in patients with ITP.

Atorvastatin restores imbalance of cluster of differentiation 4 (CD4)+ T cells in immune thrombocytopenia in vivo and in vitro.

Immune thrombocytopenia (ITP) is an autoimmune haemorrhagic disease, in which the overactivation of T cells is crucial in the pathogenesis. Atorvastatin (AT), a lipid-lowering medicine, has shown promising immunomodulatory effects in certain inflammatory conditions. However, the immunoregulatory role of AT in ITP remains elusive. To investigate the effect of AT in the treatment of ITP, cluster of differentiation 4 (CD4)+ T cells were isolated from patients with ITP and cultured with different dosages of AT. We found that AT significantly inhibited cell proliferation, led to cell cycle arrest, induced apoptosis, and repressed the activation of CD4+ T cells in vitro. ITP murine models were then established, and results showed that AT treatment led to faster recovery of the platelet count to normal and exhibited comparable immunomodulatory function. Furthermore, we found the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), as well as activation of rat sarcoma virus (RAS) were all reduced dramatically after AT treatment in vitro. In conclusion, our present study demonstrated that AT could reinstate the functions of CD4+ T cells by inhibiting the excessive activation, proliferation, and survival of CD4+ T cells in ITP via the RAS/mitogen-activated protein kinase kinase (MEK)/ERK and the mTOR/phosphatidylinositol-3 kinase (PI3K)/AKT pathway. Therefore, we propose that AT could be used as a potential therapeutic option for ITP by restoring the over-activated cellular immunity.

Low-dose decitabine modulates T-cell homeostasis and restores immune tolerance in immune thrombocytopenia.

Our previous clinical study showed that low-dose decitabine exhibited sustained responses in nearly half of patients with refractory immune thrombocytopenia (ITP). The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T-cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T-cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA-sequencing and cytokine analysis showed that low-dose decitabine rebalanced T-cell homeostasis, decreased proinflammatory cytokines, and downregulated phosphorylated STAT3 in patients with ITP. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicate that the immunomodulatory effect of decitabine provides one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18ß-glycyrrhetinic acid (18ß-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18ß-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18ß-GA in Treg of ITP patients. Furthermore, we found that 18ß-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18ß-GA treatment. In addition, 18ß-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18ß-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

The mTORC1 pathway participate in hyper-function of B cells in immune thrombocytopenia.

B cell hyper-function plays an important role in the pathogenesis of immune thrombocytopenia (ITP), but the molecular mechanisms underlying such changes remain unclear. We sought to identify regulators of B cell dysfunction in ITP patients through transcriptome sequencing and the use of inhibitors. B cells were isolated from PBMC of 25 ITP patients for B cell function test and transcriptome sequencing. For the potential regulatory factors identified by transcriptome sequencing, the corresponding protein inhibitors were used to explore the regulatory effect of the regulatory factors on B cell dysfunction in vitro. In this study, increased antibody production, enhanced terminal differentiation and highly expressed costimulatory molecules CD80 and CD86 were found in B cells of patients with ITP. In addition, RNA sequencing revealed highly activated mTOR pathway in these pathogenic B cells, indicating that the mTOR pathway may be involved in B cell hyper-function. Furthermore, mTOR inhibitors rapamycin or Torin1 effectively blocked the activation of mTORC1 in B cells, resulting in reduce antibody secretion, impaired differentiation of B cells into plasmablasts and downregulation of costimulatory molecules. Interestingly, as an unspecific inhibitor of mTORC2 besides mTORC1, Torin1 did not show a stronger capacity to modulate B cell function than rapamycin, suggesting that the regulation of B cells by Torin1 may depend on blockade of mTORC1 rather than mTORC2 pathway. These results indicated that the activation of mTORC1 pathway is involved in B cell dysfunction in patients with ITP, and inhibition of mTORC1 pathway might be a potential therapeutic approach for ITP.

Single-dose versus low-dose rituximab in corticosteroid-resistant or relapsed ITP: A multicenter, randomized, controlled study.

Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, in which rituximab (RTX) induces the best long-term effect among recommended second-line treatments. Nevertheless, the optimal regimen of RTX remains unclear. We herein conducted a prospective, multicenter, open-label, randomized controlled trial to compare the efficacy and safety of RTX at two different dosage regimens in patients with corticosteroid-resistant or relapsed ITP. Recruited patients were randomly assigned (1:1) to receive either RTX at a repeated low dose (100 mg weekly for 4 weeks, LD-RTX) or at a single dose (375 mg/m2 , S-RTX). Overall response was achieved in 64.3% of patients who received LD-RTX versus 67.4% of those receiving S-RTX (p = .759). The complete response (CR) rate was 23.8% after LD-RTX and 28.3% after S-RTX (p = .635). In health-related quality of life, S-RTX improved patients' psychological status, quality of life, social activities, and work compared with LD-RTX. Furthermore, S-RTX significantly reduced physician visits without compromising efficacy. Our findings demonstrate that a S-RTX is comparable to LD-RTX in effectiveness and safety for treatment of corticosteroid-resistant or relapsed ITP. The single-dosage regimen optimizes the use of medical resources, improves the cost-effectiveness of RTX, and represents a promising and more convenient replacement for LD-RTX in ITP. This study has been completed and is registered with ClinicalTrials.gov, number NCT03258866.

