Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22-/- mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide-treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.

The STAT3 inhibitor stattic overcome bortezomib-resistance in multiple myeloma via decreasing PSMB6.

Bortezomib, an FDA approved drug in 2003 for newly diagnosed and relapsed/refractory MM, had showed great efficacy in different clinical settings. However, many patients still developed resistance to Bortezomib, and the mechanism of action remains unelucidated. Here, we showed that Bortezomib resistance can be partially overcome by targeting a different subunit of 20 S complex - PSMB6. PSMB6 knock down by shRNA increased sensitivity to Bortezomib in resistant and sensitive cell line. Interestingly, a STAT3 inhibitor, Stattic, is shown to selectively inhibit PSMB6 and induce apoptosis in Bortezomib resistant and sensitive MM cells, even with IL-6 induction. Therefore, PSMB6 is a novel target for Bortezomib resistance and Stattic may offer a potential therapeutic strategy.

PD-1 antibody and ruxolitinib enhances graft-versus-lymphoma effect without increasing acute graft-versus-host disease in mice.

Boosting T cell immune response posttransplant with checkpoint inhibitors increases graft-versus-lymphoma (GVL) effects at the cost of increasing acute graft-versus-host disease (aGVHD). A combined targeted therapy is needed to decrease checkpoint inhibitors-induced aGVHD without impairing GVL. We studied whether this competition could be avoided by giving concurrent anti-PD-1 antibody and ruxolitinib in allotransplant mouse models in which recipients were challenged with A20 or EL4 lymphoma cells. Given alone the PD-1 antibody increased GVL but did not improve survival of recipients challenged with A20 cells because of increased deaths from aGVHD. Adding ruxolitinib decreased levels of effector T cells and related cytokines. Tbx21- T cells had higher PD-1 levels compared with Tbx21+ T cells. Ruxolitinib increased PD-1 levels on donor T cells by suppressing Tbx21 expression. Ruxolitinib increased apoptosis of T cells which was reversed by the PD-1 antibody. PD-1 antibody preserved expression of granzyme B and cytotoxicity of T cells which were decreased by ruxolitinib. The net result of combined therapy was increased GVL, no increase in aGVHD and increased survival. The combined therapy improved survival of recipients challenged by A20 cells which expressed high level of PD-L1, but not EL4 cells which do not express PD-L1.

Dimethyl fumarate inhibits antibody-induced platelet destruction in immune thrombocytopenia mouse.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disease characterized as a low platelet count resulting from immune-mediated platelet destruction. Dimethyl fumarate (DMF) is widely applied for the treatment of several autoimmune diseases with immunosuppressive effect. However, whether it ameliorates ITP is unclear. This study aims to evaluate whether DMF has a preventive effect on ITP in mice. METHODS: DMF (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice followed by injection of rat anti-mouse integrin GPIIb/CD41antibody to induce ITP. Peripheral blood was isolated to measure platelet count and spleen mononuclear cells were extracted to measure Th1 and Treg cells along with detecting the levels of IFN-γ, and TGFß-1 in plasma and CD68 expression in spleen by immuohistochemical staining. Additionally, macrophage cell line RAW264.7 was cultured and treated with DMF followed by analysis of cell apoptosis and cycle, and the expression of FcγRI, FcγRIIb and FcγRIV mRNA. RESULTS: DMF significantly inhibited antiplatelet antibody-induced platelet destruction, decreased Th1 cells and the expression of T-bet and IFN-γ, upregulated Treg cells and the expression of Foxp3 and TGF-ß1 as well as reduced CD68 expression in the spleen of ITP mouse. DMF-treated RAW264.7 cells showed S-phase arrest, increased apoptosis and downregulated expression of FcγRI and FcγRIV. Meanwhile, in vitro treatment of DMF also decreased the expression of cyclin D1 and E2, reduced Bcl-2 level and increased Bax expression and caspase-3 activation. CONCLUSIONS: In conclusion, DMF prevents antibody-mediated platelet destruction in ITP mice possibly through promoting apoptosis, indicating that it might be used as a new approach for the treatment of ITP.

