Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

N-Acetylcysteine overcomes epalrestat-mediated increase of toxic 4-hydroxy-2-nonenal and potentiates the anti-arthritic effect of epalrestat in AIA model.

Epalrestat, an aldose reductase inhibitor (ARI), has been clinically adopted in treating diabetic neuropathy in China and Japan. Apart from the involvement in diabetic complications, AR has been implicated in inflammation. Here, we seek to investigate the feasibility of clinically approved ARI, epalrestat, for the treatment of rheumatoid arthritis (RA). The mRNA level of AR was markedly upregulated in the peripheral blood mononuclear cells (PBMCs) of RA patients when compared to those of healthy donors. Besides, the disease activity of RA patients is positively correlated with AR expression. Epalrestat significantly suppressed lipopolysaccharide (LPS) induced TNF-α, IL-1ß, and IL-6 in the human RA fibroblast-like synoviocytes (RAFLSs). Unexpectedly, epalrestat treatment alone markedly exaggerated the disease severity in adjuvant induced arthritic (AIA) rats with elevated Th17 cell proportion and increased inflammatory markers, probably resulting from the increased levels of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA). Interestingly, the combined treatment of epalrestat with N-Acetylcysteine (NAC), an anti-oxidant, to AIA rats dramatically suppressed the production of 4-HNE, MDA and inflammatory cytokines, and significantly improved the arthritic condition. Taken together, the anti-arthritic effect of epalrestat was diminished or even overridden by the excessive accumulation of toxic 4-HNE or other reactive aldehydes in AIA rats due to AR inhibition. Co-treatment with NAC significantly reversed epalrestat-induced upregulation of 4-HNE level and potentiated the anti-arthritic effect of epalrestat, suggesting that the combined therapy of epalrestat with NAC may sever as a potential approach in treating RA. Importantly, it could be regarded as a safe intervention for RA patients who need epalrestat for the treatment of diabetic complications.

Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.

Modified artery-first approach for distal pancreatectomy with celiac axis resection.

BACKGROUND: The superior mesenteric artery-first approach has been proved superior in pancreatoduodenectomy compared with the standard procedure. It is unclear whether similar benefits could be obtained in distal pancreatectomy with celiac axis resection. METHODS: Perioperative and survival outcomes of patients who underwent distal pancreatectomy with celiac axis resection with the modified artery-first approach or traditional approach between January 2012 and September 2021 were compared. RESULTS: The entire cohort comprised 106 patients (modified artery-first approach, n = 35; traditional approach, n = 71). The most common complication was postoperative pancreatic fistula (n = 18, 17.0 per cent), followed by ischaemic complications (n = 17, 16.0 per cent) and surgical site infection (n = 15, 14.0 per cent). Intraoperative blood loss (400 versus 600 ml, P = 0.017) and intraoperative transfusion rate (8.6 versus 29.6 per cent, P = 0.015) were lower in the modified artery-first approach group compared with the traditional approach group. A higher number of harvested lymph nodes (18 versus 13, P = 0.030) and R0 resection rate (88.6 versus 70.4 per cent, P = 0.038) and a lower incidence of ischaemic complications (5.7 versus 21.1 per cent, P = 0.042) was observed in the modified artery-first approach group compared with the traditional approach group. In multivariable analysis, the modified artery-first approach (OR 0.006, 95 per cent c.i., 0 to 0.447; P = 0.020) was protective against ischaemic complications. CONCLUSIONS: Compared with the traditional approach, the modified artery-first approach was associated with lower blood loss and fewer ischaemic complications, and a higher number of harvested lymph nodes and R0 resection rate. Thus, it might improve the safety, staging and prognosis of distal pancreatectomy with celiac axis resection for pancreatic cancer.

Involvement of endothelial progenitor cells in blood flow recovery through activation of the Wnt/ß-catenin signaling pathway and inhibition of high oxidative stress in diabetic hindlimb ischemic rats.

