Bacterial Endotoxin Activates the Coagulation Cascade through Gasdermin D-Dependent Phosphatidylserine Exposure.

Excessive activation of the coagulation system leads to life-threatening disseminated intravascular coagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. We found that caspase-11, a cytosolic LPS receptor, activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores and subsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD pores mediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, or neutralization of phosphatidylserine or TF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α and IL-1ß, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Relationship Between Serum 25-Hydroxyvitamin D and Bone Mineral Density, Fracture Risk, and Bone Metabolism in Adults With Osteoporosis/Fractures.

OBJECTIVE: To evaluate the association of serum 25-hydroxyvitamin D (25(OH) D) levels with bone mineral density (BMD), fracture risk, and bone metabolism. METHODS: This multicenter cross-sectional study recruited menopausal females and males greater than or equal to 50 year old with osteoporosis/fractures between September 2016 and September 2021. Assessment included clinical data, 25(OH)D, intact parathyroid hormone (iPTH), procollagen type 1 amino-terminal propeptide (P1NP), carboxy-terminal collagen crosslinks (CTX), lateral thoracolumbar spine x-rays, and BMD. RESULTS: A total of 3003 individuals were stratified by 25(OH) D levels: 720 individuals (24%) <20 ng/mL, 1338 individuals (44.5%) 20 to 29 ng/mL, and 945 individuals (31.5%) ≥30 ng/mL. In unadjusted and multivariable models, BMD T-score, except spine, was significantly and positively associated with 25(OH)D levels. 25(OH) D levels were inversely associated with Fracture Risk Assessment Tool scores. Patients with 25(OH)D <20 ng/mL had significantly higher iPTH and bone turnover markers (P1NP and CTX) than patients with 25(OH)D â‰§20 ng/mL in all models. When analyzing bone-related markers and BMD, total hip and femoral neck BMD T-scores were positively correlated with 25(OH)D concentrations and BMI but negatively correlated with iPTH, P1NP, CTX, and age. In multivariate models with all bone-related markers, only 25(OH)D levels were significantly associated with total hip and femoral neck BMD. CONCLUSION: Vitamin D deficiency is significantly associated with decreased total hip and femoral neck BMD and increased fracture risk as assessed by Fracture Risk Assessment Tool. In those with osteoporosis/fractures, vitamin D is implicated in the causal relationship between bone remodeling and BMD. Assessing vitamin D status is imperative for those at risk for osteoporosis/fractures.

Clinical practice guidelines for the prevention and treatment of osteoporosis in Taiwan: 2022 update.

Osteoporosis greatly increases the risk of fractures. Osteoporotic fractures negatively impact quality of life, increase the burden of care, and increase mortality. Taiwan is an area with a high prevalence of osteoporosis. This updated summary of guidelines has been developed by experts of the Taiwan Osteoporosis Association with the intention of reducing the risks of osteoporotic fractures and improving the quality of care for patients with osteoporosis. The updated guidelines compile the latest evidence to provide clinicians and other healthcare professionals with practical recommendations for the prevention, diagnosis, and management of osteoporosis under clinical settings in Taiwan.

Asia-pacific consensus on osteoporotic fracture prevention in postmenopausal women with low bone mass or osteoporosis but no fragility fractures.

Postmenopausal women are at significant risk for osteoporotic fractures due to their rapid bone loss. Half of all postmenopausal women will get an osteoporosis-related fracture over their lifetime, with 25% developing a spine deformity and 15% developing a hip fracture. By 2050, more than half of all osteoporotic fractures will occur in Asia, with postmenopausal women being the most susceptible. Early management can halt or even reverse the progression of osteoporosis. Consequently, on October 31, 2020, the Taiwanese Osteoporosis Association hosted the Asia-Pacific (AP) Postmenopausal Osteoporotic Fracture Prevention (POFP) consensus meeting, which was supported by the Asian Federation of Osteoporosis Societies (AFOS) and the Asia Pacific Osteoporosis Foundation (APOF). International and domestic experts developed ten applicable statements for the prevention of osteoporotic fractures in postmenopausal women with low bone mass or osteoporosis but no fragility fractures in the AP region. The experts advocated, for example, that postmenopausal women with a high fracture risk be reimbursed for pharmaceutical therapy to prevent osteoporotic fractures. More clinical experience and data are required to modify intervention tactics.