Emerging role of stem cell memory-like T cell in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by autoantibody-mediated platelet destruction. Multiple factors have been implicated in ITP pathogenesis, including T-lymphocyte dysfunctions. Increasing studies have indicated that stem cell memory-like T cell (TSCM) plays an important role in the development of multiple autoimmune diseases. This study aimed to explore the clinical correlation between the TSCM subset and ITP. The percentages of peripheral blood naïve T cells (TNs), TSCMs, central memory T cells (TCMs), effector memory T cells (TEMs) and effector T cells (TEs) among CD4+ and CD8+ T cells in 20 ITP patients before and after treatment were detected using flow cytometry. Our results showed that the percentages of peripheral blood CD4+ and CD8+ T cells in ITP patients were imbalanced. The percentage of CD8+ TSCMs in peripheral blood before treatment in ITP patients was significantly higher than that in healthy controls, whereas the percentages of the other T cell subsets did not exhibit significant differences. Our study further analysed the correlation between the change in the percentage of CD8+ TSCMs and the treatment efficacy. The results showed that the percentage of peripheral blood CD8+ TSCMs in ITP patients after glucocorticoid treatment significantly decreased and the changes of the percentages of CD8+ TSCMs before and after treatment in complete response (CR) and response (R) patients were obvious. Our finding showed that the imbalance of the percentage of CD8+ TSCMs might be involved in the development of ITP and might serve as a novel indicator of efficacy.

Constitutively Photomorphogenic 1 Reduces the Sensitivity of Chronic Lymphocytic Leukemia Cells to Fludarabine Through Promotion of Ubiquitin-Mediated P53 Degradation.

BACKGROUND/AIMS: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. METHODS: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell's apoptosis, and HG3 transplant mice survival with drug treatment. RESULTS: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. CONCLUSION: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

Platycodin D inhibits platelet function and thrombus formation through inducing internalization of platelet glycoprotein receptors.

BACKGROUND: Platycodin D (PD) is one of the major bioactive components of the roots of Platycodon grandiflorum and possesses multiple biological and pharmacological properties, such as antiviral, anti-inflammatory, and anti-cancer activities. However, whether it affects platelet function remains unclear. This study aims to evaluate the role of PD in platelet function and thrombus formation. METHODS: Platelets were treated with PD followed by measuring platelet aggregation, activation, spreading, clot retraction, expression of glycoprotein receptors. Moreover, mice platelets were treated with PD and infused into wild-type mice for analysis of in vivo hemostasis and arterial thrombosis. RESULTS: Platycodin D treatment significantly inhibited platelet aggregation in response to collagen, ADP, arachidonic acid and epinephrine, reduced platelet P-selectin expression, integrin αIIbß3 activation, spreading on fibrinogen as well as clot retraction, accompanied with decreased phosphorylation of Syk and PLCγ2 in collagen-related peptide or thrombin-stimulated platelets. Moreover, PD-treated mice platelets presented significantly impaired in vivo hemostasis and arterial thrombus formation. Interestingly, PD induced internalization of glycoprotein receptors αIIbß3, GPIbα and GPVI. However, GM6001, cytochalasin D, BAPTA-AM and wortmannin did not prevent PD-induced internalization of receptors. CONCLUSIONS: Our study demonstrates that PD inhibits platelet aggregation, activation and impairs hemostasis and arterial thrombosis, suggesting it might be a potent anti-thrombotic drug.

Recipient-derived IL-22 alleviates murine acute graft-versus-host disease in association with reduced activation of antigen presenting cells.

Acute graft-versus-host disease (aGVHD) remains a major challenging complication of patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). CD4+ effector T cells and their related cytokines mediate pathogenesis of aGVHD, in which donor-T-cell derived interleukin-22 (IL-22) was recently indicated to play a role. The role of recipient-derived IL-22 in aGVHD remains to be elucidated. By applying IL-22 knock out (IL-22KO) mice as recipients of allotransplant, we found recipient derived IL-22 alleviated aGVHD and improved survival of allotransplant recipients. Knock out of IL-22 in recipient increased levels of T-helper (Th1) 1 cells but decreased levels of regulatory T cells (Tregs) in target tissues of aGVHD. Levels of IL-22 increased in aGVHD mice. Recipient antigen presenting cells (APCs) are important sources of IL-22. IL-22 reduced activation of APCs in vitro. Defect of IL-22 in APCs resulted in increased polarization of Th1 cells but decreased level of Tregs in an in vitro co-culture system. Our data highlight an immunoregulatory function of recipient-derived IL-22 in aGVHD.

Cytotoxicity of different sized TiO2 nanoparticles in mouse macrophages.

With large-scale production and wide application of nano-titanium oxide (TiO2), its health hazard has attracted extensive attention worldwide. In this study, mouse macrophages (Ana-1 and MH-S cells) were used to evaluate the cytotoxicity of different sized TiO2 nanoparticles. The results showed that TiO2 nanoparticles caused low toxicity, especially in MH-S cells. There was a difference in the cytotoxicity induced by different sized TiO2 particles. The 25 nm anatase particles induced the strongest cytotoxicity and oxidative stress, followed by 5 and 100 nm anatase particles; in contrast, 100 nm rutile particles induced the lowest toxicity. Although TiO2 nanoparticles induced high levels of intracellular reactive oxygen species (ROS), the determination of ROS demonstrated that the inherent oxidative capacity of TiO2 nanoparticles was lower in the absence of photoactivation. Therefore, the generation of intracellular ROS could not completely depend on inherent oxidative capacity of TiO2 nanoparticles. Toxicity of TiO2 nanoparticles could mainly depend on the structural characteristics.

MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis.

Human MutT Homolog 1 (MTH1) is a nucleotide pool sanitization enzyme that hydrolyzes oxidized nucleotides to prevent their mis-incorporation into DNA under oxidative stress. Expression and functional roles of MTH1 in platelets are not known. Here, we show MTH1 expression in platelets and its deficiency impairs hemostasis and arterial/venous thrombosis in vivo. MTH1 deficiency reduced platelet aggregation, phosphatidylserine exposure and calcium mobilization induced by thrombin but not by collagen-related peptide (CRP) along with decreased mitochondrial ATP production. Thrombin but not CRP induced Ca2+-dependent mitochondria reactive oxygen species generation. Mechanistically, MTH1 deficiency caused mitochondrial DNA oxidative damage and reduced the expression of cytochrome c oxidase 1. Furthermore, MTH1 exerts a similar role in human platelet function. Our study suggests that MTH1 exerts a protective function against oxidative stress in platelets and indicates that MTH1 could be a potential therapeutic target for the prevention of thrombotic diseases.

Molecular basis of JAK2 H608Y and H608N mutations in the pathology of acute myeloid leukemia.

Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML.

Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice.

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from wild type or PDE5A-/- mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (ROS) generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK, and AKT. FeCl3-induced arterial thrombosis and venous thrombosis were assessed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A-deficient mice presented significantly prolonged tail bleeding time and delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression, and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production, and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK, and AKT. In conclusion, PDE5A modulates platelet activation and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

Inhibition of IL-1 Receptor-Associated Kinase 1 Decreases Murine Acute Graft-versus-Host Disease While Preserving the Graft-versus-Lymphoma Effect.

Activation of antigen-presenting cells (APCs) is crucial in initiating inflammation and alloreaction during acute graft-versus-host disease (aGVHD), a common life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). IL-1 receptor-associated kinase 1 (IRAK1) regulates the activation of APCs in inflammatory settings, and inhibition of IRAK1 might decrease APC activation and aGVHD. This study was conducted to explore the impact of IRAK1 inhibition on APC activation and aGVHD in mice. We administered a selective IRAK1 inhibitor, Jh-X-119-01, to recipient mice undergoing allo-HCT or co-challenged by A20 lymphoma cells. We assessed aGVHD and the graft-versus-lymphoma (GVL) effect. T cell and APC activations were analyzed as well. Jh-X-119-01 was associated with increased survival and decreased aGVHD of recipients. Jh-X-119-01 decreased the proportions of Th1 cells and Tc1 cells in the aGVHD model and in the in vitro mixed lymphocyte reaction. The IRAK1 inhibitor reduced production of TNFα and IFNγ in macrophages of recipient mice. In in vitro cultured bone marrow dendritic cells (BMDCs), Jh-X-119-01 decreased productions of inflammatory cytokines, reduced expression levels of CD80 and CD86, and decreased protein levels of antiapoptotic Bcl2 and phosphorylated NF-κB p65. RNA-seq analysis showed that Jh-X-119-01 had an impact on several pathophysiologic processes of BMDCs, including reduction of GVHD-related genes and regulation of helper T cell differentiation. Importantly, IRAK1 inhibition did not impair cytotoxic function of T cells or the allo-HCT-related GVL effect against A20 lymphoma cells. In addition, the IRAK1 inhibitor did not retard recovery of hematopoietic cells in blood or bone marrow. Our findings show that selective IRAK1 inhibition ameliorates murine aGVHD but preserves the GVL effect. Our findings may have implications for the use of an IRAK1 inhibitor in allo-HCT.

Prolonged hematological toxicity in patients receiving BCMA/CD19 CAR-T-cell therapy for relapsed or refractory multiple myeloma.