BACKGROUND: Diabetes mellitus (DM) often causes stenosis and occlusion of hindlimb blood vessels, which are also the main cause for hindlimb ischemia in elderly people. OBJECTIVES: To investigate the therapeutic effect of endothelial progenitor cell (EPC) transplantation on diabetic hindlimb ischemia. MATERIAL AND METHODS: Endothelial progenitor cells were separated, labeled with PKH-26 and transplanted into rat models (107 cells/100 g). Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect any oxidative stress. Streptozotocin (STZ) was injected to establish a diabetic rat model and hindlimb ischemia model was established via operation. Western blotting was used to detect total ß-catenin (T-ß-catenin) and non-phospho-ß-catenin (NP-ß-catenin) levels. The malondialdehyde (MDA), superoxide dismutase (SOD), Wnt3a, Wnt5a and Wnt7a levels were detected using enzyme-linked immunosorbent assay (ELISA). Oxidative stress was measured using DCFH-DA and dihydroethidium (DHE). The endothelial biomarker CD31 was observed to highlight vessels, and PKH-26 to trace migration/adhesion of EPCs. RESULTS: Endothelial progenitor cells were successfully isolated and identified, and diabetic hindlimb ischemic rat models were created. Tempol remarkably improved blood flow in diabetic hindlimb ischemic rats compared to DM+EPCs rats at 14 days (p < 0.001) and 28 days post-operation (p < 0.001). High oxidative stress was observed in diabetic hindlimb ischemic rats. Tempol significantly inhibited oxidative stress levels in diabetic hindlimb ischemic rats. Furthermore, Tempol significantly promoted angiogenesis in diabetic hindlimb ischemic rats compared to DM+EPCs rats. The ß-catenin inhibitor, XAV (DM+EPCs+Tempol+XAV group), significantly suppressed blood flow recovery and angiogenesis in diabetic hindlimb ischemic rats when compared to the DM+EPCs+Tempol group at 14 days (p = 0.026) and 28 days (p < 0.001). The XAV remarkably reduced T-ß-catenin (p < 0.001) and N-ß-catenin (p = 0.030) levels in Tempol-treated diabetic hindlimb ischemic rats, as compared to the DM+EPCs+Tempol group. The Wnt5a participated in the pathology of diabetic hindlimb ischemia. CONCLUSIONS: There are high oxidative stress levels in both EPCs in high-glucose environments and diabetic hindlimb ischemia, which can lead to limited blood flow recovery. The high oxidative stress caused the inhibition of Wnt/ß-catenin signaling pathway, leading to limited blood flow recovery in diabetic hindlimb ischemia. At the same time, Wnt5a participated in the EPC-mediated blood flow recovery.

The Role and Mechanisms of Action of Natural Compounds in the Prevention and Treatment of Cancer and Cancer Metastasis.

Cancer has emerged as one of the world's most concerning health problems. The progression and metastasis mechanisms of cancer are complex, including metabolic disorders, oxidative stress, inflammation, apoptosis, and intestinal microflora disorders. These pose significant challenges to our efforts to prevent and treat cancer and its metastasis. Natural drugs have a long history of use in the prevention and treatment of cancer. Many effective anti-tumor drugs, such as Paclitaxel, Vincristine, and Camptothecin, have been widely prescribed for the prevention and treatment of cancer. In recent years, a trend in the field of antitumor drug development has been to screen the active antitumor ingredients from natural drugs and conduct in-depth studies on the mechanisms of their antitumor activity. In this review, high-frequency keywords included in the literature of several common Chinese and English databases were analyzed. The results showed that five Chinese herbal medicines (Radix Salviae, Panax Ginseng C. A. Mey, Hedysarum Multijugum Maxim, Ganoderma, and Curcumaelongae Rhizoma) and three natural compounds (quercetin, luteolin, and kaempferol) were most commonly used for the prevention and treatment of cancer and cancer metastasis. The main mechanisms of action of these active compounds in tumor-related research were summarized. Finally, we found that four natural compounds (dihydrotanshinone, sclareol, isoimperatorin, and girinimbin) have recently attracted the most attention in the field of anti-cancer research. Our findings provide some inspiration for future research on natural compounds against tumors and new insights into the role and mechanisms of natural compounds in the prevention and treatment of cancer and cancer metastasis.

