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Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467–80. ©2018 AACR.

Supplementary Video 1. Time-lapse sequence of a serial killer NK cell attacking a cluster of six 221 cells in a 50Ã-50Ã-300 μm3 microwell. The movie plays the same sequence simultaneously side-by-side in fluorescence (left) and bright field (right). The NK cell is shown in blue (in both panels), live targets are shown in green and dead targets in red. At the end of the movie five target cells have been killed. Total imaging time was 4.5 hours with 10 minutes between frames.

Drawing upon the job demands-resources model (JD-R) and self-determination theory, this study investigates whether a set of fringe benefit initiatives taken by the police organization was able to relieve perceived work–family conflict and further reduce policemen’s intention to leave the job. A survey of 421 respondents from Beijing City Police Bureau revealed a positive relationship between work pressure, work–family conflict (WFC), and turnover intention (TI), showing that the satisfaction of fringe benefits (SFB) can moderate the linkage between WFC and TI. However, the moderating role of SFB would be crowded out when the participants possess higher level of public service motivation (PSM). This suggests that organizational support via fringe-benefit policy helps to decrease turnover intention only among employees who possess lower PSM. These findings demonstrate a special interaction of external benefits and PSM in the context of retaining police forces.

According to shock index, adult patients with HSP-related different severity gastrointestinal bleeding of this study were classified into different severity groups, aiming at analyzing and preliminarily discussing the clinical vaule of the above common blood indicators in such kinds of patient. Adult patients with HSP-related different severity gastrointestinal bleeding were retrospectively selected. Meanwhile, the healthy subjects were included as the healthy control group. According to the shock index grouping criteria, those patients were divided into three groups: no shock group, mild shock group and moderate shock group. The general data, laboratory indicators of all patients and healthy subjects were recorded in detail, and calculating NLR,PLR, then comparing the differences in the above common indicators among groups by statistical analysis. Comparing to healthy control group, WBC, N, PLR and NLR in no shock, mild shock and moderate shock groups were higher (P＜0.05); the WBC, N, hs-CRP, NLR and PLR in moderate shock group were higher than no shock and mild shock group(P＜0.05). Comparing to healthy control group, PLT, FIB and D-D in no shock, mild shock and moderate shock groups were higher (P＜0.05), and PT, INR in moderate shock group were prolonged while MPV decreased (P＜0.05); comparing to no shock group, FIB and D-D in moderate shock group were higher, while MPV decreased(P＜0.05); D-D in moderate shock group was higher than mild shock group(P＜0.05);difference in immunization and complement indicators among no shock, mild shock and moderate shock groups had no statistical significance(P＞0.05); PLR in moderate shock group was positively correlated with shock index(P＜0.05), r=0.662.There are differences on common coagulation indicators in adult patients with HSP-related different severity gastrointestinal bleeding, especially D-D is higher in moderate shock group than any other group;WBC, N, hs-CRP, PLR and NLR are different among different groups, and PLR in moderate shock group is positively correlated with shock index, higher PLR indicating more severe disease, so the above clinical indicators can be used to assess the severity of adult patients with HSP-related different severity gastrointestinal bleeding.

OBJECTIVE To evaluate the effects of low-molecular-weight heparin (LMWH) combined with low-dose aspirin (LDA) in pregnant women with a history of pregnancy-related hypertensive disorders. METHODS In the current retrospective stratified cohort study, 33 women with previous hypertensive disorders of pregnancy treated with LMWH and LDA were compared with 37 control women who did not undergo LMWH or LDA treatment. Rates of pre-eclampsia recurrence, placental abruption, and other adverse outcomes for the fetuses and pregnant women were compared in the two groups. RESULTS The pre-eclampsia recurrence rates were 12/33 (36.4%) in the LMWH + LDA group and 28/37 (75.7%) in the control group (P

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.Engineered T cells kill leukemic cells with little off-target toxicity.

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians.

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.