The role of type 1 interferons in coagulation induced by gram-negative bacteria.

Bacterial infection not only stimulates innate immune responses but also activates coagulation cascades. Overactivation of the coagulation system in bacterial sepsis leads to disseminated intravascular coagulation (DIC), a life-threatening condition. However, the mechanisms by which bacterial infection activates the coagulation cascade are not fully understood. Here we show that type 1 interferons (IFNs), a widely expressed family of cytokines that orchestrate innate antiviral and antibacterial immunity, mediate bacterial infection-induced DIC by amplifying the release of high-mobility group box 1 (HMGB1) into the bloodstream. Inhibition of the expression of type 1 IFNs and disruption of their receptor IFN-α/ßR or downstream effector (eg, HMGB1) uniformly decreased gram-negative bacteria-induced DIC. Mechanistically, extracellular HMGB1 markedly increased the procoagulant activity of tissue factor by promoting the externalization of phosphatidylserine to the outer cell surface, where phosphatidylserine assembles a complex of cofactor-proteases of the coagulation cascades. These findings not only provide novel insights into the link between innate immune responses and coagulation, but they also open a new avenue for developing novel therapeutic strategies to prevent DIC in sepsis.

Efficacy, safety and bioequivalence of the human-derived B-domain-deleted recombinant factor VIII TQG202 for prophylaxis in severe haemophilia A patients.

INTRODUCTION: Current treatment of severe haemophilia A includes prophylaxis with factor VIII (FVIII) replacement. The supply of plasma-derived FVIII is short in China. PURPOSE: To evaluate the efficacy and safety of a new B-domain deleted (BDD) recombinant FVIII (TQG202) produced by human-derived cells for prophylaxis in severe haemophilia A patients and compare the bioequivalence with Xyntha. METHODS: This multicentre, clinical trial consisted of an open-label, randomized, two-period cross-over trial assessing single-dose pharmacokinetics (PK), and a single-arm clinical trial evaluating the efficacy and safety of 24 weeks of TQG202 prophylaxis, and repeated PK were assessed after prophylaxis phase. The single-dose was 50 IU/kg in PK assessment, and the initial dose was 30 ± 5 IU/kg for prophylaxis. The primary endpoints of prophylaxis were the annualized bleeding rate (ABR) and the incremental recovery rate of the first administration. Adverse events (AEs) were recorded. RESULTS: Twenty-six participants were enrolled in the PK assessment and 81 participants in the prophylaxis phase. Mean age was 25.9 ± 10.8 years and all participants were male. The results of PK assessment showed TQG202 is bioequivalent to Xyntha. The total ABR was 2.0 (95% CI: 1.2-2.9) in prophylaxis phase. The mean incremental recovery rate of the first administration was .027 (95% CI: .026-.028) (IU/ml)/(IU/kg). AEs occurred in 42 participants, with an incidence of 51.9%. One severe AE not related to TQG202 occurred. No participants developed FVIII inhibitors. CONCLUSION: TQG202 shows bioequivalence with Xyntha. The promising efficacy and tolerability in the severe haemophilia A prophylaxis support the use of TQG202in clinical practice.

Pyruvate dehydrogenase kinase 4-mediated metabolic reprogramming is involved in rituximab resistance in diffuse large B-cell lymphoma by affecting the expression of MS4A1/CD20.