Integrated profiling of human pancreatic cancer organoids reveals chromatin accessibility features associated with drug sensitivity.

Chromatin accessibility plays an essential role in controlling cellular identity and the therapeutic response of human cancers. However, the chromatin accessibility landscape and gene regulatory network of pancreatic cancer are largely uncharacterized. Here, we integrate the chromatin accessibility profiles of 84 pancreatic cancer organoid lines with whole-genome sequencing data, transcriptomic sequencing data and the results of drug sensitivity analysis of 283 epigenetic-related chemicals and 5 chemotherapeutic drugs. We identify distinct transcription factors that distinguish molecular subtypes of pancreatic cancer, predict numerous chromatin accessibility peaks associated with gene regulatory networks, discover regulatory noncoding mutations with potential as cancer drivers, and reveal the chromatin accessibility signatures associated with drug sensitivity. These results not only provide the chromatin accessibility atlas of pancreatic cancer but also suggest a systematic approach to comprehensively understand the gene regulatory network of pancreatic cancer in order to advance diagnosis and potential personalized medicine applications.

Large-cohort humanized NPI mice reconstituted with CD34+ hematopoietic stem cells are feasible for evaluating preclinical cancer immunotherapy.

Cancer immunotherapy has achieved impressive therapeutic effects in many cancers, while only a small subset of patients benefit from it and some patients even have experienced severe toxicity. It is urgent to develop a feasible large-cohort humanized mouse model to evaluate the pre-clinical efficacy and safety of cancer immunotherapy. Furthermore, developing potentially effective combination therapy between cancer immunotherapy and other therapies also needs humanized mouse model to adequately mimic clinical actual setting. Herein, we established a humanized mouse model engrafted with less human CD34+ HSCs than ever before and then evaluated reconstitution efficiency and the profiles of human immune cells in this humanized mouse model. Also, this humanized mouse model was used to evaluate the preclinical efficacy and safety of cancer immunotherapy. For each batch of CD34+ HSCs humanized mouse model, a relatively-large cohort with over 25% human CD45+ cells in peripheral blood was established. This humanized mouse model could efficiently reconstitute human innate and adaptive immune cells. This humanized mouse model supported patient-derived xenograft tumor growth and tumor infiltration of PD-1+ human T cells. Furthermore, therapeutic efficacy, re-activation of tumor-infiltrated T cells, and side effects of checkpoint blockade therapy could be monitored in this humanized mouse model. Human T cells from this humanized mouse model were successfully engineered with CD19-CAR. CD19 CAR-T cells could effectively deplete B cells and suppress tumor growth of acute lymphoblastic leukemia in vivo in this humanized mouse model. This humanized mouse model also could be used to demonstrate the efficacy of bispecific antibodies, such as anti-CD19/CD3. Overall, our work provides a feasible large-cohort humanized mouse model for evaluating a variety of cancer immunotherapy approaches including checkpoint inhibitors, adoptive cell therapy, and bispecific antibody therapy, and demonstrates that human T cells from this humanized mouse model possess anti-tumor activities in vitro and in vivo.

The Landscape of Genetic Alterations Stratified Prognosis in Oriental Pancreatic Cancer Patients.

