Search

Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)–based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)–specific miRNAs and miRNA-related epigenetic modulations.Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo.Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor–AKT signaling.Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel “DNMT1–miR-126 epigenetic circuit” is involved in ESCC progression. Consequently, miR-126–based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics. Clin Cancer Res; 21(4); 854–63. ©2014 AACR.

Supplementary Figure S3. Suppressive effect of miR-126 on ESCC cell migration and growth.

The file included six supplementary tables as following: Table S1.Clinicopathological Characteristics; Table S2. Differently expressed miRNAs between ECs and NMs; Table S3. Univariate and Multivariate analysis of factors associated with disease-specific survival; Table S4. A short list of functionally relevant genes significantly downredulated in 126-EC cells vs. NC-EC cells; Table S5. Primer sequences (5'-3'), Table S6. The primary antibodies for western blot and IHC.

This file contained the supplementary materials and methods including RNA extraction and quantitative real-time PCR, miRNA overexpression and RNA interference (RNAi), Cell proliferation and migration assays, Immunohistochemistry (IHC) and Western blotting, EGFR inhibitor (AG1478) and 5-aza-2'-deoxycytidine (5-aza-dC) treatments, ISH scoring, DNA methylation analysis, Target prediction, 3'-UTR vector preparation, and luciferase reporter assay, Tumor xenograft in nude mice, mRNA sequencing. As well as, legends of the six supplementary figures were included in this file.

B lymphocytes are the source of autoantibodies against the thyroid-stimulating hormone receptor (TSHR) in Graves’ disease (GD). Characterization of autoimmune B-cell expression profiles might enable a better understanding of GD pathogenesis. To reveal this, the expression levels of long noncoding RNAs (lncRNAs) and mRNAs (genes) in purified B cells from patients with newly diagnosed GD and healthy individuals were compared using microarrays, which elucidated 604 differentially expressed lncRNAs (DE-lncRNAs) and 410 differentially expressed genes (DEGs). GO and pathway analyses revealed that the DEGs are mainly involved in immune response. A protein–protein interaction network presented experimentally validated interactions among the DEGs. Two independent algorithms were used to identify the DE-lncRNAs that regulate the DEGs. Functional annotation of the deregulated lncRNA–mRNA pairs identified 14 pairs with mRNAs involved in cell proliferation. The lncRNAs TCONS_00022357-XLOC_010919 and n335641 were predicted to regulate TCL1 family AKT coactivator A (TCL1A), and the lncRNA n337845 was predicted to regulate SH2 domain containing 1A (SH2D1A). TCL1A and SH2D1A are highly involved in B-cell proliferation. The differential expression of both genes was validated by qRT-PCR. In conclusion, lncRNA and mRNA expression profiles of B cells from patients with GD indicated that the lncRNA–mRNA pairs n335641–TCL1A, TCONS_00022357-XLOC_010919–TCL1A, and n337845–SH2D1A may participate in GD pathogenesis by modulating B-cell proliferation and survival. Therefore, the identified lncRNA and mRNA may represent novel biomarkers and therapeutic targets for GD.

Hui Wang,* Xiaoye Liu,* Xuechao Jiang, Yongchun Song, Xiaoguang Wang, Jingsheng Wang, Yang Dong, Fengtong Li, Zhiqiang Wu, Yuhan Zhang, Zhiyong Yuan Department of Radiation Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, China *These authors contributed equally to this work Purpose: Traditionally, radiosurgery was considered less effective for patients with cystic brain metastases. However, comparisons of prognosis between cystic and solid brain metastases in cancer patients have been seldom reported. We aimed to compare the survival between cystic and solid brain metastases and assess risk factors for overall survival after brain metastases (BMOS) in patients who underwent radiosurgery treatment. Patients and methods: The Kaplan–Meier method and multivariate Cox regression analysis were used to compare survival time and evaluate risk factors for BMOS. Results: A total of 356 patients (including 498 brain metastases) were analyzed in our study, including 67 patients (67/356, 18.8%) with 75 cystic brain metastases. There is no statistical significance in BMOS between patients with cystic (17 months, range: 3–64 months) and solid (17.5 months, range: 1–65 months) brain metastases (P=0.148). However, the volume of cystic brain metastases decreased more slowly than solid brain metastases (P