Diffuse large B cell lymphoma (DLBCL) heterogeneity promotes recurrence and anti-CD20-based therapeutic resistance. Previous studies have shown that downregulation of MS4A1/CD20 expression after chemoimmunotherapy with rituximab leads to rituximab resistance. However, the mechanisms of CD20 loss remain unknown. We identified that pyruvate dehydrogenase kinase 4 (PDK4) is markedly elevated in DLBCL cells derived from both patients and cell lines with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) resistance. We found that overexpression of PDK4 in DLBCL cells resulted in cell proliferation and resistance to rituximab in vitro and in vivo. Furthermore, loss of PDK4 expression or treatment with the PDK4 inhibitor dichloroacetate was able to significantly increase rituximab-induced cell apoptosis in DLBCL cells. Further studies suggested PDK4 mediates a metabolic shift, in that the main energy source was changed from oxidative phosphorylation to glycolysis, and the metabolic changes could play an important role in rituximab resistance. Importantly, by knocking down or overexpressing PDK4 in DLBCL cells, we showed that PDK4 has a negative regulation effect on MS4A1/CD20 expression. Collectively, this is the first study showing that targeting PDK4 has the potential to overcome rituximab resistance in DLBCL.

PRICKLE1, a Wnt/PCP signaling component, is overexpressed and associated with inferior prognosis in acute myeloid leukemia.

BACKGROUND: Prickle planar cell polarity protein 1 (PRICKLE1), a core component of the non-canonical Wnt/planar cell polarity (PCP) pathway, was recently reported to be upregulated and correlated with poor prognosis in solid cancers. However, the effect of PRICKLE1 on acute myeloid leukemia (AML) remains unknown. This study aims to characterize the prognostic significance of PRICKLE1 expression in patients with AML. METHODS: RNA-seq was performed to compare mRNA expression profiles of AML patients and healthy controls. qRT-PCR and western blotting were used to analyze the expression of PRICKLE1 in AML patients and cell lines, and two independent datasets (TCGA-LAML and TARGET-AML) online were used to validate the expression results. The correlations between the expression of PRICKLE1 and clinical features were further analyzed. RESULTS: Our data showed that PRICKLE1 expression levels were markedly high in AML patients at the time of diagnosis, decreased after complete remission and increased again at relapse. Of note, PRICKLE1 was highly expressed in drug resistant AML cells and monocytic-AML patients. High PRICKLE1 expression was found in FLT3/DNMT3A/IDH1/IDH2-mutant AML and associated with poor prognosis. Furthermore, high expression of PRICKLE1 may be correlated with migration and invasion components upregulation in AML patients. CONCLUSIONS: These results indicated that high PRICKLE1 expression may be a poor prognostic biomarker and therapeutic target of AML.

Vertebral Fractures in Type 2 Diabetes Patients: Utility of Trabecular Bone Score and Relationship With Serum Bone Turnover Biomarkers.

BACKGROUND: Patients with type 2 diabetes (T2D) have an increased risk for vertebral fracture (VF). The aim of this study is to determine the utility of trabecular bone score (TBS) in T2D patients with VF and the relationship of TBS with serum bone turnover biomarkers (SBTBs). METHODOLOGY: Postmenopausal T2D female patients were prospectively enrolled. All patients received: (1) dual-energy X-ray absorptiometry exam for bone mineral density (BMD), T-score, and TBS values; (2) lateral lumbar spine radiographs for VF assessment; and (3) SBTBs: bone specific alkaline phosphatase and Beta-C-Terminal telopeptides. BMD, T-score, TBS, and SBTBs were tested for association with VF. RESULTS: The study included 285 T2D patients (mean age = 61.1 years) and 32 patients had VF (11.2%). TBS had the strongest association with VF in T2D patients (area under curve 0.775). The TBS cutoff values for VF are 1.279 in T-score ≥1 and 1.236 in T-score <-1. In patients without VF, all sites of BMD and TBS are significantly associated with SBTBs, but in patients with VF, no associations are found between SBTBs and all sites of BMD and TBS. CONCLUSIONS: TBS can assess bone quality in the spine. The low TBS cutoff values for T2D patients with VF imply T2D does impair bone quality. Thus, TBS should be incorporated in VF risk assessment in T2D patients. In addition, a dissociated relationship between BMD and TBS with SBTBs represents imbalanced bone turnover rate and results in bone fragility and VF.

Effect of Chemotherapy on Dual-Energy X-ray Absorptiometry (DXA) Body Composition Precision Error in Head and Neck Cancer Patients.

BACKGROUND: Precision error in dual-energy X-ray absorptiometry (DXA) is defined as difference in results due to instrumental and technical factors given no biologic change. The aim of this study is to compare precision error in DXA body composition scans in head and neck cancer patients before and 2 months after chemotherapy. METHODOLOGY: A total of 34 male head and neck cancer patients with normal body mass index (BMI) were prospectively enrolled and all patients received 2 consecutive DXA scans both before and after 2 months of chemotherapy for a total of 4 scans. The precision error of 3 DXA body composition values (lean mass, fat mass, and bone mineral content) was calculated for total body and 5 body regions (arms, legs, trunk, android, and gynoid). Precision errors before and after treatment were compared using generalized estimating equation model. RESULTS: There was no significant change in precision error for the DXA total body composition values following chemotherapy; lean mass (0.33%-0.40%, p = 0.179), total fat mass (1.39%-1.70%, p = 0.259) and total bone mineral content (0.42%-0.56%, p = 0.243). However, there were significant changes in regional precision error; trunk lean mass (1.19%-1.77%, p = 0.014) and android fat mass (2.17%-3.72%, p = 0.046). CONCLUSIONS: For head and neck cancer patients, precision error of DXA total body composition values did not change significantly following chemotherapy; however, there were significant changes in fat mass in the android and lean mass in the trunk. Caution should be exercised when interpreting longitudinal DXA body composition data in those body parts.

Effects of phytoestrogens on the activity and growth of primary breast cancer cells ex vivo.

AIM: To explore the ex vivo effects of phytoestrogens on primary human breast cancer cells. METHODS: Breast cancer cells were obtained from patients who underwent primary breast cancer surgery, which were treated with 10-8 M 17ß-estradiol (E2 ), one of three phytoestrogens (genistein, resveratrol and quercetin, 10-7 M), and a combination of E2 and one of the three phytoestrogens for 48 h. These cells were then extracted for viability and apoptosis assay. The proteins involved in the proliferative and apoptotic pathways were evaluated by western blot analysis. RESULTS: Human breast cancer cell viability was inhibited by all phytoestrogens but induced by E2 with or without phytoestrogen. Apoptotic cells, as well as the proteins involved in apoptotic pathway and estrogen receptor (ER) ß, were significantly increased in the cells treated with phytoestrogen alone. The use of E2 with or without a phytoestrogen revealed completely opposite results. The proteins involved in the proliferative pathway and ER α expression were all increased in the cultures with E2 with or without phytoestrogens. CONCLUSION: In the presence of E2 , these phytoestrogens lose the effects of suppressing breast cancer cells; contrastingly, induce growth stimulatory effects by inhibiting apoptosis and stimulating proliferation in primary breast cancer cells. Thus, the effects of phytoestrogens on breast cancer should be considered as E2 still present in breast cancer tissue.

HMG-CoA synthase 1 is a synthetic lethal partner of BRAFV600E in human cancers.