BACKGROUND: Pancreatic cancer is a life-threatening malignant disease with significant diversity among geographic regions and races leading to distinct carcinogenesis and prognosis. Previous studies mainly focused on Western patients, while the genomic landscape of Oriental patients, especially Chinese, remained less investigated. METHODS: A total of 408 pancreatic cancer patients were enrolled. A panel containing 436 cancer-related genes was used to detect genetic alterations in tumor samples. RESULTS: We profiled the genomic alteration landscape of pancreatic duct adenocarcinoma (PDAC), intraductal papillary mucinous neoplasm (IPMN), periampullary carcinoma (PVC), and solid-pseudopapillary tumor (SPT). Comparison with a public database revealed specific gene mutations in Oriental PDAC patients including higher mutation rates of DNA damage repair-related genes. Analysis of mutational signatures showed potential heterogenous carcinogenic factors caused by diabetes mellitus. KRAS mutation, especially KRAS G12D mutation, was associated with poor survival, while patients not harboring the 17 significant copy number variations (CNVs) had a better prognosis. We further identified multiple correlations between clinicopathologic variables and genetic mutations, as well as CNVs. Finally, by network-based stratification, three classes of PDAC patients were robustly clustered. Among these, class 1 (characterized by the Fanconi anemia pathway) achieved the best outcome, while class 2 (involved in the platinum drug resistance pathway) suffered from the worst prognosis. CONCLUSIONS: In this study, we reported for the first time the genetic alteration landscape of Oriental PDAC patients identifying many Oriental-specific alterations. The relationship between genetic alterations and clinicopathological factors as well as prognosis demonstrated important genomic impact on tumor biology. This study will help to optimize clinical treatment of Oriental PDAC patients and improve their survival.

AGEs promote calcification of HASMCs by mediating Pi3k/AKT-GSK3ß signaling.

This study aimed to investigate the effects of advanced glycation end products (AGEs) on the calcification of human arterial smooth muscle cells (HASMCs) and to explore whether AGEs can promote the calcification of HASMCs by activating the phosphoinositide 3-kinase (PI3K)/AKT-glycogen synthase kinase 3 beta (GSK3-ß) axis. Cultured HASMCs were divided into five groups: blank control group, dimethyl sulfoxide (vehicle) group, AGEs group, LY294002 (AKT inhibitor) group, and TWS119 (GSK3-ß inhibitor) group. Cells were pretreated with either vehicle, LY294002, or TWS119 for 2 hours followed by incubation with AGEs (25 µg/mL) for 5 days, and the expression levels of proteins in each group were analyzed by western blotting. AGE treatment promoted HASMC calcification, which coincided with increased expression of p-AKT and p-GSK3-ß (serine 9). Also, AGEs upregulated the expression of osteoprotegerin and bone morphogenetic protein, and these effects were suppressed by LY294002 but enhanced by TWS119. In conclusion, AGEs promote calcification of HASMCs, and this effect is ameliorated by inhibition of AKT activity but potentiated by inhibition of GSK3-ß activity. Hence, AGEs trigger HASMC calcification by regulating PI3K/AKT-GSK3-ß signaling.

Comparison of 4- and 4 plus-courses S-1 administration as adjuvant chemotherapy for pancreatic ductal adenocarcinoma.

PURPOSE: The study aimed to investigate the potential benefit of more than 4 courses of S1 adjuvant chemotherapy for patients with pancreatic ductal adenocarcinoma (PDAC) after surgery. METHOD: Data were retrospectively collected from consecutive patients who underwent S-1 adjuvant chemotherapy following curative pancreatectomy between January 2016 and December 2018. Four-courses and > 4 courses cohorts were compared for overall survival (OS) as a primary outcome, and relapse-free survival (RFS) and adverse event incidence as secondary outcomes. RESULTS: Four-courses and > 4 courses cohorts comprised 99 patients and 64 ones, respectively. TNM stage (stage II vs. I: HR, 2.125; 95% CI, 1.164-4.213; P = 0.015), duration of S-1 administration (4 vs. > 4 courses: HR, 3.113; 95% CI, 1.531-6.327; P = 0.002) and tumor grade (G3 vs. G1/2: HR, 3.887; 95% CI, 1.922-7.861; P < 0.001) were independent prognostic factors. Under the condition of patients' survival time beyond 8 months, the OS of patients in > 4 courses cohort was significantly prolonged compared with that of 4 courses cohort (4 vs. > 4 courses: HR, 2.284; 95% CI, 1.197-4.358; P = 0.012), especially for patients in TNM stageII (4 vs. > 4 courses: HR, 2.906; 95% CI, 1.275-6.623; P = 0.011).RFS and adverse events incidence did not signifcantly difer between both cohorts. CONCLUSION: Prolonged duration of S-1 intake is beneficial to prognosis of patients with PDAC resection.