Background: Thoracic radiation therapy plays an important role in the treatment of inoperable lung cancer. However, radiotherapy-induced toxicity poses an important challenge for radiation oncologists. Research has shown that Compound Kushen Injection (CKI) has anti-inflammatory activity and can reduce toxicity when given with chemotherapy. In the present trial, we investigated whether CKI also can decrease chemoradiotherapy-induced thoracic toxicity in lung cancer patients.Methods: A prospective, open, randomized, multi-center study was performed, in which a total of 296 patients were enrolled and randomly divided 1:1 into experimental and control groups. The control group received standard chemoradiotherapy including precise thoracic radiotherapy (60 Gy/30 fractions/6 weeks) plus concurrent platinum-based chemotherapy. The experimental group received standard chemoradiotherapy plus CKI via mainline treatments of 250 ml CKI (20 ml diluted in 0.9% normal saline) daily for 20 continuous days. The incidence of adverse events and severity of thoracic toxicity after treatment were observed according to version 4.0 of the Common Terminology Criteria for Adverse Events. Adverse drug reactions (ADRs) and quality of life (QLQ-C30) also were compared between the two groups.Results: Two hundred ninety-one qualifying patients were included in the statistical analysis. Symptomatic radiation-induced thoracic toxicity was lower in the experimental group than in the control group (14.1% vs 25.6%, p=0.017). According to QLQ-C30 questionnaire findings, CKI provided superior outcomes related to pain relief, relieving fatigue, social functioning and emotional functioning (p0.05) and severe adverse events (5.4% vs 2.8%, p>0.05) did not differ significantly between the experimental and control groups.Conclusion: CKI significantly alleviated symptomatic radiation-induced thoracic toxicity, as well as pain, when given with chemoradiotherapy for lung cancer treatment.

The endothelial-to-mesenchymal transition (EndMT) is a critical process that occurs during the development of the outflow tract (OFT). Malformations of the OFT can lead to the occurrence of conotruncal defect (CTD). SOX7 duplication has been reported in patients with congenital CTD, but its specific role in OFT development remains poorly understood. To decipher this, histological analysis showed that SRY-related HMG-box 7 (SOX7) was regionally expressed in the endocardial endothelial cells and in the mesenchymal cells of the OFT, where EndMT occurs. Experiments, using in vitro collagen gel culture system, revealed that SOX7 was a negative regulator of EndMT that inhibited endocardial cell (EC) migration and resulted in decreased number of mesenchymal cells. Forced expression of SOX7 in endothelial cells blocked further migration and improved the expression of the adhesion protein vascular endothelial (VE)-cadherin (VE-cadherin). Moreover, a VE-cadherin knockdown could partly reverse the SOX7-mediated repression of cell migration. Luciferase and electrophoretic mobility shift assay (EMSA) demonstrated that SOX7 up-regulated VE-cadherin by directly binding to the gene’s promoter in endothelial cells. The coding exons and splicing regions of the SOX7 gene were also scanned in the 536 sporadic CTD patients and in 300 unaffected controls, which revealed four heterozygous SOX7 mutations. Luciferase assays revealed that two SOX7 variants weakened the transactivation of the VE-cadherin promoter. In conclusion, SOX7 inhibited EndMT during OFT development by directly up-regulating the endothelial-specific adhesion molecule VE-cadherin. SOX7 mutations can lead to impaired EndMT by regulating VE-cadherin, which may give rise to the molecular mechanisms associated with SOX7 in CTD pathogenesis.

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19+CD24hiCD27+ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19+CD24hiCD27+ B cells (regulatory B cells, Bregs) with CD19+CD24loCD27- B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.