Indirubin modulates CD4+ T-cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4+ T-cell homeostasis with simultaneous decrease of CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell-axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose-dependent manner. Indirubin was observed to restore the expression of programmed cell-death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4+ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4+ T cells in a PD1/PTEN/AKT signalling pathway.

Activation of NMDA receptors mediated iron accumulation via modulating iron transporters in Parkinson's disease.

Increasing evidence has confirmed that nigral iron accumulation and activation of NMDA receptors (NRs) contribute to the neurodegeneration of dopamine (DA) neurons in Parkinson's disease (PD). Earlier work indicated that activation of NRs participated in iron metabolism in the hippocampus. However, the relationship between activation of NRs and iron accumulation in DA neurons of the substantia nigra in PD was unknown. In this study, our results showed that NRs inhibitors MK-801 and AP5 protected nigrostriatal projection system and reduced nigral iron levels of 6-hydroxydopamine (6-OHDA)-induced PD rats. In vitro studies demonstrated that NMDA treatment increased the expression of iron importer divalent metal transporter 1 (DMT1) and decreased the expression of iron exporter ferropotin 1 (Fpn1), which were dependent on iron regulatory protein 1 (IRP1). This led to increased intracellular iron levels and intensified the decrease in mitochondrial transmembrane potential in MES23.5 dopaminergic neurons. In addition, we reported that MK801 and neuronal nitric oxide synthase inhibitor could antagonize 6-OHDA-induced up-regulation of IRP1 and DMT1 and down-regulation of Fpn1, thus attenuating 6-OHDA-induced iron accumulation in MES23.5 cells. This suggested that 6-OHDA-induced activation of NRs might modulate the expression of DMT1 and Fpn1 via the neuronal nitric oxide synthase-IRP1 pathway.-Xu, H., Liu, X., Xia, J., Yu, T., Qu, Y., Jiang, H., Xie, J., Activation of NMDA receptors mediated iron accumulation via modulating iron transporters in Parkinson's disease.

Structural and biochemical studies of the extracellular domain of Myelin protein zero-like protein 1.

Myelin protein zero-like protein 1 (MPZL1) is a member of the immunoglobulin superfamily, and is also a receptor of concanavalin A (ConA). MPZL1 is upregulated in hepatocellular carcinoma (HCC) and accelerates migration of HCC cells. However, function of MPZL1 as a receptor of ConA and its role in HCC development are largely unknown. To elucidate the functional basis, we have determined the crystal structure of the extracellular domain of MPZL1 at 2.7 Šresolution. Overall, it folds like a typical immunoglobulin variable-like domain that is much like MPZ. Unexpectedly, we found Asn50 is a unique glycosylation site and the glycosylation mediates its interaction with ConA. Furthermore, we also found that MPZL1 exists as a homodimer in the crystal, in which hydrogen bonds between Ser86 and Val145 play an important role. Our results demonstrate that glycosylation of Asn50 is essential for its function as a receptor of ConA. We propose that dimerization of MPZL1 participates in control of its signal transmission in cell adhesion.

ASP: Learn a Universal Neural Solver!

Applying machine learning to combinatorial optimization problems has the potential to improve both efficiency and accuracy. However, existing learning-based solvers often struggle with generalization when faced with changes in problem distributions and scales. In this paper, we propose a new approach called ASP: Adaptive Staircase Policy Space Response Oracle to address these generalization issues and learn a universal neural solver. ASP consists of two components: Distributional Exploration, which enhances the solver's ability to handle unknown distributions using Policy Space Response Oracles, and Persistent Scale Adaption, which improves scalability through curriculum learning. We have tested ASP on several challenging COPs, including the traveling salesman problem, the vehicle routing problem, and the prize collecting TSP, as well as the real-world instances from TSPLib and CVRPLib. Our results show that even with the same model size and weak training signal, ASP can help neural solvers explore and adapt to unseen distributions and varying scales, achieving superior performance. In particular, compared with the same neural solvers under a standard training pipeline, ASP produces a remarkable decrease in terms of the optimality gap with 90.9% and 47.43% on generated instances and real-world instances for TSP, and a decrease of 19% and 45.57% for CVRP.

U-Net Modelling-Based Imaging MAP Score for Tl Stage Nephrectomy: An Exploratory Study.

We evaluate the stability of the clinical application of the MAP scoring system based on anatomical features of renal tumour images, explore the relevance of this scoring system to the choice of surgical procedure for patients with limited renal tumours, and investigate the effectiveness of automated segmentation and reconstruction 3D models of renal tumour images based on U-net for interpretative cognitive navigation during laparoscopy Tl stage radical renal tumour cancer surgery. A total of 5 000 kidney tumour images containing manual annotations were applied to the training set, and a stable and efficient full CNN algorithm model oriented to clinical needs was constructed to regionalism and multistructure and to finely automate segmentation of kidney tumour images, output modelling information in STL format, and apply a tablet computer to intraoperatively display the Tl stage kidney tumour model for cognitive navigation. Based on a training sample of MR images from 201 patients with stage Tl renal tumour cancer, an adaptation of the classical U-net allows individual segmentation of important structures such as renal tumours and 3D visualisation to visualise the structural relationships and the extent of tumour invasion at key surgical sites. The preoperative CT and clinical data of 225 patients with limited renal tumours treated surgically at our hospital from August 2011 to August 2012 were retrospectively analysed by three imaging physicians using the MAP scoring system for the total score and the variables R (maximum diameter), E (exogenous/endogenous), N (distance from the renal sinus), A (ventral/dorsal), L (relationship along the longitudinal axis of the kidney), and h (whether in contact with the renal hilum). The score for each variable (contact with the renal hilum) was statistically compared with each other for the three observers. Patients were divided into three groups according to the total score-low, medium, and high-and according to the surgical procedure-radical and partial resection. The correlation between the total score and the score of each variable and the choice of surgical procedure was analysed. The agreement rate of the total score and the score of each variable for all three observers was over 90% (P ≤ 0.001). The map scoring system based on the anatomical features of renal tumour imaging was well stabilized, and the scores were significantly correlated with the surgical approach.

Research on Ecological Effect Assessment Method of Ecological Restoration of Open-Pit Coal Mines in Alpine Regions.

Open-pit mining is an important form of coal mining in China, and its damage to the ecological environment is particularly obvious in alpine regions. The ecological restoration of alpine open-pit coal mines faces severe challenges, and its restoration effect will directly affect the ecological security of China. Meanwhile, comprehensive and system-oriented evaluation of ecological restoration effects is still insufficient in current research. In this study, we selected different quantities of assessment factors on the two scales of ecological project area and ecological impact area to evaluate the ecological restoration effect of an alpine open-pit coal mine. Then, we formed a multi-scale and multi-dimensional ecological restoration effect assessment model of the alpine open-pit coal mine and used this model to analyze the implementation effect of the ecological restoration project of the Baiyinhua No. 2 Open-pit Mine. The results show that the multi-scale and multi-dimensional ecological restoration effect assessment model of alpine open-pit coal mine proposed in this study can accurately characterize the restoration effect of open-pit coal mines in alpine regions and can also be used as a significant evaluation tool in the future ecological construction of mining areas. This study hopes the multi-scale and multi-dimensional ecological restoration effect assessment model of alpine open-pit coal mine can provide a comprehensive, systematic, and scientific evaluation method for the ecological restoration of alpine open-pit coal mines and provide a scientific basis for the ecological restoration and green development of relevant mining areas.

Abnormalities of bone marrow B cells and plasma cells in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoantibody-mediated hemorrhagic disorder in which B cells play an essential role. Previous studies have focused on peripheral blood (PB), but B cells in bone marrow (BM) have not been well characterized. We aimed to explore the profile of B-cell subsets and their cytokine environments in the BM of patients with ITP to further clarify the pathogenesis of the disease. B-cell subpopulations and their cytokine/chemokine receptors were detected by using flow cytometry. Plasma concentrations of cytokines/chemokines were measured by using enzyme-linked immunosorbent assay. Messenger RNA levels of B cell-related transcription factors were determined by using quantitative polymerase chain reaction. Regulatory B cell (Breg) function was assessed by quantifying their inhibitory effects on monocytes and T cells in vitro. Decreased proportions of total B cells, naive B cells, and defective Bregs were observed in patients with ITP compared with healthy controls (HCs), whereas an elevated frequency of long-lived plasma cells was found in BM of autoantibody-positive patients. No statistical difference was observed in plasmablasts or in short-lived plasma cells between patients with ITP and HCs. The immunosuppressive capacity of BM Bregs from patients with ITP was considerably weaker than HCs. An in vivo study using an active ITP murine model revealed that Breg transfusion could significantly alleviate thrombocytopenia. Moreover, overactivation of CXCL13-CXCR5 and BAFF/APRIL systems were found in ITP patient BM. Taken together, B-cell subsets in BM were skewed toward a proinflammatory profile in patients with ITP, suggesting the involvement of dysregulated BM B cells in the development of the disease.