Advanced Glycation End Products Induce Proliferation and Migration of Human Aortic Smooth Muscle Cells through PI3K/AKT Pathway.

Advanced glycation end products (AGEs) have been widely regarded as an important inducing factor in the pathogenesis of diabetic arteriosclerosis, and the proliferation and migration of vascular smooth muscle cells (VSMCs) are also involved in this process. However, it is not clear whether AGEs promote atherosclerosis by inducing the proliferation and migration of VSMCs. To figure out this question, this study investigated the effects of AGEs on the proliferation and migration of human aorta vascular smooth muscle cells (HASMCs) and the underlying mechanisms. This study evaluated the effects of different concentrations of AGEs on cell proliferation and migration. CCK8, transwell, and western blotting assays demonstrated that AGEs significantly increased cell proliferation and migration in a concentration-dependent manner and that the optimal proproliferative and promigratory concentrations of AGEs were 10 mg/L and 20 mg/L, respectively. AGE-induced cell proliferation, migration, and expression of filament actin (F-actin) were markedly attenuated by a PI3K inhibitor (LY2940002). Additionally, the phosphorylation of AKT was reduced when the receptor of advanced glycation end product (RAGE) gene was silenced by lentivirus transfection, which led to a concomitant reduction of the expression of proliferation and migration-related proteins. These data indicate that AGEs may activate the PI3K/AKT pathway through RAGE and thus facilitate the proliferation and migration of HASMCs.

Preoperative detection of KRAS G12D mutation in ctDNA is a powerful predictor for early recurrence of resectable PDAC patients.

BACKGROUND: About 25-37% of resectable pancreatic ductal adenocarcinoma (PDAC) had a great chance of early recurrence after radical resection, which is mainly due to preoperative micrometastasis. We herein demonstrated the profiles of ctDNA in resectable PDAC and use of ctDNA to identify patients with potential micrometastasis. METHODS: A total of 113 and 44 resectable PDACs were enrolled in discovery and validation cohorts, separately. A panel containing 50 genes was used to screen ctDNA by an NGS-based assessment with high specificity. RESULTS: In the discovery cohort, the overall detection rate was 38.05% (43/113). Among positive ctDNA, KRAS mutation had the highest detection rate (23.01%, 26/113), while the others were <5%. Survival analysis showed that plasma KRAS mutations, especially KRAS G12D mutation, had significant association with OS and RFS of resectable PDAC. Plasma KRAS G12D mutation showed a strong correlation with early distant metastasis. In the validation cohort, survival analysis showed similar association between plasma KRAS G12D mutation and poor outcomes. CONCLUSIONS: This study demonstrated that plasma KRAS mutations, especially KRAS G12D mutation, served as a useful predictive biomarker for prognosis of resectable PDAC. More importantly, due to high correlation with micrometastasis, preoperative detection of plasma KRAS G12D mutation helps in optimising surgical selection of resectable PDAC.

Liver ChREBP Protects Against Fructose-Induced Glycogenic Hepatotoxicity by Regulating L-Type Pyruvate Kinase.