The efficacy of Fluorouracil (FU) in the treatment of colorectal cancer (CRC) is greatly limited by drug resistance. Autophagy has been implicated in chemoresistance, but the role of selective autophagic degradation in regulating chemoresistance remains unknown. In this study, we revealed a critical role of ABHD5 in charging CRC sensitivity to FU via regulating autophagic uracil yield. We demonstrated that ABHD5 localizes to lysosome and interacts with PDIA5 to prevent PDIA5 from interacting with RNASET2 and inactivating RNASET2. ABHD5 deficiency releases PDIA5 to directly interact with RNASET2 and leave RNASET2 in an inactivate state, which impairs RNASET2-mediated autophagic uracil yield and promotes CRC cells to uptake FU as an exogenous uracil, thus increasing their sensitivity to FU. Our findings for the first time reveal a novel role of ABHD5 in regulating lysosome function, highlighting the significance of ABHD5 as a compelling biomarker predicting the sensitivity of CRCs to FU-based chemotherapy., The mechanisms underlying differential chemotherapeutic response to 5-fluorouracil are not fully known. Here, the authors show that ABDH5 regulates sensitivity to 5-fluorouracil in colorectal cancer by regulating lysosome function.

Background: Sepsis is a system inflammation disease that can lead to liver injury. Long non-coding RNAs as crucial regulators participate in the regulation of sepsis-induced liver injury. However, the role of lncRNA CASC7 (CASC7) in the modulation of sepsis-induced liver injury remains elusive. Here, we aimed to explore the effect of CASC7 on the sepsis-induced liver injury. Methods: The sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS). The effect of CASC7 on sepsis-induced liver injury was analyzed by Hematoxylin and Eosin (HE) staining, ELISA assays, TUNEL detection kit, CCK‐8 assays, and Annexin V-FITC Apoptosis Detection Kit in vivo or in vitro. The mechanism investigation was performed using RNA pull-down, luciferase reporter gene assays, qPCR assays, and Western blot analysis.Results: The expression of CASC7 was elevated in a time-dependent manner in the liver tissues of the sepsis mice and LPS-treated LO2 cells. The depletion of CASC7 decreased the LPS treatment-induced liver injury in the sepsis mice. The treatment of LPS enhanced the apoptosis in the sepsis mice, while the depletion of CASC7 blocked this enhancement in the system. The CASC7 knockdown inhibited the LPS-enhanced expression of TNF-α and IL-1β in the mice. CASC7 served as a sponge for the miR-217 in the liver cells. CASC7 promoted the progression of sepsis-induced liver injury by sponging miR-217. MiR-217 attenuated sepsis-induced liver injury by targeting TLR4. Conclusions: Thus, we conclude that CASC7 contributes to the progression of sepsis-induced liver injury by targeting miR-217/TLR4 axis.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467–80. ©2018 AACR.

Purpose To investigate the correlation between chemoresistance of colorectal cancer to 5-fluorouracil and BNIP3 and the underlying mechanism. Methods BNIP3 protein in specimens was evaluated using immunohistochemistry. Semi-quantitative reverse transcription PCR and Western blot was employed to assay gene expression. The promoter methylation status of BNIP3 was examined by methylation-specific PCR. Drug sensitivity was assayed using MTT assay. Results Specimens from 81 patients with colorectal cancer receiving 5-fluorouracil-based chemotherapy were analyzed. BNIP3 expression was negative in 42 cancer samples. The mean score of BNIP3 in cancer was 1.8±0.2 and it was 3.7±0.5 in adjacent colorectum (p

Autophagy critically contributes to metabolic reprogramming and chromosomal stability. It has been reported that monoallelic loss of the essential autophagy gene BECN1 (encoding BECN1/Beclin 1) promotes cancer development and progression. However, the mechanism by which BECN1 is inactivated in malignancy remains largely elusive. We have previously reported a tumor suppressor role of ABHD5 (abhydrolase domain containing 5), a co-activator of PNPLA2 (patatin like phospholipase domain containing 2) in colorectal carcinoma (CRC). Here we report a noncanonical role of ABHD5 in regulating autophagy and CRC tumorigenesis. ABHD5 directly competes with CASP3 for binding to the cleavage sites of BECN1, and consequently prevents BECN1 from being cleaved by CASP3. ABHD5 deficiency provides CASP3 an advantage to cleave and inactivate BECN1, thus impairing BECN1-induced autophagic flux and augmenting genomic instability, which subsequently promotes tumorigenesis. Notably, clinical data also confirm that ABHD5 proficiency is significantly correlated with the expression levels of BECN1, LC3-II and CASP3 in human CRC tissues. Our findings suggest that ABHD5 possesses a PNPLA2-independent function in regulating autophagy and tumorigenesis, further establishing the tumor suppressor role of ABHD5, and offering an opportunity to develop new approaches aimed at preventing CRC carcinogenesis.