Contributions of metabolic changes to cancer development and maintenance have received increasing attention in recent years. Although many human cancers share similar metabolic alterations, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Using an RNAi-based screen targeting the majority of the known metabolic proteins, we recently found that oncogenic BRAFV600E up-regulates HMG-CoA lyase (HMGCL), which converts HMG-CoA to acetyl-CoA and a ketone body, acetoacetate, that selectively enhances BRAFV600E-dependent MEK1 activation in human cancer. Here, we identified HMG-CoA synthase 1 (HMGCS1), the upstream ketogenic enzyme of HMGCL, as an additional "synthetic lethal" partner of BRAFV600E Although HMGCS1 expression did not correlate with BRAFV600E mutation in human melanoma cells, HMGCS1 was selectively important for proliferation of BRAFV600E-positive melanoma and colon cancer cells but not control cells harboring active N/KRAS mutants, and stable knockdown of HMGCS1 only attenuated colony formation and tumor growth potential of BRAFV600E melanoma cells. Moreover, cytosolic HMGCS1 that co-localized with HMGCL and BRAFV600E was more important than the mitochondrial HMGCS2 isoform in BRAFV600E-expressing cancer cells in terms of acetoacetate production. Interestingly, HMGCL knockdown did not affect HMGCS1 expression levels, whereas HMGCS1 knockdown caused a compensating increase in HMGCL protein level because of attenuated protein degradation. However, this increase did not reverse the reduced ketogenesis in HMGCS1 knockdown cells. Mechanistically, HMGCS1 inhibition decreased intracellular acetoacetate levels, leading to reduced BRAFV600E-MEK1 binding and consequent MEK1 activation. We conclude that the ketogenic HMGCS1-HMGCL-acetoacetate axis may represent a promising therapeutic target for managing BRAFV600E-positive human cancers.

ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway.

BACKGROUND/AIMS: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. METHODS: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. RESULTS: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. CONCLUSION: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

LPS enhances platelets aggregation via TLR4, which is related to mitochondria damage caused by intracellular ROS, but not extracellular ROS.

Platelet is an important cell contributing to hemostasis and immunity. Bacterial lipopolysaccharide (LPS), mainly functioning by stimulating toll-like receptor 4 (TLR4), mediates platelet activation and sepsis. However, the inter-relationship between these players in sepsis remains unknown. We found that the aggregation of platelets was enhanced in complete blood of sepsis patients than that of healthy donors. PRP isolated from complete blood of healthy donors was used in the following study to filter out the interference of irrelevant cells. The results shown that the maximum aggregation rate (MAR) was significantly higher in LPS-challenged PRP model than that of controls, and administration of the specific TLR4 inhibitor, TAK242, reduced the MAR in this model. LPS promoted P-selectin expression and intracellular ROS production, and both TAK242 and N-acetyl-L-cysteine (NAC) could depressed the LPS-induced increase of P-selectin and intracellular ROS. H2O2 administration increased P-selectin expression partially but had little effect on intracellular ROS, thought it increased mitochondrial damage. In vivo, LPS increased both intracellular ROS and CD62P comparing with that of controls, effects that were prevented by TAK242. Furthermore, platelet aggregation through LPS-TLR4 pathway was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. In conclusion, this study shows that intracellular ROS, not extracellular ROS such as H2O2, plays a crucial role in facilitating platelet aggregation via LPS/TLR4 pathway, and this process was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. The results would helpful for understanding the role of intracellular ROS and LPS-TLR4 pathway in platelet aggregation.

Correlation of quality of life with risk factors for first-incident hip fracture in postmenopausal women.

AIM: The aim of this study was to identify the correlation between health-related quality of life (HRQOL) and risk factors of first-incidence hip fracture in postmenopausal women. METHODS: This case-control study included 99 postmenopausal women with first-incident hip fracture and 101 women without hip fracture who were matched according to age. Evaluation consisted of clinical factors, 36-item Short-Form Health Survey (SF-36) and dual-energy X-ray absorptiometry for bone mineral density of hip and spine. RESULTS: The mean age of patients with an accidental first-incident hip fracture was 78.0 years. Patients with hip fractures had significantly lower scores for SF-36 domains at enrollment and 4-month follow up compared with the controls. Mental health also deteriorated significantly 4 months after hip fracture. Aside from lower HRQOL, clinical factors, including increased body height, no experience with estrogen therapy, rheumatoid arthritis, use of walking aids, less weight-bearing exercise, and diuretics use, were significant risk factors for hip fracture in univariate analysis. After multivariate adjustment, only the use of walking aids and decreased physical component summary were independent risk factors of hip fracture. Besides aging, use of walking aids, weight-bearing exercise, and psychological medication were the main factors affecting HRQOL when considering their relationship with hip fractures. CONCLUSION: The occurrence of first-incidence hip fracture is highly associated with HRQOL. Aside from aging, clinical factors affecting HRQOL correlated with hip fracture incidence. Thus, in elderly women, exercise or physical therapy to improve physical function and mental support is crucial and should be considered the most important factors for first-incidence hip fracture prevention.

[SIRT1 deficiency in CD4+T cells induces acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To study the relationship between acute graft-versus-host disease (aGVHD) and the SIRT1 expression in peripheral blood CD4+T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
 Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors. SIRT1 expression level in CD4+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4+T cells was analyzed by co-immunoprecipitation and Western blot. Over-expression of SIRT1 in aGVHD CD4+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The mRNA levels of RORγt, IL-17A, IL-17F related to Th17 were detected by real-time PCR.
 Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation (r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4+T cells from patients with aGVHD compared with patients without aGVHD. After SIRT1 over-expression in aGVHD CD4+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17A, and IL-17F related to Th17 were significantly down-regulated (P<0.05).
 Conclusion: SIRT1 deficiency in CD4+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aGVHD after allogeneic hematopoietic stem cell transplantation.

[Regulation of DNA demethylation of STAT3 promoter in CD4+ T cells from aGVHD patients by HMGB1/GADD45A].

OBJECTIVE: To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease (aGVHD) patients.
 Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-identical sibling donors. GADD45A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4+ T cells was analyzed by co-immunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively.
 Results: GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4+ T cells only with overexpression of HMGB1.
 Conclusion: The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from aGVHD patients.

Inhibitory effects of ethyl pyruvate on platelet aggregation and phosphatidylserine exposure.

Ethyl pyruvate (EP) is a stable lipophilic pyruvate derivative. Studies demonstrated that EP shows potent anti-oxidation, anti-inflammatory and anti-coagulant effects. Inflammation and coagulation are closely interacted with platelet activation. However, it is unclear whether EP has anti-platelet effects. Therefore, we investigated the anti-platelet effect of EP in this study in vitro. We found that EP inhibited agonists induced platelets aggregation, ATP release and adhesion to collagen. Flow cytometric analysis revealed that EP inhibited agonist induced platelets PAC-1 binding, as well as P-selectin and CD40L expression. The underlying mechanism of action may involve the inhibition of platelet PI3K/Akt and Protein Kinase C (PKC) signaling pathways. Additionally, EP dose dependently inhibited platelet PS exposure induced by high concentration thrombin. Lactate dehydrogenase (LDH) activity assay and mice platelet count implied that EP may have no toxic effect on platelets. Therefore, we are the first to report that EP has potent anti-platelet activity and attenuates platelet PS exposure in vitro, suggesting that the inhibitory effects of EP on platelets may also play important roles in improvement of inflammation and coagulation disorder in related animal models.

Effect of NPM1 type B mutation on the proliferation, invasion and chemosensitivity of THP-1 leukemia cells.

Acute myeloid leukemia (AML) is the most malignant myeloid disorder in adults. AML with mutated nucleophosmin (NPM1) is regarded as an independent leukemia subtype. According to previous studies, the role of NPM1 gene A mutation in AML has been well established; however, another major type, NPM1 gene B type mutation (NPM1 MutB) has been rarely reported. In the present study, we found that overexpression of NPM1 MutB enhanced the proliferation and invasion of THP-1 AML cells through the regulation of TIMP-2, MMP-2, Ang-1, c-myc and CCND1; led to no significant change of apoptosis rate with the absence of chemotherapy agents, while enhanced the chemosensitivity of THP-1 AML cells to chemotherapy agents DNR and Ara-C through the regulation of Bax, Bcl-2 and caspase-3. Further, we revealed that NPM1 MutB overexpression reduced the NF-κB activity of THP-1 cells upon drug treatment. Taken together, we demonstrated the detailed functions of NPM1MutB in THP-1 proliferation, invasion, apoptosis and chemo-sensitivity. We provided a novel understanding of prognosis of patients carrying the NPM1 B mutation.