Although chimeric antigen receptor T (CAR-T) cell therapy has been indicated to be effective in treating relapsed or refractory multiple myeloma (R/R MM), severe hematological toxicity (HT) remains an intractable issue. This study enrolled 54 patients with R/R MM following combined infusion of anti-CD19 and anti-BCMA CAR-T cells. The results showed that the rates of severe cytopenia were high, including severe neutropenia (28/54, 52%), severe anemia (15/54, 28%), and severe thrombocytopenia (18/54, 33%). Moreover, the incidence of prolonged HT (PHT) on Day 28 post-infusion was 52% (28/54), including 46% for severe neutropenia, 30% for severe anemia, and 31% for severe thrombocytopenia. Patients with PHT had a poorer median progression-free survival (PFS) and overall survival (OS) than patients without PHT (P=0.011; P=0.007). Furthermore, Cox regression analyses showed that PHT was an independent risk factor for PFS and OS. Univariate analyses showed that IFNγ (OR: 1.046; 95% CI: 1.002-1.093, P=0.042) and severe HT after lymphodepletion chemotherapy (OR: 0.082; 95% CI: 0.017-0.404; P=0.002) were independent risk factors for PHT. In conclusion, these results indicated that PHT was associated with poor outcomes following CAR-T-cell therapy in MM patients. Early detection and management of PHT would be beneficial for the prevention of life-threatening complications and improvement in the survival of patients after CAR-T-cell therapy. Clinical trial registration: This trial was registered on 1 May 2017 at http://www.chictr.org.cn as ChiCTR-OIC-17011272.

Dynamic of plasma IL-22 level is an indicator of thymic output after allogeneic hematopoietic cell transplantation.

AIMS: Interleukin-22 (IL-22) promotes thymus recovery and improves T-cell recovery in preclinical allogeneic hematopoietic cell transplant models. However, the correlation between IL-22 and thymus recovery is unknown in human transplant. MATERIALS AND METHODS: In this study, plasma IL-22 levels of transplanted humans were analyzed peri-transplant. Thymic output was assessed by detecting blood signal joint T-cell receptor excision circles (TRECs). Flow cytometry was applied to measure T-cell subsets. KEY FINDINGS: Plasma IL-22 level positively correlated with blood TRECs level at days 14 and 28 posttransplant. Multiple linear regression analysis showed plasma IL-22 level, occurrence of acute graft-versus-host disease (aGVHD) and age were significantly associated with blood TRECs level at day 28 after allotransplant. An increase of plasma IL-22 level during day 14 and day 28 correlated with faster recovery of blood TRECs and naïve T-cell levels in allotransplant recipients. Recipients with high TRECs levels at day 28 had lower incidence of aGVHD comparing with those who with low TRECs levels according to a median split of their TRECs levels, an effect also seen in the high IL-22 level and low IL-22 level cohorts. Other factors such as age and infection had impacts on plasma IL-22 level in allotransplants. SIGNIFICANCE: Our findings suggest that dynamic change of plasma IL-22 level is an indicator of thymic output and occurrence of aGVHD. Monitoring plasma IL-22 level might help to assess recovery of thymus function in human allotransplants.

Plumbagin reduces chronic lymphocytic leukemia cell survival by downregulation of Bcl-2 but upregulation of the Bax protein level.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and mainly originates from an accumulation of abnormal B cells caused by the dysregulation of cell proliferation and apoptosis rates. The aberration of apoptosis-related genes in CLL cells results in defective apoptosis of CLL cells in response to traditional therapeutic medicine. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a natural compound from Plumbago zeylinica, has been shown to exhibit pro-apoptotic activities in tumor cells. In the present study, we report that plumbagin effectively inhibited CLL cell viability with a lower dose compared to fludarabine, and inhibited cell proliferation in a dose-dependent manner. In addition, plumbagin promoted accumulation of MEC-1 cells in the S phase, and blocked cell cycle transition of HG3 cells from G0/G1 to S phase. Molecularly, plumbagin markedly induced CLL cell apoptosis through reduction of Bcl-2, but through an increase in the Bax protein level. These results suggest that plumbagin may be considered as a potential anticancer agent for CLL therapy.

Decreased level of cytotoxic T lymphocyte antigen-4 (CTLA-4) in patients with acute immune thrombocytopenia (ITP).

INTRODUCTION: Previously, we demonstrated the importance of T-cell immune response cDNA 7 (TIRC7) in acute immune thrombocytopenia (ITP). As the downstream molecule of TIRC7, cytotoxic T lymphocyte antigen-4 (CTLA-4) has been verified its negative regulation of acute ITP. This study aimed to investigate the exact role of CTLA-4 and its relationship with TIRC7 in acute ITP. PATIENTS AND METHODS: 37 patients with acute ITP were enrolled and received dexamethasone (40mg/day) for 4 consecutive days. Patients who had platelet counts more than 50×10(9)/L or less were defined as responders or non-responders after treatment. The plasma, protein and mRNA levels of CTLA-4 and TIRC7 were monitored by ELISA, western blot and q-PCR, respectively. RESULTS: After high-dose dexamethasone therapy, CTLA-4 levels were significantly elevated not only in acute ITP patients (P<0.001; P<0.0001) but also in acute ITP responders (P<0.0001; P<0.0001). The levels of CTLA-4 were negatively correlated with the levels of TIRC7 before and after treatment; IFN-γ (Th1), IL-17 (Th17) and IL-22 (Th22) levels were all elevated, which were decreased after treatment not only in patients with acute ITP (P<0.01) but also in acute ITP responders (P<0.01). CONCLUSIONS: CTLA-4 level might reflect treatment efficacy and it might be associated with the pathogenesis of acute ITP.

Study on cytotoxicity and structure-activity relationship of HL-7702 cell exposed to naphthoquinones.

The acute cytotoxicities of six naphthoquinone compounds, including Atovaquone, Buparvaquone, Menadione, 2-acetoxy-1,4-naphthoquinone and 2-ethoxy-1,4-naphthoquinone, to HL-7702 cells were determined. The results showed that the toxicities of these naphthoquinones were characterized by a steep response pattern except for 2-hydroxy-1,4-naphthoquinone. Meanwhile, the cellular injuries were unrecoverable. Several molecular descriptors, such as the octanol-water partition coefficients (LogP), diameter (Dia) and topological index (TIndx), played an important role in the toxicity of naphthoquinones to HL-7702 cell. Our results provide a foundation for further investigation using 3D-QSAR and HQSAR to evaluate the aquatic ecological risk and the possible mechanisms of toxicity of naphthoquinones.

Toxic effect of different ZnO particles on mouse alveolar macrophages.

To study the toxicity mechanism of ZnO nanoparticles on mouse macrophages, the toxic effect of different ZnO nanoparticles on mouse alveolar macrophages (MH-S) was investigated in this study. The results showed that the 24h IC(50) of four ZnO particles were 48.53, 47.37, 45.43 and 26.74 µg/ml for bulk ZnO, 100 nm, 30 nm and 10-30 nm ZnO particles, respectively. At the concentration of 10 µg/ml and below, dissolved zinc ions induced metallothionein synthesis, enhanced cellular resistance to oxidative stress. ZnO particles mainly induced cell apoptosis. When the concentration of ZnO particles was 20 µg/ml and above, excessive zinc destroyed mitochondrial function and cell membrane, caused cell necrosis. Dissolved zinc ions first cause toxicity in MH-S cells. However, the toxic effect of dissolved zinc ions may exist a threshold on mouse macrophages, inducing about 50% cell death. The toxic difference of different ZnO particles mainly depended on the effect of nondissolved ZnO particles.

Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles.

With large-scale production and wide application of nanoscale ZnO, its health hazard has attracted extensive worldwide attention. In this study, cytotoxicity of different sized and shaped ZnO nanoparticles in mouse macrophage Ana-1 was investigated. And contribution of dissolved Zn(2+) and ROS in toxicity of ZnO particles was analyzed. The results indicated that ZnO particles manifested dose-dependent toxic effect on Ana-1 cells without size-dependence, and the particles shape may impact cytotoxicity of ZnO particles. When the concentration of dissolved Zn(2+) tended to equilibrium in the complete cell medium, the zinc ion concentration was approximately 10 µg/ml, inducing about 50% cell death, which was close to the cytotoxicity of ZnCl(2) (IC(50)=13.33 µg Zn/ml). The Zn(2+) concentration had significant correlations with cell viability and LDH level induced by the supernatant of ZnO particle suspensions (incubation at 37°C for 24h). Thus, the dissolved Zn(2+) played the main role in toxic effect of ZnO particles. Moreover, ROS generation assays demonstrated that ZnO particles produced intrinsically a small quantity of ROS, intracellular ROS was mainly produced after ZnO particles or the dissolved Zn(2+) entered into the cells. Although intracellular ROS had significant correlations with cell viability and LDH induced by ZnO particles, intracellular ROS may not be a major factor in cytotoxicity of ZnO nanoparticles, but the cytotoxic response.