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8

When a T-cell Receptor (TcR) interacts with its cognate peptide-MHC (pMHC), it triggers activation of a signaling cascade that results in the elicitation of a T cell effector function. Different models have been proposed to understand which parameters are needed to obtain an optimal activation of the signaling. It was speculated that improving the binding of a TcR could bring a stronger pMHC recognition, hence a stronger stimulation of the T cell. However, it was recently shown that an increase in affinity does not seem to be sufficient to guarantee improved functionality. A combination of factors is necessary to place the modified TcR in an optimal functional window. We here compared the binding parameters of two mutants of the melanoma antigen peptide MART-127-35 specific TcR DMF5. The first mutant was previously isolated by others in a screen for improved TcR. It was reported to have an increased CD8-independent activity. We confirmed these data and showed that the enhancement was neither due to change in half life (t1/2) nor Kd of the pMHC-TcR complex. The second mutant was designed based on a previous report claiming that a particular polymorphic residue in the TRAV12-2 chain was stabilizing the TcR. We created a DMF5 mutant for this residue and showed that, unexpectedly, this TcR had acquired a reduced overall activity although the TcR-pMHC complex was more stable when compared to the TcR wild type complex (increased t1/2). In addition, the soluble TcR form of this mutant bound target cells less efficiently. From this we concluded that kinetic parameters do not always predict the superior functionality of mutant TcRs.

Amniotic fluid embolism (AFE) is an unpredictable and unpreventable complication of maternity. The presentation may range from relatively subtle clinical events to sudden maternal cardiac arrest. However, the neglected diagnosis of non-classical form of AFE (atypical AFE) is very common. The aim of this study was to examine population-based regional data from Suzhou, China. Based on the analysis of all available case reports, we put forward an outline of atypical AFE and investigate whether any variation identified could be ascribed to methodology. Retrospective study from January 2004 to December 2013, 53 cases was identified from the database of Center for Disease Control (CDC) in the city of Suzhou. We investigated the presentations of atypical AFE and maternal characteristics with potential factors underlying AFE. Multiple-regression analysis was used to calculate adjusted odds ratios (ORs) and 95 % confidence intervals (CIs). The incidence of AFE was 6.91 per 100,000 deliveries (53/766,895). Seventeen deaths occurred, a mortality rate of 32 %. Atypical AFE may as the earlier stage or mild form of AFE, there was no death case in the study with timely remedy. The atypical AFE appear is obstetric hemorrhage and/or pulmonary and renal dysfunction postpartum. Hyperfibrinolysis and coagulopathy may the early laboratory findings of atypical AFE. Atypical and classical AFE shared the same risks, such as advanced maternal age, placental abnormalities, operative deliveries, eclampsia, cervical lacerations, and induction of labor. Staying alert to premonitory symptoms of AFE is critical to turn it to a remediable disease. Patient complaints such as breathlessness, chest pain, feeling cold, distress, panic, a feeling of nausea, and vomiting should elicit close attention. The management of a suspected episode of amniotic fluid embolism is generally considered to be supportive. Hysterectomy must be performed if there is further progression of symptoms. Due to advances in acute care, mortality has decreased in recent years, highlighting the importance of early detection and treatment.