Low-Dose Decitabine Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PD-1 Methylation in Immune Thrombocytopenia.

Cytotoxic T lymphocytes (CTLs)-mediated platelet destruction plays an important role in the pathogenesis of primary immune thrombocytopenia (ITP). The programmed cell death protein 1 (PD-1) signaling can turn off autoreactive T cells and induce peripheral tolerance. Herein, we found that the expression of PD-1 and its ligand PD-L1 on CD8+ T cells from ITP patients was decreased. Activating PD-1 pathway by PD-L1-Fc fusion protein inhibited CTLs-mediated platelet destruction in ITP in vitro. PD-1 promoter hypermethylation in CD8+ T cells was found in ITP patients, resulting in decreased PD-1 expression. The demethylating agent decitabine at a low dose was proved to restore the methylation level and expression of PD-1 on CD8+ T cells and reduce the cytotoxicity of CTLs of ITP patients. The phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and AKT in CD8+ T cells were significantly downregulated by low-dose decitabine. Furthermore, blocking PD-1 could counteract the effect of low-dose decitabine on CTLs from ITP patients. Therefore, our data suggest that the aberrant PD-1/PD-L1 pathway is involved in the pathophysiology of ITP and enhancing PD-1/PD-L1 signaling is a promising therapeutic approach for ITP management. Our results reveal the immunomodulatory mechanism of low-dose decitabine in ITP by inhibiting CTLs cytotoxicity to autologous platelets through PD-1 pathway.

Structural Insights into Stimulation of Ash1L's H3K36 Methyltransferase Activity through Mrg15 Binding.

The evolutionarily conserved Trithorax group protein Ash1 is a SET domain histone methyltransferase that mono- and dimethylates lysine 36 of histone H3 (H3K36). Ash1 forms a complex with Mrg15 and Nurf55, and the binding of Mrg15 greatly stimulates the catalytic activity of Ash1, yet the underlying molecular mechanisms remain unknown. Here we report the crystal structure of the tandem Mrg15-interacting and SET domains of human Ash1L in complex with Mrg15. Ash1L interacts with Mrg15 principally via a segment located N-terminal to the catalytic SET domain. Surprisingly, an autoinhibitory loop in the post-SET region of Ash1L is destabilized on Mrg15 binding despite no direct contact. Dynamics of the autoinhibitory loop can be attributed to subtle structural changes of the S-adenosylmethionine (SAM) binding pocket induced by Mrg15 binding, implicating a mechanism of conformational coupling between SAM and substrate binding sites. The findings broaden the understanding of regulation of H3K36 methyltransferases.

Disrupted balance of CD4+ T-cell subsets in bone marrow of patients with primary immune thrombocytopenia.

Disequilibrium of CD4+ T-cell subpopulations in peripheral blood (PB) of patients with primary immune thrombocytopenia (ITP) has been well established, whereas the profile of CD4+ T-cell subpopulations in bone marrow (BM) remains elusive. In the present study, the frequencies of T helper 22 (Th22), Th17, Th1, Th2, follicular T helper (Tfh) cells and regulatory T cells (Tregs) as well as their effector cytokines in BM and PB from active ITP patients and healthy controls (HCs) were determined. Results showed that the frequencies of Th22, Th17, Th1, and Tfh cells were significantly higher, but Treg number was remarkably lower in BM from ITP patients than from HCs. In the ITP group, it was notable that the numbers of BM Th22, Th17, Th1, Th2, and Tfh cells were significantly elevated compared with the matched PB counterparts, while Treg number in BM was considerably reduced compared with that in PB. In consistence with the BM Th subset pattern, plasma levels of interleukin (IL)-22, IL-17A, and interferon (INF)-γ in BM from ITP patients were significantly increased compared with that from HCs. Therefore, the balance of CD4+ T-cell subsets was disrupted in both BM and PB of ITP patients, suggesting that this might play important roles in the pathophysiological process of ITP.

Image region duplication detection based on circular window expansion and phase correlation.

Region duplication forgery is one of the tampering techniques that are frequently used, where a part of an image is copied and pasted into another part of the same image. In this paper, a phase correlation method based on polar expansion and adaptive band limitation is proposed for region duplication forgery detection. Our method starts by calculating the Fourier transform of the polar expansion on overlapping windows pair, and then an adaptive band limitation procedure is implemented to obtain a correlation matrix in which the peak is effectively enhanced. After estimating the rotation angle of the forgery region, a searching algorithm in the sense of seed filling is executed to display the whole duplicated region. Experimental results show that the proposed approach can detect duplicated region with high accuracy and robustness to rotation, illumination adjustment, blur and JPEG compression while rotation angle is estimated precisely for further calculation.