Excessive fructose consumption is closely linked to the pathogenesis of metabolic disease. Carbohydrate response element-binding protein (ChREBP) is a transcription factor essential for fructose tolerance in mice. However, the functional significance of liver ChREBP in fructose metabolism remains unclear. Here, we show that liver ChREBP protects mice against fructose-induced hepatotoxicity by regulating liver glycogen metabolism and ATP homeostasis. Liver-specific ablation of ChREBP did not compromise fructose tolerance, but rather caused severe transaminitis and hepatomegaly with massive glycogen overload in mice fed a high-fructose diet, while no obvious inflammation, cell death, or fibrosis was detected in the liver. In addition, liver ATP contents were significantly decreased by ChREBP deficiency in the fed state, which was rendered more pronounced by fructose feeding. Mechanistically, liver contents of glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase, were markedly increased in the absence of liver ChREBP, while fasting-induced glycogen breakdown was not compromised. Furthermore, hepatic overexpression of LPK, a ChREBP target gene in glycolysis, could effectively rescue glycogen overload and ATP reduction, as well as mitigate fructose-induced hepatotoxicity in ChREBP-deficient mice. Taken together, our findings establish a critical role of liver ChREBP in coping with hepatic fructose stress and protecting from hepatotoxicity by regulating LPK.

Melatonin suppresses TLR9-triggered proinflammatory cytokine production in macrophages by inhibiting ERK1/2 and AKT activation.

Toll-like receptor (TLR) signaling plays major roles in innate immune response in macrophages. Melatonin regulates TLR3- and TLR4-mediated innate immune responses in macrophages. However, it remains unknown whether melatonin regulates TLR9-mediated innate immune responses in macrophages. Here we demonstrated that melatonin suppressed TLR9 ligand-induced proinflammatory cytokines mRNA and protein production in peritoneal macrophages without interrupting the viability of peritoneal macrophages. Using a melatonin membrane receptors MT1/MT2 antagonist luzindole, we found that MT1 and MT2 were dispensable for melatonin's inhibitory effects on TLR9-mediated proinflammatory cytokines production, even though melatonin upregulated mRNA expression of MT1 and MT2 in macrophages. Furthermore, melatonin did not affect mRNA expressions of TLR9 and MyD88 but attenuated TLR9 ligand-induced ERK1/2 and AKT phosphorylation without affecting p38 and NF-κB p65 phosphorylation. Also, melatonin inhibited TLR9-mediated proinflammatory cytokines production in vivo. Taken together, our results demonstrate that melatonin suppresses TLR9-triggered proinflammatory cytokines production in macrophages via melatonin membrane receptor-independent manners and probably through inhibiting ERK1/2 and AKT activation, which further elucidates the roles of melatonin in regulating TLR-mediated innate immune responses in macrophages.

ZBTB20 regulates EGFR expression and hepatocyte proliferation in mouse liver regeneration.

Liver has a unique regenerative capacity, however, its regulatory mechanism is not fully defined. We have established the zinc-finger protein ZBTB20 as a key transcriptional repressor for alpha-fetoprotein (AFP) gene in liver. As a marker of hepatic differentiation, AFP expression is closely associated with hepatocyte proliferation. Unexpectedly, here we showed that ZBTB20 acts as a positive regulator of hepatic replication and is required for efficient liver regeneration. The mice specifically lacking ZBTB20 in hepatocytes exhibited a remarkable defect in liver regeneration after partial hepatectomy, which was characterized by impaired hepatocyte proliferation along with delayed cyclin D1 induction and diminished AKT activation. Furthermore, we found that epithelial growth factor receptor (EGFR) expression was dramatically reduced in the liver in the absence of ZBTB20, thereby substantially attenuating the activation of EGFR signaling pathway in regenerating liver. Adenovirus-mediated EGFR overexpression in ZBTB20-deficient hepatocytes could largely restore AKT activation in response to EGFR ligands in vitro, as well as hepatocyte replication in liver regeneration. Furthermore, ZBTB20 overexpression could significantly restore hepatic EGFR expression and cell proliferation after hepatectomy in ZBTB20-deficient liver. Taken together, our data point to ZBTB20 as a critical regulator of EGFR expression and hepatocyte proliferation in mouse liver regeneration, and may serve as a potential therapeutic target in clinical settings of liver regeneration.