INTRODUCTION:To investigate the effect of low-molecular-weight heparin (LMWH) added low dose aspirin (LDA) in the subsequent pregnancy of the women who have the previous history of hypertensive disorder of pregnancy.METHODS:Prophylactically adding LMWH and LDA in 33 women who has previous hypertensi

Our aim was to identify a T-cell receptor (TCR) that recognizes a leukemia-associated self-antigen that is highly expressed in acute myeloid leukemia and that is restricted by HLA-A2 (expressed in approximately 50% of Caucasians) for future adoptive T-cell therapy. Tumor-associated self-antigens (self-TAA) make attractive targets for adoptive T-cell therapy in cancer. However, autologous T-cells are tolerant to self-TAA expressed on self-HLA if antigen expression is sufficiently high. In contrast, T-cells are not tolerant to self-antigens presented on non-self HLA. By identifying alloreactive T-cells reactive to self-peptide in complex with foreign HLA and subsequently cloning their TCR, it is possible to redirect patient T-cells against self-TAA. The advantage of targeting self-antigens is the potential application of TCR-engineered T-cells in all cancer patients with a certain cancer type and expressing the restricting HLA molecule. We identified a protein that is selectively expressed in normal myeloid cells and highly expressed by leukemic cells in patients with acute myeloid leukemia (AML). HLA-A2 negative CD8 T-cells from healthy donors were stimulated with HLA-A2 positive dendritic cells that were expressing the full-length target antigen. CD8 T-cells recognizing peptides from the target antigen in complex with HLA-A2 were subsequently identified and sorted using fluorescently labeled HLA-A2-peptide-multimers. T-cell clones obtained from sorted multimer-positive T-cells responded to HLA-A2 positive patient leukemia cells and cell lines expressing the antigen, whereas no response was seen to HLA-A2 positive antigen negative targeT-cells, unless loaded with the relevant peptide. Responses were measured as secretion of interferon gamma, expression of the T-cell activation marker CD137 or expression of the degranulation marker CD107. The TCR sequences were identified, and we next evaluated specificity and functionality of TCR-engineered cells. Indeed, TCR-transduced healthy donor T-cells were able to selectively kill HLA-A2 positive AML patient-derived leukemic cells as well as antigen positive cell lines in vitro. We also performed a comprehensive peptide scan to determine potential cross reactivity of the obtained TCR. For this scan, the amino acids (AA) in each position of the target peptide-epitope were individually replaced by every other natural amino acid (19 AA for each position x 9 postions = 171 peptides). In addition, potential responses to other variants of the 9mer target peptide based on the natural antigen protein sequence were evaluated (8mer, 10mers, 11mers and 12mers). Based on the results obtained with TCR-transduced T-cells (interferon gamma ELISA), candidate peptides naturally occurring in the proteome to which our TCR could cross-react were not identified. These data warrant further evaluation of the TCR for in vivo efficacy and specificity. Citation Format: Maxi-Lu Böschen, Weiwen Yang, Erlend Strønen, Johanna Olweus. Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A024.

Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4+ T cells. Here, we investigated if Ii could similarly activate human CD8+ T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8+ T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naive CD8+ T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

// Jianming He 1, 2, * , Jinming Zhou 1, * , Weiwen Yang 1 , Qi Zhou 1 , Xi Liang 3 , Xueli Pang 1 , Jianjun Li 1 , Feng Pan 1 and Houjie Liang 1 1 Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China 2 Department of Radiotherapy, Hebei Provincial Hospital of Traditional Chinese Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050011, China 3 Department of Radiology, Hebei Provincial Hospital of Traditional Chinese Medicine, Hebei University of Chinese Medicine, Shijiazhuang 050011, China * These authors have contributed equally to this work Correspondence to: Houjie Liang, email: lianghoujie@sina.com Keywords: glucocorticoid, glucocorticoid receptor, NF-κB, colon cancer, lymphoma Received: November 11, 2016 Accepted: June 02, 2017 Published: June 28, 2017 ABSTRACT Glucocorticoids are effective to treat lymphoma and leukemia. Their effect in colon cancer remains far from clear. Here, we found that glucocorticoid receptor (GR) α protein level was dramatically lower in colon cancer than in lymphoma. Colon cell lines LoVo and HCT116 were GRα-rich and GRα was not detectable in HT29 or SW480. Dexamethasone significantly inhibited cell growth of GRα-rich cell lines and did not significantly affect GRα-negative cell lines. Dexamethasone induced apoptosis and increased chemosensitivity of GRα-rich cell lines. Knockdown of GRα significantly attenuated dexamethasone effects on cell growth, apoptosis and chemosensitivity. NF-κB p65 significantly correlated with GRα in colon cancer samples. Dexamethasone decreased NF-κB p65 activity. Knockdown of NF-κB p65 increased apoptosis. Our data demonstrate GRα protein level is dramatically lower in colon cancer than in lymphoma. Dexamethasone inhibits cell growth, induces apoptosis and enhances chemosensitivity in colon cancer, at least partly, via GRα and NF-κB.

Outsourcing cancer immunotherapy Successful cancer immunotherapy depends on a patient's T cells recognizing tumor-specific mutations and then waging a lethal attack. Despite tumors harboring many mutations, most individuals have very few T cells that respond to these so-called “neo-antigens.” Strønen et al. isolated T cells from healthy donors that responded to predicted neo-antigens expressed by melanomas taken from three patients, sometimes including neo-antigens that the patient's own T cells ignored (see the Perspective by Yadav and Delamarre). Testing whether such an outsourcing strategy could improve clinical outcomes will be an important next step. Science , this issue p. 1337 ; see also p. 1275

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.

Objective: The aim of this study was to investigate the potential role of ADAMTS13 analysis in the early recognition and management of thrombotic thrombocytopenic purpura (TTP) in pregnant women. Methods: Five cases of TTP were evaluated retrospectively. Clinical and laboratory findings, von Willebrand factor (vWF)-cleaving metalloprotease (ADAMTS13) activity and maternal and neonatal outcome were recorded and analysed. Results: Five cases were all nulliparous. ADAMTS13 assay was performed and the enzyme activity was less than 5% of the normal controls in three cases. Gene mutation in the 9th exon resulting in amino acid exchange 349ArgCys in ADAMTS13 was identified in one patient. After treatment including transfusion of fresh-frozen plasma (n = 5), packed red blood cells (n = 5), platelet transfusions (n = 2) and/or continued renal replacement therapy (CRRT) (n = 1) and plasma exchange (n = 2), three patients were alive, one died on postpartum day 6 in hospital without plasma exchange and one of familial TTP died three months after discharge. Conclusion: With increasing awareness, extra-attention must be paid to patients with thrombotic microangiopathy and to measurement of ADAMTS13 activity for early diagnosis. Although severe ADAMTS13 deficiency may be helpful for TTP, it may not be sensitive enough to identify all TTP patients. Therefore, despite ADAMTS13 result positive or negative, prompt aggressive management should include early termination of pregnancy, plasma transfusion and/or plasma exchange.

INTRODUCTION:To evaluate plasma levels of platelet auto-antibodies in patients with thrombocytopenia in pregnancy and assess the clinical significance.METHODS:A total of 54 pregnant women with thrombocytopenia, 44 normal pregnant women and 28 women with immune thrombocytopenia (ITP), as well as 31 h

Herein, we show that both exogenously transfected and endogenously activated p53 repress promoter activity and expression of PDGFRB. p53 binds the proximal promoter containing the CCAAT motif as examined by EMSA and chromatin immunoprecipitation. However, gradual induction of p53 in tet-onSAOS2 cells resulted in a transient increase of the PDGFRB-promoter activity and its expression. As binding of p53 to the promoter increased, previously bound p73, DeltaNp73, c-Myc, HDAC1 and HDAC4 were dismissed from the repressed promoter, and p300 was recruited. The transient increase of the promoter activity was therefore induced by the release of the p73, Myc and HDACs, previously shown to act as repressors to this promoter. Along with further increase of p53, p300 was replaced by HDAC1 and HDAC4, resulting in decreased PDGFRB expression. For the repression, acetylation of the C-terminal lysines of p53 is important, and both acetyl-K373p53 and methyl-K370p53 became bound to the promoter. The acetyl-K373p53 was accumulated in the nucleus and colocalized with promyelocytic leukemia protein. Mitomycin treatment of MEF induced similar epigenetic modification of p53 and its binding to the promoter chromatin. Addition of a PDGFR tyrosine-kinase inhibitor to p53-inducing tet-onSAOS2 increased the number of apoptotic cells. These results suggest that p53 represses the PDGFRB promoter, facilitating the p53-induced apoptosis, whereas tumor cells with p53 mutation or a high level of DeltaNp73 or Myc could become refractory to the regulation.

The Epstein-Barr virus (EBV) tumor-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We previously reported that an E-box element at the LMP1 regulatory sequence (LRS) represses transcription of the LMP1 gene through the recruitment of a Max-Mad1-mSin3A complex. In the present study, using deletion/mutation analysis, and electrophoretic mobility shift assays, we show that the promoter region adjacent to the E-box (-59/-67) is required for the full repression conferred by E-box binding proteins. The repressive effect of these factors was overcome by an inhibitor of histone deacetylation, Trichostatin A (TSA), concurring with the reports that histone deacetylation plays an important role in repression mediated by Max-Mad1-mSin3A complex. Furthermore, ChIP analyses showed that histones at the transcriptionally active LMP1 promoter were hyperacetylated, whereas in the absence of transcription they were hypoacetylated. EBNA2 activation of the promoter required a consensus AP-2 sequence in the -103/-95 LRS region. While EMSA results and the low level of AP-2 factors expression in B cells argue against known AP-2 factors binding to this site, several pieces of evidence point to a similar mechanism of promoter activation as seen by AP-2 factors. We conclude that an AP-2 site-binding factor and EBNA2 act in concert to overcome the repression of the LMP1 promoter via the consensus AP-2 site. This activation showed strong correlation with histone hyperacetylation at the promoter, indicating this to be a major mechanism for the EBNA2 mediated LMP1 transactivation.

Electroacupuncture (EA) delivered to the acupoint (AP) called Zusanli (ST36) was administered on the bilateral hindlimb. This experiment resulted in strong expression of c-Fos immunoreactivity in the ventrolateral to lateral subdivision throughout the periaqueductal gray (PAG) compared to the non-AP and sham cases. On the other hand, it was of particular interest in the experiment of the AP that strong expression of gamma aminobutylic acid (GABA) frequently showed similar pattern of distribution to that of c-Fos in the PAG. This overlapped pattern of distribution, demonstrated in the present study, suggests that the PAG neurons activated by EA at the AP might play an important role in the descending pain control system involving the GABA since the PAG has special reference to the dorsal horn of the spinal cord and function of pain control.

Neuroblastoma is one of the most common solid tumors in childhood. With the aim of developing a targeting vector for neuroblastoma, we cloned and characterized an enhancer in the 5'-flanking regions of the MASH1 gene by a random-trap method from a 36 kb cosmid DNA. The enhancer-containing clone was identified by the expression of GFP when transfected into neuroblastoma cell lines. The enhancer-luciferase activity is higher in neuroblastoma cell lines, IMR32, BE2 and SH-SY5Y, compared with those in non-neuroblastoma cell lines, U1242 glioma, N417 small cell lung cancer and EOMA hemangioma. The core enhancer was determined within a 0.2 kb fragment, yielding three- to fourfold higher activity than that of the MASH1 promoter alone in IMR32 and BE2. This area possesses GATA- and CREB-binding sites, as well as the E-box. EMSA on this area demonstrated that CREB/ATF could bind the DNA. Chromatin immunoprecipitation assay revealed that N-myc, CREB, and co-activators CBP and PCAF, but not HDAC1, are bound to the core enhancer at the same time as the co-activators and N-myc bind to the promoter. This supports the idea that the commonly overexpressed genes HASH1 and N-myc are regulated in concert, confirming their importance as prognostic markers or targets for therapy.

Sensor devices are promising to revolutionize our interaction with the physical world by allowing continuous monitoring and reaction to natural and artificial processes at an unprecedented level of spatial and temporal resolution. As sensors become smaller, cheaper and more configurable, systems incorporating large numbers of them become feasible. Besides the technological aspects of sensor design, a critical factor enabling future sensor-driven applications will be the availability of an integrated infrastructure taking care of the onus of data management. Ideally, accessing sensor data should be no difficult or inconvenient than using simple SQL.In this paper we investigate some of the issues that such an infrastructure must address. Unlike conventional distributed database systems, a sensor data architecture must handle extremely high data generation rates from a large number of small autonomous components. And, unlike the emerging paradigm of data streams, it is infeasible to think that all this data can be streamed into the query processing site, due to severe bandwidth and energy constraints of battery-operated wireless sensors. Thus, sensing data architectures must become quality-aware , regulating the quality of data at all levels of the distributed system, and supporting user applications' quality requirements in the most efficient manner possible.

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.

Limited digestion of fructose 1, 6-bisphosphatase with subtilisin produces an S-peptide with an about 60-residue peptide fragment that is non-covalently associated with the enzyme. The 60-residue peptide fragment consists of the most part of allosteric site for AMP binding. It could be separated from S-protein by gel filtration with a Sephadex G-75 column equilibrated with 9% formic acid. According to X-ray diffraction results the S-peptide consists of two alpha-helices without beta-strand and the alpha-helix content is about 60% in the 60-residue-peptide fragment. When the enzyme is subjected to limited proteolysis with subtilisin, the secondary structure of the enzyme does not show a detectable change in CD spectrum. The CD spectra of the isolated S-peptide were measured under different concentrations. In the absence of GuHCl, S-peptide had 30% alpha-helix and 38.5% turn-like structure but had no beta-strand, suggesting that the N-terminal 60-residue fragment, which is synthesized initially by ribosome, would form a conformation spontaneously similar to that of the isolated 60-residue-peptide, i.e. about 30% alpha-helix and 30% turn-like structure. As the elongation of the peptide chain of the enzyme proceeds, the newly synthesized segment or the final entire enzyme, in turn, affects the conformation of prior peptide segment and adjusts its conformation to the final native state. The content of alpha-helix did not increase as perturbing the conformation of S-peptide by adding ethanol, cyclohexane or a small amount of SDS. On the contrary, the ordered structure was slightly decreased, indicating that the difference of conformations of S-peptide in the isolated form and in the associated protein was not an artifact produced by isolation process.

HLA molecules presenting peptides derived from tumor-associated self-antigens (self-TAA) are attractive targets for T-cell-based immunotherapy of cancer. However, detection of such epitopes is hampered by self-tolerance and limitations in the sensitivity of mass spectrometry. Here, we used T cells from HLA-A2-negative donors as tools to detect HLA-A2-bound peptides from two leukemia-associated differentiation antigens; CD20 and the previously undescribed cancer target myeloperoxidase. A high-throughput platform for epitope discovery was designed using dendritic cells cotransfected with full-length transcripts of self-TAA and HLA-A2 to allow presentation of all naturally processed peptides from a predefined self-protein on foreign HLA. Antigen-reactive T cells were directly detected using panels of color-coded peptide-HLA multimers containing epitopes predicted by a computer algorithm. Strikingly, cytotoxic T cells were generated against 37 out of 50 peptides predicted to bind HLA-A2. Among these, 36 epitopes were previously undescribed. The allorestricted T cells were exquisitely peptide- and HLA-specific and responded strongly to HLA-A2-positive leukemic cells with endogenous expression of CD20 or myeloperoxidase. These results indicate that the repertoire of self-peptides presented on HLA class I has been underestimated and that a wealth of self-TAA can be targeted by T cells when using nontolerized T-cell repertoires.

Online reviews greatly impact consumers' purchasing decisions. A slight difference in a business' rating on a review website can significantly change the company's bottom line in some cases. By the same token, review websites are often targeted by spammers with fraudulent reviews, either to exaggerate the positive features of a business itself or to defame a competitor with negative ratings/comments. Many consumers' online reviews contain such information as rating value, customer name, and descriptions about a product or service. This paper discusses methods that help web administrators and/or business managers identify the legitimate versus illegitimate customers, use auto regression moving average (ARMA) to predict ratings, and more importantly, detect fraudulent reviews by comparing the differences among customer class, predicted rating, and the actual rating by the customer. In the end, this paper reports an experiment using online restaurant reviews to test the proposed algorithms. The results suggest that our method can yield high accuracy in detecting fraudulent restaurant reviews.

Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

Objective To investigate factors associated with acute maternal morbidity and mortality in Kowloon Hospital, Suzhou, China. Methods Data from cases of near-miss and maternal death between January 2008 and December 2012 were reviewed retrospectively. Maternal characteristics and related factors were identified, and multiple regression analysis was used to calculate adjusted odds ratios (aORs) and 95% confidence intervals (CIs). Results During the study period, there were 18 104 deliveries, 69 near-miss cases, and 3 maternal deaths. Women who had no health insurance (aOR, 4.55; 95% CI, 0.87–21.8), had fewer than 6 prenatal consultations (aOR, 6.76; 95% CI, 0.76–45.8), were part of a migrant population (aOR, 2.34; 95% CI, 0.45–24.9), or delayed seeking healthcare (aOR, 4.76; 95% CI, 0.89–13.6) had a greater risk of near-miss morbidity or death. Admission to intensive care (aOR, 6.75; 95% CI, 0.89–34.6) and blood transfusion within 30 min (aOR, 3.79; 95% CI, 0.65–8.67) were protective factors in disease progression. Conclusion The factors associated with maternal near-miss morbidity and mortality were closely related to health insurance and socioeconomic status, suggesting that the government should take an active role in the community in preventing morbidity and mortality in pregnancy.

T cells engineered to express chimeric antigen receptors (CARs) targeted to CD19 are effective in treatment of B-lymphoid malignancies. However, CARs recognize all CD19 positive (pos) cells, and durable responses are linked to profound depletion of normal B cells. Here, we designed a strategy to specifically target patient B cells by utilizing the fact that T-cell receptors (TCRs), in contrast to CARs, are restricted by HLA. Two TCRs recognizing a peptide from CD20 (SLFLGILSV) in the context of foreign HLA-A*02:01 (CD20p/HLA-A2) were expressed as 2A-bicistronic constructs. T cells re-directed with the A23 and A94 TCR constructs efficiently recognized malignant HLA-A2(pos) B cells endogenously expressing CD20, including patient-derived follicular lymphoma and chronic lymphocytic leukemia (CLL) cells. In contrast, a wide range of HLA-A2(pos)CD20(neg) cells representing different tissue origins, and HLA-A2(neg)CD20(pos) cells, were not recognized. Cytotoxic T cells re-directed with CD20p/HLA-A2-specific TCRs or CD19 CARs responded with similar potencies to cells endogenously expressing comparable levels of CD20 and CD19. The CD20p/HLA-A2-specific TCRs recognized CD20p bound to HLA-A2 with high functional avidity. The results show that T cells expressing CD20p/HLA-A2-specific TCRs efficiently and specifically target B cells. When used in context of an HLA-haploidentical allogeneic stem cell transplantation where the donor is HLA-A2(neg) and the patient HLA-A2(pos), these T cells would selectively kill patient-derived B cells and allow reconstitution of the B-cell compartment with HLA-A2(neg) donor cells. These results should pave the way for clinical testing of T cells genetically engineered to target malignant B cells without permanent depletion of normal B cells.

Computer manufacturers consist of multiple departments, and each department has its own duties and functions. As email is often used as the primary communication tool for computer industries, the customer service departments of the computer manufacturers receive a large number of emails daily. The emails need to be forwarded to the corresponding personnel and processed. Excessive resources and time are spent in manually reading and answering customers' emails. An automatic reply tool is crucial in reducing company resources for manually processing customers' emails. This paper discusses how to improve processing emails automatically for computer manufacturers. Specifically, emails from senders are classified into multiple categories by machine learning algorithms. The reply for each email category is predefined. The Automatic Email Responding System (AERS) replies to an email based on the predefined, corresponding category. This experiment shows that the automatic tool can process and answer emails efficiently.