Xuechao Jiang Department of Radiation Oncology, Binzhou Center Hospital Affiliated to Binzhou Medical College, Binzhou, Shandong, People’s Republic of ChinaCorrespondence: Xuechao JiangDepartment of Radiation Oncology, Binzhou Center Hospital Affiliated to Binzhou Medical College, Binzhou, Shandong, People’s Republic of ChinaTel/Fax +86 543-5325652Email 13696307005@163.comPurpose: This retrospective study aimed to determine the prognostic factors associated with overall survival after intracranial local and distant recurrence in patients undergoing stereotactic radiosurgery (SRS) for brain metastases.Patients and Methods: Clinical characteristics and therapeutic parameters of 251 patients, who were treated with initial stereotactic radiosurgery for brain metastases and later experienced intracranial recurrence, were analyzed to identify prognostic factors of post-recurrence overall survival (PROS). A Cox proportional hazard model was applied for univariate and multivariate analyses.Results: Among the 251 patients, the median post-recurrence overall survival was 8 months, and the six-month PROS rate was 60.2%. The interval from initial radiosurgery treatment to intracranial recurrence (hazard ratio [HR]:0.970), the number of brain recurrent tumors (HR:1.245), the number of extracranial metastatic organs (HR:1.183), recursive partition analysis (RPA) (HR:1.778), and Eastern Cooperative Oncology Group Performance Status (ECOG PS) (HR:2.442) were identified as independent prognostic factors. The patients who received local treatment for solitary brain recurrence achieved better survival (the median survival time after recurrence was 22 months). In patients without extracranial metastasis, the median post-recurrence overall survival of the local treatment group was longer than that in the whole brain radiation therapy (WBRT) group (P< 0.001) and the systemic therapy group (P< 0.001).Conclusion: A shorter interval from initial stereotactic radiosurgery to recurrence, an increasing number of brain recurrences and extracranial metastatic organs, and poor RPA and ECOG PS values are associated with poor post-recurrence prognosis. When the number of brain recurrent tumors and extracranial metastatic organs was limited, local treatment including stereotactic radiosurgery, surgery or intensity-modulated radiation therapy (IMRT) improved the post-recurrence overall survival.Keywords: stereotactic radiosurgery, brain metastases, prognostic factors, intracranial recurrence, salvage treatment

Conotruncal heart defects (CTD) are an important subtype of congenital heart disease that occur due to abnormality in the development of the cardiac outflow tract (OFT). FOXH1 is a transcription factor that participates in the morphogenesis of the right ventricle and OFT. In this study, we confirmed the expression of FOXH1 in mouse and human embryos during OFT development. We also scanned the coding exons and splicing regions of the FOXH1 gene in 605 patients with sporadic CTD and 300 unaffected controls, from which we identified seven heterozygous FOXH1 gene mutations. According to bioinformatics analysis results, they were predicted potentially deleterious at conserved amino acid sites. Western blot was used to show that all the variants decreased the expression of FOXH1 protein, while dual-luciferase reporter assay showed that six of them, with an exception of p.P35R, had enhanced abilities to modulate the expression of MEF2C, which interacts with NKX2.5 and is involved in cardiac growth. The electrophoretic mobility shift assays result showed that two mutations altered DNA-binding abilities of mutant FOXH1 proteins. Phenotype heterogeneity was found in patients with the same mutation. These results indicate that FOXH1 mutations lead to disease-causing functional changes that contribute to the occurrence of CTD.

Objectives The purpose of this research was to study the outcome of brain metastases in a cohort of patients undergoing a second course of stereotactic image-guided robotic radiosurgery and to identify predictors corelated with survival. Methods A total of 63 patients with primary malignancies underwent a second course of CyberKnife radiosurgery for intracranial progression, including recurrence and new metastases after initial stereotactic radiosurgery (SRS). Overall survival (OS) and control rate were calculated by the Kaplan-Meier method. A Cox proportional hazards model was used to analyze predictive factors for survival. Results With a median follow-up duration of 12 months after second SRS, the median OS of the second course of radiosurgery was 18 months. On multivariate analysis, the sum of total planned target volume (hazard ratio, 2.112; 95% confidence interval, 1.069–4.173) and minimum dose (hazard ratio, 1.990, 95% confidence interval, 1.017–3.892) were significantly associated with OS. Median intracranial progression-free survival was 23 months. The 6-month and 12-month local control rates of the targets were 97.0% and 94.4%, respectively. Univariate analysis showed that only tumor number significantly influenced intracranial progression-free survival (P = 0.012). Nine patients (14.2%) developed brain necrosis. Median time to brain necrosis in regions in which brain necrosis occurred after a single course of SRS was not reached, compared with 16 months for those treated with repeat SRS (P = 0.041). Conclusions A second course of CyberKnife radiosurgery seems to be an effective salvage option for brain progression after initial SRS. The total planned target volume shows prediction for OS. Tumor volume of initial SRS may influence selection of the potential population that may benefit from salvage radiosurgery.

Background & Aims B cells infiltrate tumors, but little is known about how they affect tumor growth and progression. microRNA15A (MIR15A or miRNA15A) and microRNA16-1 (MIR16-1 or miRNA16-1) regulate cell proliferation, apoptosis, and drug resistance. We investigated their involvement in B-cell–mediated immune suppression by colorectal tumors. Methods Mice with disruptions of the gene cluster that encodes MIR15A and MIR16–1 (knockout mice), and control (C57BL/B6) mice were given azoxymethane with dextran sodium sulfate (AD) to induce formation of colorectal tumors. Mice were given anti-CD20 to delete B cells, or injections of agomir to increase MIR15A and MIR16-1. Proliferation of CD8 + T cells was measured by carboxyfluorescein-succinimidyl-ester analysis. Colon tissues were collected from mice and analyzed by flow cytometry, microRNA (miRNA) sequencing, and for cytokine production. Intestinal epithelial cells (IECs) were isolated and transfected with miRNA mimics, to identify their targets. We analyzed miRNA expression patterns and quantified B cells in colorectal cancer tissue microarrays derived from 90 patients who underwent surgical resection, from July 2006 through April 2008, in Shanghai, China; expression data were compared with clinical outcomes. Results Tumors that developed in knockout mice following administration of AD were larger and contained greater numbers of B cells than tumors that grew in control mice. Most of the B cells in the tumors were positive for immunoglobulin A (IgA + ). IgA + B cells expressed high levels of immune regulatory molecules (programmed death ligand 1, interleukin 10, and transforming growth factor beta), and repressed the proliferation and activation of CD8 + T cells. Levels of MIR15A and MIR16-1 were reduced in colon tumors from mice, compared with nontumor colon tissue. Incubation of IECs with IL17A reduced expression of MIR15A and MIR16-1. Transgenic expression of MIR15A and MIR16-1 in IECs decreased activation of NF-κB and STAT1 by reducing expression of I-kappaB kinases; this resulted in reduced production of chemokine (C-X-C motif) ligands 9 and 10 and decreased chemotaxis of IgA + B cells. Tumors in mice injected with AD and agomir grew more slowly than tumors in mice not given in agomir and contained fewer IgA + B cells. We found a negative correlation between levels of MIR15A and MIR16-1 and numbers of IgA + B cells in human colorectal tumor tissues; high levels of MIR15A and MIR16-1 and low numbers of IgA + B cells were associated with longer survival times of patients. Conclusions We found increased levels of MIR15A and MIR16-1 to reduce numbers of IgA + B cells in colorectal tumor tissues and correlate with increased survival time of patients. In mice that lack MIR15A and MIR16-1, colon tumors grow more rapidly and contain increased numbers of IgA + B cells. MIR15A and MIR16-1 appear to activate signaling pathways required for B-cell–mediated immune suppression.

Paclitaxel (PTX) is the front-line chemotherapeutic agent against human non-small cell lung cancer (NSCLC). However, its therapeutic efficacy is restricted by the increasing frequency of chemotherapeutic resistance in NSCLC. Accumulating evidence has shown the potential role of microRNAs (miRNAs) in the chemotherapeutic sensitivity of cancer cells. Previously it was reported that microRNA-7 (miR-7) acts as an important tumor suppressor in NSCLC. Therefore, the present study was conducted to determine the regulatory role of miR-7 in PTX chemotherapy for NSCLC. Four NSCLC cell lines were used to analyze the correlation of the PTX-sensitivity and endogenoaus miR-7 expression. miR-7 expression was up- and downregulated using miR-7 mimics and inhibitors respectively, and the role of miR-7 in sensitizing NSCLC cells to PTX was assessed by cell viability and apoptosis assays. The molecular mechanism of PTX sensitivity was determined by quantitative polymerase chain reaction and western blotting. It was found that the sensitivity of NSCLC cells to PTX was dependent on endogenous miR-7. Upregulation of miR-7 enhanced the PTX-sensitivity of NSCLC cells by suppressing cell proliferation and promoting cell apoptosis, while the inhibition of miR-7 abrogated the antiproliferative proapoptotic effects of PTX. Pretreatment of miR-7 mimics enhanced the PTX-mediated downregulation of epidermal growth factor receptor (EGFR) in NSCLC cells. These results have identified miR-7 as a potential EGFR-targeting sensitizer in PTX therapy. These data may facilitate the development of novel chemotherapeutic approaches for NSCLC.

Biological response modifiers (BRMs) emerge as a lay of new compounds or approaches used in improving cancer immunotherapy. Evidences highlight that cytokines, Toll-like receptor (TLR) signaling, and noncoding RNAs are of crucial roles in modulating antitumor immune response and cancer-related chronic inflammation, and BRMs based on them have been explored. In particular, besides some cytokines like IFN-α and IL-2, several Toll-like receptor (TLR) agonists like BCG, MPL, and imiquimod are also licensed to be used in patients with several malignancies nowadays, and the first artificial small noncoding RNA (microRNA) mimic, MXR34, has entered phase I clinical study against liver cancer, implying their potential application in cancer therapy. According to amounts of original data, this chapter will review the regulatory roles of TLR signaling, some noncoding RNAs, and several key cytokines in cancer and cancer-related immune response, as well as the clinical cases in cancer therapy based on them.

Biological response modifiers (BRMs) emerge as a lay of new compounds or approaches used in improving cancer immunotherapy. Evidences highlight that cytokines, Toll-like receptor (TLR) signaling, and noncoding RNAs are of crucial roles in modulating antitumor immune response and cancer-related chronic inflammation, and BRMs based on them have been explored. In particular, besides some cytokines like IFN-α and IL-2, several Toll-like receptor (TLR) agonists like BCG, MPL, and imiquimod are also licensed to be used in patients with several malignancies nowadays, and the first artificial small noncoding RNA (microRNA) mimic, MXR34, has entered phase I clinical study against liver cancer, implying their potential application in cancer therapy. According to amounts of original data, this chapter will review the regulatory roles of TLR signaling, some noncoding RNAs, and several key cytokines in cancer and cancer-related immune response, as well as the clinical cases in cancer therapy based on them.

Increasing evidence in recent years has suggested that B cells act as a crucial regulator in autoimmune diseases. However, little is known about their role in autoimmune hepatitis (AIH) and the underlying regulatory mechanisms. In this study, we show that B cells ameliorated experimental AIH (EAH) by suppressing CD4+ T-cell responses and that CD11b expression on B cells was required for the regulatory function of B cells. In vitro studies reveal that the suppressive function of CD11b was mediated by the impairment of T-cell antigen receptor (TCR) signaling transduction and the promotion of TCR down-regulation. Moreover, we show that the increased CD11b expression on B cells was interleukin (IL)−10 dependent and that additional IL-10 stimulation promoted CD11b expression on B cells, thereby enhancing B-cell regulatory effects. Conclusion: These findings reveal a previously unrecognized role for CD11b in B-cell regulatory function and its protective effect on EAH. (Hepatology 2015;62:1563–1575)

Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)–based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)–specific miRNAs and miRNA-related epigenetic modulations. Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo. Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor–AKT signaling. Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel “DNMT1–miR-126 epigenetic circuit” is involved in ESCC progression. Consequently, miR-126–based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics. Clin Cancer Res; 21(4); 854–63. ©2014 AACR.

Dextran sodium sulphate (DSS) induced colitis is commonly used to simulate human ulcerative colitis (UC). However, the mucosal immune responses related to the severity of disease have not been comprehensively documented. We used different concentration of DSS, induced various severities of colitis, and simultaneously examined the frequency of immune cells, antibodies and cytokine production. We found that T regulatory cells (Tregs), B cells, and IgA secretion increased on the recovery phase of mild colitis, accompanied by CD11b(+) cells, interleukin (IL)-6 and tumor necrosis factor (TNF)-α accumulated mildly. While during severe and irreversible colitis, the CD11b(+) cells, IL-6, and TNF-α infiltrated severely with Tregs, B cells, and IgA increased inconspicuously. These results demonstrate that Tregs, B cells, and IgA may play a significant role in maintaining the homeostasis of gut, by suppressing CD11b(+) cells and the pro-inflammatory cytokines.

Systemic lupus erythematosus (SLE) is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA) and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells in vitro. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation in vitro and in vivo. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138+ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1) and the X-box binding protein 1 (XBP1). Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern) that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.

In smart power substations, the application of precision time protocol defined in IEEE1588 becomes more and more popular. As a distributed network application, PTP is a perfect service to unify all the clocks of smart subsystems in the power substation. It will make great sense to research and improve PTP protocol in order to let it be practical and applicable for power automation applications. This paper focuses the performance of PTP. For convenience to analyze the performance of PTP, the basic elements, the messages, and protocol procedures are detailedly described in this paper. Formulas related with PTP performance are deduced. Frequency difference is the main factor to degrade the accuracy of PTP. Frequency difference among mater clock, slave clock and transparent clock is fully discussed in this paper. The influence in directly connected PTP system and in transparent clock participated PTP system is analyzed from various aspects. In the end of the paper, some countermeasures to cope with degradation of PTP performance are suggested.