CD13hi Neutrophil-like myeloid-derived suppressor cells exert immune suppression through Arginase 1 expression in pancreatic ductal adenocarcinoma.

Perineural invasion and immunosuppressive tumor microenvironment are the distinct features of pancreatic ductal adenocarcinoma (PDAC). Heterogeneous myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity, posing obstacles for cancer immunotherapy. Increasing evidences have demonstrated the accumulation of MDSCs in PDAC patients. However, the role of MDSCs in perineural invasion of PDAC and the existence of novel MDSC subsets during PDAC remain unclear. This study found that lymphocytic perineural cuffs were frequently present in chronic pancreatitis (CP) tissues and adjacent non-neoplastic pancreatic tissues (ANPTs), but not in PDAC with perineural invasion. Meanwhile, we found that neutrophil-like MDSCs (nMDSCs), but not monocyte-like MDSCs (mMDSCs), were significantly increased in PBMCs and tumor tissues of PDAC patients. Further observation identified two distinct subsets of nMDSCs, CD13hi and CD13low nMDSCs in PDAC patients, which have not been reported previously. Despite a similar morphology, CD13hi nMDSCs expressed higher levels of CD11b, CD33, CD16 and arginase 1 but lower levels of CD66b than CD13low nMDSCs. Importantly, CD13hi MDSCs, compared with CD13low nMDSCs, more effectively suppressed alloreactive T cell responses via an arginase-1-related mechanism. After tumor resection, the circulating CD13hi nMDSCs were decreased markedly. PDAC patients with more CD13hi nMDSCs had a shorter overall survival than those with less CD13hi nMDSCs. To conclude, we identified two novel MDSC subsets with different characteristics and functions in PDAC, demonstrated the association of the two MDSC subsets with cancer progression, and explored their roles in perineural invasion and immune escape of PDAC.

AGE/RAGE promotes thecalcification of human aortic smooth muscle cells via the Wnt/ß-catenin axis.

OBJECTIVE: The present study aims to investigate whether RAGE promotes the calcification of human arterial smooth muscle cells (HASMCs) and determine the relationshipbetween RAGE and the Wnt/ß-catenin signaling pathway in this process. METHOD: In this study,there were four groups, namelythe blank control group, the non-transfection group, the empty vector group, and the RAGE transfection group.Cells were co-cultured with 10 mmol/L ß-glycerophosphoric acid, pyruvate and 20 mg/L AGE. The expression of osteogenic proteins in each group before and after the intervention wasdetected using Western blotting. Short interfering RNA (siRNA) targeting ß-catenin was used toinhibitthe expression of ß-catenin. HASMCs cultured under normal conditions were usedas the blank control. RESULTS: (1) High RAGE expression was successfully induced in HASMCs according to the results of GFP detection, flow cytometry and Western blotting. (2) Compared with the blank control group, non-transfection group and empty vector group, RAGE transfection enhanced the calcification of cells when incubated with calcification medium plus AGE. (3) The expression of RAGE, ß-catenin, OPG and Cbfa1 proteins in the blank control group, empty vector group and RAGE transfection group wasnot significantly enhanced after intervention. However, expression of the proteins in the RAGE transfection group was much higher than those of the other groups. (4) Compared with the RAGE transfection group and control siRNA group, the cells transfected with ß-catenin siRNA and cultured with interventional drugs showed significant inhibition of the expression of the downstream Cbfa1 and OPG genes. CONCLUSION: Increased expression of RAGE promoted calcification in HASMCs and up regulated the ß-catenin, OPG and Cbfa1 genes. RAGE may activate the downstream genes via the Wntß-catenin pathway, thereby promoting HASMC differentiation into osteogenic cells and calcification.

The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I-peptide complex formation.

CD8α(+) dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α(+) DCs, and Siglec-G deficient (Siglecg(-/-)) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I-peptide complexes were more abundant on Siglecg(-/-) CD8α(+) DCs than on Siglecg(+/+) CD8α(+) DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47(phox) and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I-peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity.