Sample records for activated b-cell abc

  1. Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma.

    PubMed

    Lenz, Georg; Nagel, Inga; Siebert, Reiner; Roschke, Anna V; Sanger, Warren; Wright, George W; Dave, Sandeep S; Tan, Bruce; Zhao, Hong; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D; Campo, Elias; Jaffe, Elaine S; Smeland, Erlend B; Fisher, Richard I; Kuehl, W Michael; Chan, Wing C; Staudt, Louis M

    2007-03-19

    To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB.

  2. Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1.

    PubMed

    Gu, Yongzhong; Bian, Yuehong; Xu, Xiaofei; Wang, Xietong; Zuo, Changting; Meng, Jinlai; Li, Hongyan; Zhao, Shigang; Ning, Yunnan; Cao, Yongzhi; Huang, Tao; Yan, Junhao; Chen, Zi-Jiang

    2016-12-01

    Placenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. Expression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. Expression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3' untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. Our results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta. Copyright © 2016 Elsevier Ltd. All rights

  3. Comparison of the algorithms classifying the ABC and GCB subtypes in diffuse large B-cell lymphoma.

    PubMed

    Boltežar, Lučka; Prevodnik, Veronika Kloboves; Perme, Maja Pohar; Gašljević, Gorana; Novaković, Barbara Jezeršek

    2018-05-01

    Different immunohistochemical algorithms for the classification of the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of diffuse large B-cell lymphoma (DLBCL) are applied in different laboratories. In the present study, 127 patients with DLCBL were investigated, all treated with rituximab and cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP) or CHOP-like regimens between April 2004 and December 2010. Multi-tumor tissue microarrays were prepared and were tested according to 4 algorithms: Hans; modified Hans; Choi; and modified Choi. For 39 patients, the flow cytometric quantification of CD19 and CD20 antigen expression was performed and the level of expression presented as molecules of equivalent soluble fluorochrome units. The Choi algorithm was demonstrated to be prognostic for OS and classified patients into the GCB subgroup with an HR of 0.91. No difference in the expression of the CD19 antigen between the ABC and GCB groups was observed, but the ABC subtype exhibited a decreased expression of the CD20 antigen compared with the GCB subtype.

  4. A Phase I Clinical Trial of Systemically Delivered NEMO Binding Domain Peptide in Dogs with Spontaneous Activated B-Cell like Diffuse Large B-Cell Lymphoma

    PubMed Central

    Habineza Ndikuyeze, Georges; Gaurnier-Hausser, Anita; Patel, Reema; Baldwin, Albert S.; May, Michael J.; Flood, Patrick; Krick, Erika; Propert, Kathleen J.; Mason, Nicola J.

    2014-01-01

    Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL. PMID:24798348

  5. Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation.

    PubMed

    Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B; Lenz, Georg; Ruland, Jürgen

    2015-12-29

    The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL.

  6. NEMO Binding Domain peptide inhibits constitutive NF-κB activity and reduces tumor burden in a canine model of relapsed, refractory Diffuse Large B-Cell Lymphoma

    PubMed Central

    Gaurnier-Hausser, Anita; Patel, Reema; Baldwin, Albert S.; May, Michael J.; Mason, Nicola J.

    2011-01-01

    Purpose Activated B-Cell Diffuse Large B-Cell Lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via over-expression of anti-apoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL. Experimental Design Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by qRT-PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor Nemo Binding Domain (NBD) peptide, and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intra-nodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre and post treatment. Results Constitutive canonical NF-κB activity and increased NF-κB target gene expression was detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intra-tumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden. Conclusions This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and demonstrates the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients. PMID:21610150

  7. Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT.

    PubMed

    Paul, Juliane; Soujon, Maurice; Wengner, Antje M; Zitzmann-Kolbe, Sabine; Sturz, Andrea; Haike, Katja; Keng Magdalene, Koh Hui; Tan, Sze Huey; Lange, Martin; Tan, Soo Yong; Mumberg, Dominik; Lim, Soon Thye; Ziegelbauer, Karl; Liu, Ningshu

    2017-01-09

    Compared with follicular lymphoma, high PI3Kα expression was more prevalent in diffuse large B cell lymphoma (DLBCL), although both tumor types expressed substantial PI3Kδ. Simultaneous inhibition of PI3Kα and PI3Kδ dramatically enhanced the anti-tumor profile in ABC-DLBCL models compared with selective inhibition of PI3Kδ, PI3Kα, or BTK. The anti-tumor activity was associated with suppression of p-AKT and a mechanism of blocking nuclear factor-κB activation driven by CD79 mut , CARD11 mut , TNFAIP3 mut , or MYD88 mut . Inhibition of PI3Kα/δ resulted in tumor regression in an ibrutinib-resistant CD79B WT /MYD88 mut patient-derived ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79B mut -ABC-DLBCL to show a robust response to PI3K and BTK inhibitor monotherapies. A combination of ibrutinib with the PI3Kα/δ inhibitor copanlisib produced a sustained complete response in vivo in CD79B mut /MYD88 mut ABC-DLBCL models. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. CD79B limits response of diffuse large B cell lymphoma to ibrutinib.

    PubMed

    Kim, Joo Hyun; Kim, Won Seog; Ryu, Kyungju; Kim, Seok Jin; Park, Chaehwa

    2016-01-01

    Blockage of B cell receptor signaling with ibrutinib presents a promising clinical approach for treatment of B-cell malignancies. However, many patients show primary resistance to the drug or develop secondary resistance. In the current study, cDNA microarray and Western blot analyses revealed CD79B upregulation in the activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) that display differential resistance to ibrutinib. CD79B overexpression was sufficient to induce resistance to ibrutinib and enhanced AKT and MAPK activation, indicative of an alternative mechanism underlying resistance. Conversely, depletion of CD79B sensitized primary refractory cells to ibrutinib and led to reduced phosphorylation of AKT or MAPK. Combination of the AKT inhibitor or the MAPK inhibitor with ibrutinib resulted in circumvention of both primary and acquired resistance in ABC-DLBCL. Our data collectively indicate that CD79B overexpression leading to activation of AKT/MAPK is a potential mechanism underlying primary ibrutinib resistance in ABC-DLBCL, and support its utility as an effective biomarker to predict therapeutic response to ibrutinib.

  9. Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

    PubMed

    Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

    2016-08-08

    Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. ABC-B transporter genes in Dirofilaria immitis.

    PubMed

    Bourguinat, Catherine; Che, Hua; Mani, Thangadurai; Keller, Kathy; Prichard, Roger K

    2016-08-01

    Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite. Copyright © 2016. Published by Elsevier Ltd.

  11. High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell–like diffuse large B-cell lymphoma cells

    PubMed Central

    Mathews Griner, Lesley A.; Guha, Rajarshi; Shinn, Paul; Young, Ryan M.; Keller, Jonathan M.; Liu, Dongbo; Goldlust, Ian S.; Yasgar, Adam; McKnight, Crystal; Boxer, Matthew B.; Duveau, Damien Y.; Jiang, Jian-Kang; Michael, Sam; Mierzwa, Tim; Huang, Wenwei; Walsh, Martin J.; Mott, Bryan T.; Patel, Paresma; Leister, William; Maloney, David J.; Leclair, Christopher A.; Rai, Ganesha; Jadhav, Ajit; Peyser, Brian D.; Austin, Christopher P.; Martin, Scott E.; Simeonov, Anton; Ferrer, Marc; Staudt, Louis M.; Thomas, Craig J.

    2014-01-01

    The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug–drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell–like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton’s tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL. PMID:24469833

  12. Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

    PubMed

    Havranek, Ondrej; Xu, Jingda; Köhrer, Stefan; Wang, Zhiqiang; Becker, Lisa; Comer, Justin M; Henderson, Jared; Ma, Wencai; Man Chun Ma, John; Westin, Jason R; Ghosh, Dipanjan; Shinners, Nicholas; Sun, Luhong; Yi, Allen F; Karri, Anusha R; Burger, Jan A; Zal, Tomasz; Davis, R Eric

    2017-08-24

    We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL. © 2017 by The American Society of Hematology.

  13. Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma.

    PubMed

    Yang, Yibin; Kelly, Priscilla; Shaffer, Arthur L; Schmitz, Roland; Yoo, Hee Min; Liu, Xinyue; Huang, Da Wei; Webster, Daniel; Young, Ryan M; Nakagawa, Masao; Ceribelli, Michele; Wright, George W; Yang, Yandan; Zhao, Hong; Yu, Xin; Xu, Weihong; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa; Staudt, Louis M

    2016-04-11

    Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activateB kinase (IKK) and the classical NF-κB pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-κB activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-κB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. The minimal-ABC trees with B1-branches.

    PubMed

    Dimitrov, Darko; Du, Zhibin; Fonseca, Carlos M da

    2018-01-01

    The atom-bond connectivity index (or, for short, ABC index) is a molecular structure descriptor bridging chemistry to graph theory. It is probably the most studied topological index among all numerical parameters of a graph that characterize its topology. For a given graph G = (V, E), the ABC index of G is defined as [Formula: see text], where di denotes the degree of the vertex i, and ij is the edge incident to the vertices i and j. A combination of physicochemical and the ABC index properties are commonly used to foresee the bioactivity of different chemical composites. Additionally, the applicability of the ABC index in chemical thermodynamics and other areas of chemistry, such as in dendrimer nanostars, benzenoid systems, fluoranthene congeners, and phenylenes is well studied in the literature. While finding of the graphs with the greatest ABC-value is a straightforward assignment, the characterization of the tree(s) with minimal ABC index is a problem largely open and has recently given rise to numerous studies and conjectures. A B1-branch of a graph is a pendent path of order 2. In this paper, we provide an important step forward to the full characterization of these minimal trees. Namely, we show that a minimal-ABC tree contains neither 4 nor 3 B1-branches. The case when the number of B1-branches is 2 is also considered.

  15. Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma

    PubMed Central

    Nagel, Daniel; Bognar, Miriam; Eitelhuber, Andrea C.; Kutzner, Kerstin; Vincendeau, Michelle; Krappmann, Daniel

    2015-01-01

    Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances. PMID:26540570

  16. Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

    2011-01-25

    Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

  17. Activation of the STAT3 Signaling Pathway Is Associated With Poor Survival in Diffuse Large B-Cell Lymphoma Treated With R-CHOP

    PubMed Central

    Huang, Xin; Meng, Bin; Iqbal, Javeed; Ding, B. Belinda; Perry, Anamarija M.; Cao, Wenfeng; Smith, Lynette M.; Bi, Chengfeng; Jiang, Chunsun; Greiner, Timothy C.; Weisenburger, Dennis D.; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Gascoyne, Randy D.; Cook, James R.; Tubbs, Raymond R.; Jaffe, Elaine S.; Armitage, James O.; Vose, Julie M.; Staudt, Louis M.; McKeithan, Timothy W.; Chan, Wing C.; Ye, B. Hilda; Fu, Kai

    2013-01-01

    Purpose We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. Patients and Methods By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. Results PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non–germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). Conclusion STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype. PMID:24220563

  18. Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

    PubMed Central

    Greene, Nicholas P.; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

    2018-01-01

    The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking. PMID:29892271

  19. Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

    PubMed

    Greene, Nicholas P; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

    2018-01-01

    The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

  20. ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma

    PubMed Central

    2013-01-01

    Background Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. Methods Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. Results Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. Conclusions ABC transporter inhibitors are already being

  1. ABCE1 is essential for S phase progression in human cells

    PubMed Central

    Toompuu, Marina; Kärblane, Kairi; Pata, Pille; Truve, Erkki; Sarmiento, Cecilia

    2016-01-01

    ABSTRACT ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion. PMID:26985706

  2. Targeting receptor-activator of nuclear kappaB ligand in aneurysmal bone cysts: verification of target and therapeutic response.

    PubMed

    Pelle, Dominic W; Ringler, Jonathan W; Peacock, Jacqueline D; Kampfschulte, Kevin; Scholten, Donald J; Davis, Mary M; Mitchell, Deanna S; Steensma, Matthew R

    2014-08-01

    Aneurysmal bone cyst (ABC) is a benign tumor of bone presenting as a cystic, expansile lesion in both the axial and appendicular skeleton. Axial lesions demand special consideration, because treatment-related morbidity can be devastating. In similar lesions, such as giant cell tumor of bone (GCTB), the receptor-activator of nuclear kappaB ligand (RANKL)-receptor-activator of nuclear kappaB (RANK) signaling axis is essential to tumor progression. Although ABC and GCTB are distinct entities, they both contain abundant multinucleated giant cells and are osteolytic characteristically. We hypothesize that ABCs express both RANKL and RANK similarly in a cell-type specific manner, and that targeted RANKL therapy will mitigate ABC tumor progression. Cellular expression of RANKL and RANK was determined in freshly harvested ABC samples using laser confocal microscopy. A consistent cell-type-specific pattern was observed: fibroblastlike stromal cells expressed RANKL strongly whereas monocyte/macrophage precursor and multinucleated giant cells expressed RANK. Relative RANKL expression was determined by quantitative real-time polymerase chain reaction in ABC and GCTB tissue samples; no difference in relative expression was observed (P > 0.05). In addition, we review the case of a 5-year-old boy with a large, aggressive sacral ABC. After 3 months of targeted RANKL inhibition with denosumab, magnetic resonance imaging demonstrated tumor shrinkage, bone reconstitution, and healing of a pathologic fracture. Ambulation, and bowel and bladder function were restored at 6 months. Denosumab treatment was well tolerated. Post hoc analysis demonstrated strong RANKL expression in the pretreatment tumor sample. These findings demonstrate that RANKL-RANK signal activation is essential to ABC tumor progression. RANKL-targeted therapy may be an effective alternative to surgery in select ABC presentations. Copyright © 2014 Mosby, Inc. All rights reserved.

  3. Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

    PubMed

    Pfeifer, M; Zheng, B; Erdmann, T; Koeppen, H; McCord, R; Grau, M; Staiger, A; Chai, A; Sandmann, T; Madle, H; Dörken, B; Chu, Y-W; Chen, A I; Lebovic, D; Salles, G A; Czuczman, M S; Palanca-Wessels, M C; Press, O W; Advani, R; Morschhauser, F; Cheson, B D; Lenz, P; Ott, G; Polson, A G; Mundt, K E; Lenz, G

    2015-07-01

    Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

  4. Responses to the Selective Bruton's Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines.

    PubMed

    Kozaki, Ryohei; Vogler, Meike; Walter, Harriet S; Jayne, Sandrine; Dinsdale, David; Siebert, Reiner; Dyer, Martin J S; Yoshizawa, Toshio

    2018-04-23

    Bruton's tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL.

  5. ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Mingli; Yin, Huancai; Bai, Pengli

    This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity ofmore » QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.« less

  6. Advanced patient age at diagnosis of diffuse large B-cell lymphoma is associated with molecular characteristics including ABC-subtype and high expression of MYC.

    PubMed

    Paul, Ulrike; Richter, Julia; Stuhlmann-Laiesz, Christiane; Kreuz, Markus; Nagel, Inga; Horn, Heike; Staiger, Annette M; Aukema, Sietse M; Hummel, Michael; Ott, German; Spang, Rainer; Rosenwald, Andreas; Feller, Alfred C; Cogliatti, Sergio; Stein, Harald; Hansmann, Martin-Leo; Moller, Peter; Szczepanowski, Monika; Burkhardt, Birgit; Pfreundschuh, Michael; Schmitz, Norbert; Loeffler, Markus; Trümper, Lorenz; Siebert, Reiner; Klapper, Wolfram

    2018-05-01

    The incidence of diffuse large B-cell lymphoma (DLBCL) increases with age being patient age at diagnosis an adverse prognostic factor. However, elderly patients are often underrepresented in common studies. To investigate the effect between age and biological characteristics in DLBCL, we analyzed data of 1534 patients encompassing all adult age groups, enriched for the age ≥75 years. Follicular lymphoma (FL) grade 3B with histopathological characteristics of DLBCLs were included. Gender, centroblastic cytology, FL grade 3B morphology, CD10 expression, and ABC/non-GCB-subtype were significantly associated with age after correction for multiple testing and after adjusting for cohorts. Analysis of a subgroup points towards an association of MYC expression with age. Our data indicate that biological features of DLBCL and FL grade 3B are associated with increasing age among adult patients. The prevalence of the ABC/non-GCB-subtype in elderly patients suggests that therapies targeting this molecular subtype should be specifically explored in this subgroup.

  7. Responses to the Selective Bruton’s Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines

    PubMed Central

    Vogler, Meike; Jayne, Sandrine; Dinsdale, David; Siebert, Reiner

    2018-01-01

    Bruton’s tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL. PMID:29690649

  8. Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

    PubMed

    Johnsen, Hans Erik; Bergkvist, Kim Steve; Schmitz, Alexander; Kjeldsen, Malene Krag; Hansen, Steen Møller; Gaihede, Michael; Nørgaard, Martin Agge; Bæch, John; Grønholdt, Marie-Louise; Jensen, Frank Svendsen; Johansen, Preben; Bødker, Julie Støve; Bøgsted, Martin; Dybkær, Karen

    2014-06-01

    Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy.

  9. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fuchs, Dominik; Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg; Daniel, Volker

    2010-04-16

    Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity ofmore » P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.« less

  10. Induction of the c-myc protooncogene following antigen binding to hapten-specific B cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Snow, E.C.; Fetherston, J.; Zimmer, S.

    1986-03-01

    Considerable controversy has centered on the role that the surface immunoglobulin (sIg) receptor for antigen plays during the induction of B cell activation. Stimulation by anti-Ig reagents has been shown to activate G/sub 0/ B cells to enter the cell cycle. The binding of thymus-dependent antigens to hapten-specific B cell populations apparently does not result in the movement of the antigen-binding cells (ABC) into the G/sub 1/ stage of the cell cycle. However, the authors have recently demonstrated that antigen binding to such hapten-specific B cells does result in the initiation of the membrane phosphatidylinositol cycle. In the present experiments,more » hapten-specific B cells (80-90% ABC, 99% in G/sub 0/) were incubated with either the correct hapten-carrier conjugate, with the carrier protein, or only media for 2 hours at 37/sup 0/C. At that time, total cellular RNA was isolated and subsequently analyzed by either dot blots or Northern gel techniques. The blots were probed with a (/sup 32/P)-c-myc SstI-Xhol fragment. The results indicate that hapten carrier stimulation of the hapten-specific B cells induces enhanced transcription of the c-myc gene. These observations lend further support to the premise that antigen binding to the sIg receptor results in the transduction to the cell of important signals and implicates the active participation of sIg during the process of antigen-mediated B cell activation.« less

  11. Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

    PubMed

    Xu, Dan-Dan; Zhou, Peng-Jun; Wang, Ying; Zhang, Li; Fu, Wu-Yu; Ruan, Bi-Bo; Xu, Hai-Peng; Hu, Chao-Zhi; Tian, Lu; Qin, Jin-Hong; Wang, Sheng; Wang, Xiao; Li, Yi-Cheng; Liu, Qiu-Ying; Ren, Zhe; Zhang, Rong; Wang, Yi-Fei

    2016-05-17

    Recent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear. Serum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b. Sphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b. The mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other's expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.

  12. Antitumor effect of the novel sphingosine kinase 2 inhibitor ABC294640 is enhanced by inhibition of autophagy and by sorafenib in human cholangiocarcinoma cells.

    PubMed

    Ding, Xiwei; Chaiteerakij, Roongruedee; Moser, Catherine D; Shaleh, Hassan; Boakye, Jeffrey; Chen, Gang; Ndzengue, Albert; Li, Ying; Zhou, Yanling; Huang, Shengbing; Sinicrope, Frank A; Zou, Xiaoping; Thomas, Melanie B; Smith, Charles D; Roberts, Lewis R

    2016-04-12

    Sphingosine kinase 2 (Sphk2) has an oncogenic role in cancer. A recently developed first-in-class Sphk2 specific inhibitor ABC294640 displays antitumor activity in many cancer models. However, the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in cholangiocarcinoma. We investigated the potential of targeting Sphk2 for the treatment of cholangiocarcinoma. We found that Sphk2 is overexpressed in five established human cholangiocarcinoma cell lines (WITT, HuCCT1, EGI-1, OZ and HuH28) and a new patient-derived cholangiocarcinoma cell line (LIV27) compared to H69 normal cholangiocytes. Inhibition of Sphk2 by ABC294640 inhibited proliferation and induced caspase-dependent apoptosis. Furthermore, we found that ABC294640 inhibited STAT3 phosphorylation, one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival. ABC294640 also induced autophagy. Inhibition of autophagy by bafilomycin A1 or chloroquine potentiated ABC294640-induced cytotoxicity and apoptosis. In addition, ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of cholangiocarcinoma cells. Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCT1 cells exposed to the ABC294640 and sorafenib combination. These findings provide novel evidence that Sphk2 may be a rational therapeutic target in cholangiocarcinoma. Combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.

  13. SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity

    PubMed Central

    Care, Matthew A.; Cocco, Mario; Laye, Jon P.; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R.; Doody, Gina M.; Tooze, Reuben M.

    2014-01-01

    Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

  14. Application of activity-based costing (ABC) for a Peruvian NGO healthcare provider.

    PubMed

    Waters, H; Abdallah, H; Santillán, D

    2001-01-01

    This article describes the application of activity-based costing (ABC) to calculate the unit costs of the services for a health care provider in Peru. While traditional costing allocates overhead and indirect costs in proportion to production volume or to direct costs, ABC assigns costs through activities within an organization. ABC uses personnel interviews to determine principal activities and the distribution of individual's time among these activities. Indirect costs are linked to services through time allocation and other tracing methods, and the result is a more accurate estimate of unit costs. The study concludes that applying ABC in a developing country setting is feasible, yielding results that are directly applicable to pricing and management. ABC determines costs for individual clinics, departments and services according to the activities that originate these costs, showing where an organization spends its money. With this information, it is possible to identify services that are generating extra revenue and those operating at a loss, and to calculate cross subsidies across services. ABC also highlights areas in the health care process where efficiency improvements are possible. Conclusions about the ultimate impact of the methodology are not drawn here, since the study was not repeated and changes in utilization patterns and the addition of new clinics affected applicability of the results. A potential constraint to implementing ABC is the availability and organization of cost information. Applying ABC efficiently requires information to be readily available, by cost category and department, since the greatest benefits of ABC come from frequent, systematic application of the methodology in order to monitor efficiency and provide feedback for management. The article concludes with a discussion of the potential applications of ABC in the health sector in developing countries.

  15. Variables affecting the quantitation of CD22 in neoplastic B cells.

    PubMed

    Jasper, Gregory A; Arun, Indu; Venzon, David; Kreitman, Robert J; Wayne, Alan S; Yuan, Constance M; Marti, Gerald E; Stetler-Stevenson, Maryalice

    2011-03-01

    Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory for diagnosis, prognosis, and assessment of patients receiving antibody-based therapy. ABC values and the effect of technical variables on CD22 quantitation in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FCL), hairy cell leukemia (HCL) and normal B cells were studied. The QuantiBrite System® was used to determine the level of CD22 expression (mean antibody bound per cell, ABC) by malignant and normal B cells. The intra-assay variability, number of cells required for precision, effect of delayed processing as well as shipment of peripheral blood specimens (delayed processing and exposure to noncontrolled environments), and the effect of paraformaldehyde fixation on assay results were studied. The QuantiBRITE method of measuring CD22 ABC is precise (median CV 1.6%, 95% confidence interval, 1.2-2.3%) but a threshold of 250 malignant cells is required for reliable CD22 ABC values. Delayed processing and overnight shipment of specimens resulted in significantly different ABC values whereas fixation for up to 12 h had no significant effect. ABC measurements determined that CD22 expression is lower than normal in ALL, CLL, FCL, and MCL but higher than normal in HCL. CD22 expression was atypical in the hematolymphoid malignancies studied and may have diagnostic utility. Technical variables such as cell number analyzed and delayed processing or overnight shipment of specimens impact significantly on the measurement of antigen expression by QFCM in the clinical laboratory. Published 2010 Wiley-Liss, Inc.

  16. Harnessing Drug Resistance: Using ABC Transporter Proteins To Target Cancer Cells

    PubMed Central

    Leitner, Heather M.; Kachadourian, Remy; Day, Brian J.

    2007-01-01

    The ATP-binding cassette (ABC) class of proteins is one of the most functionally diverse transporter families found in biological systems. Although the abundance of ABC proteins varies between species, they are highly conserved in sequence and often demonstrate similar functions across prokaryotic and eukaryotic organisms. Beginning with a brief summary of the events leading to our present day knowledge of ABC transporters, the purpose of this review is to discuss the potential for utilizing ABC transporters as a means for cellular glutathione (GSH) modulation. GSH is one of the most abundant thiol antioxidants in cells. It is involved in cellular division, protein and DNA synthesis, maintenance of cellular redox status and xenobiotic metabolism. Cellular GSH levels are often altered in many disease states including cancer. Over the past two decades there has been considerable emphasis on methods to sensitize cancer cells to chemotherapeutics and ionization radiation therapy by GSH depletion. We contend that ABC transporters, particularly multi-drug resistant proteins (MRPs), may be used as therapeutic targets for applications aimed at modulation of GSH levels. This review will emphasize MRP-mediated modulation of intracellular GSH levels as a potential alternative and adjunctive approach for cancer therapy. PMID:17585883

  17. Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas.

    PubMed

    O'Steen, Shyril; Green, Damian J; Gopal, Ajay K; Orozco, Johnnie J; Kenoyer, Aimee L; Lin, Yukang; Wilbur, D Scott; Hamlin, Donald K; Fisher, Darrell R; Hylarides, Mark D; Gooley, Theodore A; Waltman, Amelia; Till, Brian G; Press, Oliver W

    2017-07-15

    Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

    PubMed

    Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

    2016-10-18

    The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

  19. Investigation of the quaternary structure of an ABC transporter in living cells using spectrally resolved resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Singh, Deo Raj

    Forster resonance energy transfer (FRET) has become an important tool to study proteins inside living cells. It has been used to explore membrane protein folding and dynamics, determine stoichiometry and geometry of protein complexes, and measure the distance between two molecules. In this dissertation, we use a method based on FRET and optical micro-spectroscopy (OptiMiS) technology, developed in our lab, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of an ABC transporter in living cells. Specifically, the transporter we investigate originates from the pathogen Pseudomonas aeruginosa, which is a Gram-negative bacterium with several virulence factors, lipopolysaccharides being one of them. This pathogen coexpresses two unique forms of lipopolysaccharides on its surface, the A- and B-bands. The A-band polysaccharides, synthesized in the cytoplasm, are translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. In P. aeruginosa, all of the biochemical studies of A-band LPS are concentrated on the stages of the synthesis and ligation of polysaccharides (PSs), leaving the export stage involving ABC transporter unexplored. The mode of PS export through ABC transporters is still unknown. This difficulty is due to the lack of information about sub-unit composition and structure of this bi-component ABC transporter. Using the FRET-OptiMiS combination method developed by our lab, we found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the

  20. Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

    PubMed

    Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

    2017-11-06

    Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

  1. Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

    PubMed

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

  2. Independent Activity of the Homologous Small Regulatory RNAs AbcR1 and AbcR2 in the Legume Symbiont Sinorhizobium meliloti

    PubMed Central

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I.

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia. PMID:23869210

  3. Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

    PubMed

    Pletzer, Daniel; Weingart, Helge

    2014-07-11

    The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in

  4. Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora

    PubMed Central

    2014-01-01

    Background The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Results To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. Conclusions The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the

  5. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

    Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

  6. The ABCs of Activity-Based Costing: A Cost Containment and Reallocation Tool.

    ERIC Educational Resources Information Center

    Turk, Frederick J.

    1992-01-01

    This article describes activity-based costing (ABC) and how this tool may help management understand the costs of major activities and identify possible alternatives. Also discussed are the traditional costing systems used by higher education and ways of applying ABC to higher education. (GLR)

  7. An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a

    PubMed Central

    2011-01-01

    Background Efflux pumps belonging to the resistance-nodulation-division (RND) superfamily in bacteria are involved in antibiotic resistance and solvent tolerance but have an unknown physiological role. EmhABC, a RND-type efflux pump in Pseudomonas fluorescens strain cLP6a, extrudes hydrophobic antibiotics, dyes and polycyclic aromatic hydrocarbons including phenanthrene. The effects of physico-chemical factors such as temperature or antibiotics on the activity and expression of EmhABC were determined in order to deduce its physiological role(s) in strain cLP6a in comparison to the emhB disruptant strain, cLP6a-1. Results Efflux assays conducted with 14C-phenanthrene showed that EmhABC activity is affected by incubation temperature. Increased phenanthrene efflux was measured in cLP6a cells grown at 10°C and decreased efflux was observed at 35°C compared with cells grown at the optimum temperature of 28°C. Membrane fatty acids in cLP6a cells were substantially altered by changes in growth temperature and in the presence of tetracycline. Changed membrane fatty acids and increased membrane permeability were associated with ~30-fold increased expression of emhABC in cLP6a cells grown at 35°C, and with increased extracellular free fatty acids. Growth of P. fluorescens cLP6a at supra-optimal temperature was enhanced by the presence of EmhABC compared to strain cLP6a-1. Conclusions Combined, these observations suggest that the EmhABC efflux pump may be involved in the management of membrane stress effects such as those due to unfavourable incubation temperatures. Efflux of fatty acids replaced as a result of membrane damage or phospholipid turnover may be the primary physiological role of the EmhABC efflux pump in P. fluorescens cLP6a. PMID:22085438

  8. Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG.

    PubMed

    Luo, Qingshan; Yang, Xu; Yu, Shan; Shi, Huigang; Wang, Kun; Xiao, Le; Zhu, Guangyu; Sun, Chuanqi; Li, Tingting; Li, Dianfan; Zhang, Xinzheng; Zhou, Min; Huang, Yihua

    2017-05-01

    After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB 2 FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB 2 FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB 2 FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

  9. FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures

    PubMed Central

    Wong, Kah Keng; Gascoyne, Duncan M.; Soilleux, Elizabeth J.; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M.; Møller, Michael B.; Green, Tina M.; Banham, Alison H.

    2016-01-01

    FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL. PMID:27224915

  10. FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

    PubMed

    Wong, Kah Keng; Gascoyne, Duncan M; Soilleux, Elizabeth J; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M; Møller, Michael B; Green, Tina M; Banham, Alison H

    2016-08-16

    FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

  11. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

    2014-11-07

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

  12. Applying Activity Based Costing (ABC) Method to Calculate Cost Price in Hospital and Remedy Services

    PubMed Central

    Rajabi, A; Dabiri, A

    2012-01-01

    Background Activity Based Costing (ABC) is one of the new methods began appearing as a costing methodology in the 1990’s. It calculates cost price by determining the usage of resources. In this study, ABC method was used for calculating cost price of remedial services in hospitals. Methods: To apply ABC method, Shahid Faghihi Hospital was selected. First, hospital units were divided into three main departments: administrative, diagnostic, and hospitalized. Second, activity centers were defined by the activity analysis method. Third, costs of administrative activity centers were allocated into diagnostic and operational departments based on the cost driver. Finally, with regard to the usage of cost objectives from services of activity centers, the cost price of medical services was calculated. Results: The cost price from ABC method significantly differs from tariff method. In addition, high amount of indirect costs in the hospital indicates that capacities of resources are not used properly. Conclusion: Cost price of remedial services with tariff method is not properly calculated when compared with ABC method. ABC calculates cost price by applying suitable mechanisms but tariff method is based on the fixed price. In addition, ABC represents useful information about the amount and combination of cost price services. PMID:23113171

  13. Oncogenically active MYD88 mutations in human lymphoma.

    PubMed

    Ngo, Vu N; Young, Ryan M; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D; Connors, Joseph M; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Fisher, Richard I; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Denny D; Chan, Wing C; Staudt, Louis M

    2011-02-03

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

  14. Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump

    PubMed Central

    Llabrés, Salomé; Neuberger, Arthur; Blaza, James N.; Bai, Xiao-chen; Okada, Ui; Murakami, Satoshi; van Veen, Hendrik W.; Zachariae, Ulrich; Scheres, Sjors H.W.; Luisi, Ben F.

    2017-01-01

    The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain (TMD) with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism. PMID:28504659

  15. Alternative Basic Comprehensive Program (Project A.B.C.) Special Alternative Instructional Program. Final Evaluation Report 1992-93. OREA Report.

    ERIC Educational Resources Information Center

    Augustin, Marc A.

    The Alternative Basic Comprehension Program (Project A.B.C.) for bilingual high school students was a special alternative instructional program funded by Title VII for the third year at two high schools in the Bronx. In the year under review, Project A.B.C. served 260 students of limited English proficiency (LEP). Participating students received…

  16. Placental ABC Transporters: Biological Impact and Pharmaceutical Significance.

    PubMed

    Joshi, Anand A; Vaidya, Soniya S; St-Pierre, Marie V; Mikheev, Andrei M; Desino, Kelly E; Nyandege, Abner N; Audus, Kenneth L; Unadkat, Jashvant D; Gerk, Phillip M

    2016-12-01

    The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

  17. Placental ABC Transporters: Biological Impact and Pharmaceutical Significance

    PubMed Central

    Joshi, Anand A.; Vaidya, Soniya S.; St-Pierre, Marie V.; Mikheev, Andrei M.; Desino, Kelly E.; Nyandege, Abner N.; Audus, Kenneth L.; Unadkat, Jashvant D.; Gerk, Phillip M.

    2017-01-01

    The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy. PMID:27644937

  18. B-cell receptor signaling as a driver of lymphoma development and evolution.

    PubMed

    Niemann, Carsten U; Wiestner, Adrian

    2013-12-01

    The B-cell receptor (BCR) is essential for normal B-cell development and maturation. In an increasing number of B-cell malignancies, BCR signaling is implicated as a pivotal pathway in tumorigenesis. Mechanisms of BCR activation are quite diverse and range from chronic antigenic drive by microbial or viral antigens to autostimulation of B-cells by self-antigens to activating mutations in intracellular components of the BCR pathway. Hepatitis C virus infection can lead to the development of splenic marginal zone lymphoma, while Helicobacter pylori infection is associated with the development of mucosa-associated lymphoid tissue lymphomas. In some of these cases, successful treatment of the infection removes the inciting antigen and results in resolution of the lymphoma. Chronic lymphocytic leukemia has been recognized for decades as a malignancy of auto-reactive B-cells and its clinical course is in part determined by the differential response of the malignant cells to BCR activation. In a number of B-cell malignancies, activating mutations in signal transduction components of the BCR pathway have been identified; prominent examples are activated B-cell-like (ABC) diffuse large B-cell lymphomas (DLBCL) that carry mutations in CD79B and CARD11 and display chronic active BCR signaling resulting in constitutive activation of the NF-κB pathway. Despite considerable heterogeneity in biology and clinical course, many mature B-cell malignancies are highly sensitive to kinase inhibitors that disrupt BCR signaling. Thus, targeted therapy through inhibition of BCR signaling is emerging as a new treatment paradigm for many B-cell malignancies. Here, we review the role of the BCR in the pathogenesis of B-cell malignancies and summarize clinical results of the emerging class of kinase inhibitors that target this pathway. Copyright © 2013. Published by Elsevier Ltd.

  19. Activation of human B cells by phosphorothioate oligodeoxynucleotides.

    PubMed Central

    Liang, H; Nishioka, Y; Reich, C F; Pisetsky, D S; Lipsky, P E

    1996-01-01

    To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors. PMID:8787674

  20. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    PubMed

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  1. Adenosine production by human B cells and B cell–mediated suppression of activated T cells

    PubMed Central

    Saze, Zenichiro; Schuler, Patrick J.; Hong, Chang-Sook; Cheng, Dongmei; Jackson, Edwin K.

    2013-01-01

    Antibody-independent role of B cells in modulating T-cell responses is incompletely understood. Freshly isolated or cultured B cells isolated from the peripheral blood of 30 normal donors were evaluated for CD39 and CD73 coexpression, the ability to produce adenosine 5′-monophosphate (AMP) and adenosine (ADO) in the presence of exogenous adenosine triphosphate (ATP) as well as A1, A2A, A2B, and A3 adenosine receptor (ADOR) expression. Human circulating B cells coexpress ectonucleotidases CD39 and CD73, hydrolyze exogenous ATP to 5′-AMP and ADO, and express messenger RNA for A1R, A2AR, and A3R. 2-chloroadenosine inhibited B-cell proliferation and cytokine expression, and only A3R selective antagonist restored B-cell functions. This suggested that B cells use the A3R for autocrine signaling and self-regulation. Mediated effects on B-cell growth ± ADOR antagonists or agonists were tested in carboxyfluorescein diacetate succinimidyl ester assays. In cocultures, resting B cells upregulated functions of CD4+ and CD8+ T cells. However, in vitro–activated B cells downregulated CD73 expression, mainly produced 5′-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell–B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 5′-AMP, and ADO. PMID:23678003

  2. Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli.

    PubMed

    Li, Ye; Chen, Zhenya; Zhou, Zhao; Yuan, Qipeng

    2016-12-01

    Chondroitinases (ChSases) are a family of polysaccharide lyases that can depolymerize high molecular weight chondroitin sulfate (CS) and dermatan sulfate (DS). In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH of GAPDH-ChSase ABC I were 40 °C and 7.5, respectively. GAPDH-ChSase ABC I had a kcat/Km of 131 ± 4.1 L/μmol s and the catalytic efficiency was decreased as compared to ChSase ABC I. The relative activity of GAPDH-ChSase ABC I remained 89% after being incubated at 30 °C for 180 min and the thermostability of ChSase ABC I was enhanced by GAPDH when it was incubated at 30, 35, 40 and 45 °C. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma.

    PubMed

    Kuo, Hsu-Ping; Ezell, Scott A; Schweighofer, Karl J; Cheung, Leo W K; Hsieh, Sidney; Apatira, Mutiah; Sirisawad, Mint; Eckert, Karl; Hsu, Ssucheng J; Chen, Chun-Te; Beaupre, Darrin M; Versele, Matthias; Chang, Betty Y

    2017-07-01

    Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)-mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246-56. ©2017 AACR . ©2017 American Association for Cancer Research.

  4. [The ABC transporters of Saccharomyces cerevisiae].

    PubMed

    Wawrzycka, Donata

    2011-01-01

    The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

  5. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    PubMed

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  6. The Function of UreB in Klebsiella aerogenes Urease†

    PubMed Central

    Carter, Eric L.; Boer, Jodi L.; Farrugia, Mark A.; Flugga, Nicholas; Towns, Christopher L.; Hausinger, Robert P.

    2011-01-01

    Urease from Klebsiella aerogenes is composed of three subunits (UreA, UreB, and UreC) which assemble into a (UreABC)3 quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)3 or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA plus UreC to form (UreABC*)3 apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to authentic urease apoprotein, active enzyme is produced by incubation of (UreABC*)3 with Ni2+ and bicarbonate. Conversely, UreBΔ1-19, lacking the 19 residue potential hinge and tether to UreC, does not form a complex with (UreAC)3 and yields negligible levels of active enzyme when incubated under activation conditions with (UreAC)3. Comparison of activities and nickel contents for (UreAC)3, (UreABC*)3, and (UreABC)3 samples treated with Ni2+ and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)3, (UreAC)3, and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1-19. The soluble MBP-UreD:UreF:UreG complex binds in vitro to (UreABC)3, but not to (UreAC)3 or UreB. Together these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in

  7. TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

    PubMed Central

    Wang, Caixiang; Jing, Ruilian; Mao, Xinguo; Chang, Xiaoping; Li, Ang

    2011-01-01

    Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc1 complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc1 complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways. PMID:21115661

  8. Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana).

    PubMed

    Kanno, Akira; Saeki, Hiroshi; Kameya, Toshiaki; Saedler, Heinz; Theissen, Günter

    2003-07-01

    In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.

  9. Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus

    PubMed Central

    Fan, H; Liu, F; Dong, G; Ren, D; Xu, Y; Dou, J; Wang, T; Sun, L; Hou, Y

    2014-01-01

    B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19+CD27- and CD19+IgM+ B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19+ B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients. PMID:25210799

  10. Searching for the fastest dynamo: laminar ABC flows.

    PubMed

    Alexakis, Alexandros

    2011-08-01

    The growth rate of the dynamo instability as a function of the magnetic Reynolds number R(M) is investigated by means of numerical simulations for the family of the Arnold-Beltrami-Childress (ABC) flows and for two different forcing scales. For the ABC flows that are driven at the largest available length scale, it is found that, as the magnetic Reynolds number is increased: (a) The flow that results first in a dynamo is the 2 1/2-dimensional flow for which A=B and C=0 (and all permutations). (b) The second type of flow that results in a dynamo is the one for which A=B≃2C/5 (and permutations). (c) The most symmetric flow, A=B=C, is the third type of flow that results in a dynamo. (d) As R(M) is increased, the A=B=C flow stops being a dynamo and transitions from a local maximum to a third-order saddle point. (e) At larger R(M), the A=B=C flow reestablishes itself as a dynamo but remains a saddle point. (f) At the largest examined R(M), the growth rate of the 2 1/2-dimensional flows starts to decay, the A=B=C flow comes close to a local maximum again, and the flow A=B≃2C/5 (and permutations) results in the fastest dynamo with growth rate γ≃0.12 at the largest examined R(M). For the ABC flows that are driven at the second largest available length scale, it is found that (a) the 2 1/2-dimensional flows A=B,C=0 (and permutations) are again the first flows that result in a dynamo with a decreased onset. (b) The most symmetric flow, A=B=C, is the second type of flow that results in a dynamo. It is, and it remains, a local maximum. (c) At larger R(M), the flow A=B≃2C/5 (and permutations) appears as the third type of flow that results in a dynamo. As R(M) is increased, it becomes the flow with the largest growth rate. The growth rates appear to have some correlation with the Lyapunov exponents, but constructive refolding of the field lines appears equally important in determining the fastest dynamo flow.

  11. A-B-C-1-2-3 Healthy Kids in Tennessee - Let's Eat Well, Play, and Be Aware Every Day: a preliminary report.

    PubMed

    Chafin, Cynthia; Edwards, M Jo; Morgan, Debbie; Isom, Pam; Morgan, Don

    2012-01-01

    The "A-B-C-1-2-3 Healthy Kids in Tennessee - Let's Eat Well, Play, and Be Aware Every Day" project is a hands-on educational program emphasizing healthy living that targets childcare providers, the children they care for, and their families. The program was initially implemented as a pilot project in 6 middle Tennessee childcare centers. Materials were organized and developed by the Middle Tennessee Cancer Coalition's childhood action team in conjunction with staff from Middle Tennessee State University (MTSU) Center for Health and Human Services and the MTSU Center for Physical Activity and Health in Youth. The A-B-C-1-2-3 initiative served as a feasibility project to inform the conduct of field operations. Through the MTSU Center for Physical Activity and Health in Youth, an expanded 12-week pilot program took place during 2010 in 2 childcare centers. The purpose of the program is to educate childcare providers who, in turn, educate children and their parents and promote healthy lifestyles and decrease the risk of developing cancer, obesity, and other lifestyle-associated diseases and health conditions. The overall goal of the project is to decrease lifestyle and environmental cancer risk factors among Tennesseans by 2012 as detailed in the 2009-2012 Tennessee Comprehensive Cancer Control Plan and to provide educational opportunities in healthy eating and healthy weight to childcare providers detailed in the 2010-2015 Tennessee Statewide Nutrition and Physical Activity Plan using a "train the trainer approach" along with classroom and family education. In 2012, the project will partner with a statewide Tennessee Department of Health initiative, Gold Sneakers, which provides a policy piece to the A-B-C-1-2-3 Healthy Kids in Tennessee's approach to disseminate nutritional and physical activity education to childcare providers, children, and their families, offering a full-circle approach to health promotion in a childcare setting.

  12. Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

    PubMed Central

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-01-01

    SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

  13. Purification and characterization of chondroitinase ABC from Acinetobacter sp. C26.

    PubMed

    Zhu, Changliang; Zhang, Jingliang; Zhang, Jing; Jiang, Yanhui; Shen, Zhaopeng; Guan, Huashi; Jiang, Xiaolu

    2017-02-01

    An extracellular chondroitinase ABC (ChSase ABC, EC 4.2.2.4) produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0.105mg/ml, respectively. The enzyme showed the highest activity at the optimal conditions of pH 6.0 and 42 ∘C, respectively. This enzyme was stable at pH 5-10, 5-9 and 5-7 at 4°C, 37°C and 42°C, respectively. Investigation about thermal stability of ChSase ABC displayed that it was stable at 37°C. ChSase ABC activity was increased in presence of Na + , K + , Mn 2+ , 1,10-phenanthrolin and strongly inhibited by Cu 2+ , Hg 2+ , Al 3+ and SDS. These properties suggested that ChSase ABC from Acinetobacter sp. C26 bring promising prospects in medical and industry applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Effect of β-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

    PubMed

    Tang, Chao-Yuan; Zhu, Li-Xin; Yu, Jian-Dong; Chen, Zhi; Gu, Man-Cang; Mu, Chao-Feng; Liu, Qi; Xiong, Yang

    2018-07-30

    In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by β-elemene (β-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by β-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOX Fluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOX Fluc cells being treated with β-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOX Fluc was lessened when pretreated with β-ELE, which means that β-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of β-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of β-ELE. To verify the efficacy of β-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and β-ELE. MTT assay showed that β-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC 50 of the combination group was much lower than that of the single DOX or β-ELE treatment. In all, β-ELE may reverse MDR through the substrates of ABC transporters

  15. Poor concordance among nine immunohistochemistry classifiers of cell-of-origin for diffuse large B-cell lymphoma: implications for therapeutic strategies.

    PubMed

    Coutinho, Rita; Clear, Andrew James; Owen, Andrew; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; Gomes da Silva, Maria; Cabeçadas, José; Calaminici, Maria; Gribben, John G

    2013-12-15

    The opportunity to improve therapeutic choices on the basis of molecular features of the tumor cells is on the horizon in diffuse large B-cell lymphoma (DLBCL). Agents such as bortezomib exhibit selective activity against the poor outcome activated B-cell type (ABC) DLBCL. In order for targeted therapies to succeed in this disease, robust strategies that segregate patients into molecular groups with high reliability are needed. Although molecular studies are considered gold standard, several immunohistochemistry (IHC) algorithms have been published that claim to be able to stratify patients according to their cell-of-origin and to be relevant for patient outcome. However, results are poorly reproducible by independent groups. We investigated nine IHC algorithms for molecular classification in a dataset of DLBCL diagnostic biopsies, incorporating immunostaining for CD10, BCL6, BCL2, MUM1, FOXP1, GCET1, and LMO2. IHC profiles were assessed and agreed among three expert observers. A consensus matrix based on all scoring combinations and the number of subjects for each combination allowed us to assess reliability. The survival impact of individual markers and classifiers was evaluated using Kaplan-Meier curves and the log-rank test. The concordance in patient's classification across the different algorithms was low. Only 4% of the tumors have been classified as germinal center B-cell type (GCB) and 21% as ABC/non-GCB by all methods. None of the algorithms provided prognostic information in the R-CHOP (rituximab plus cyclophosphamide-adriamycin-vincristine-prednisone)-treated cohort. Further work is required to standardize IHC algorithms for DLBCL cell-of-origin classification for these to be considered reliable alternatives to molecular-based methods to be used for clinical decisions. ©2013 AACR.

  16. Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice.

    PubMed

    Kil, Laurens P; de Bruijn, Marjolein J W; van Nimwegen, Menno; Corneth, Odilia B J; van Hamburg, Jan Piet; Dingjan, Gemma M; Thaiss, Friedrich; Rimmelzwaan, Guus F; Elewaut, Dirk; Delsing, Dianne; van Loo, Pieter Fokko; Hendriks, Rudi W

    2012-04-19

    On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca(2+) influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.

  17. Comparison of mechanistic transport cycle models of ABC exporters.

    PubMed

    Szöllősi, Dániel; Rose-Sperling, Dania; Hellmich, Ute A; Stockner, Thomas

    2018-04-01

    ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane domains bind and translocate substrates and are connected to a pair of nucleotide binding domains, which bind and hydrolyze ATP to energize import or export of substrates. Over four decades of investigations into ABC transporters have revealed numerous details from atomic-level structural insights to their functional and physiological roles. Despite all these advances, a comprehensive understanding of the mechanistic principles of ABC transporter function remains elusive. The human multidrug resistance transporter ABCB1, also referred to as P-glycoprotein (P-gp), is one of the most intensively studied ABC exporters. Using ABCB1 as the reference point, we aim to compare the dominating mechanistic models of substrate transport and ATP hydrolysis for ABC exporters and to highlight the experimental and computational evidence in their support. In particular, we point out in silico studies that enhance and complement available biochemical data. "This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain." Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

    PubMed

    Chen, Lin; Duan, Kangmin

    2016-05-01

    Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. The Role of Activity Based Costing (ABC) in Educational Support Services: A White Paper.

    ERIC Educational Resources Information Center

    Edds, Daniel B.

    Many front-line managers who are assuming more financial responsibility for their organizations find traditional cost accounting inadequate for their needs and are turning to Activity Based Costing (ABC). ABC is not a financial reporting system to serve the needs of regulatory agencies, but a tool that tracks costs from the general ledger…

  20. Activity Begins in Childhood (ABC) - inspiring healthy active behaviour in preschoolers: study protocol for a cluster randomized controlled trial.

    PubMed

    Adamo, Kristi B; Barrowman, Nick; Naylor, Patti Jean; Yaya, Sanni; Harvey, Alysha; Grattan, Kimberly P; Goldfield, Gary S

    2014-07-29

    Today's children are more overweight than previous generations and physical inactivity is a contributing factor. Modelling and promoting positive behaviour in the early years is imperative for the development of lifelong health habits. The social and physical environments where children spend their time have a powerful influence on behaviour. Since the majority of preschool children spend time in care outside of the home, this provides an ideal setting to examine the ability of an intervention to enhance movement skills and modify physical activity behaviour. This study aims to evaluate the efficacy of the Activity Begins in Childhood (ABC) intervention delivered in licensed daycare settings alone or in combination with a parent-driven home physical activity-promotion component to increase preschoolers' overall physical activity levels and, specifically, the time spent in moderate to vigorous physical activity. This study is a single site, three-arm, cluster-randomized controlled trial design with a daycare centre as the unit of measurement (clusters). All daycare centres in the National Capital region that serve children between the ages of 3 and 5, expressing an interest in receiving the ABC intervention will be invited to participate. Those who agree will be randomly assigned to one of three groups: i) ABC program delivered at a daycare centre only, ii) ABC program delivered at daycare with a home/parental education component, or iii) regular daycare curriculum. This study will recruit 18 daycare centres, 6 in each of the three groups. The intervention will last approximately 6 months, with baseline assessment prior to ABC implementation and follow-up assessments at 3 and 6 months. Physical activity is an acknowledged component of a healthy lifestyle and childhood experiences as it has an important impact on lifelong behaviour and health. Opportunities for physical activity and motor development in early childhood may, over the lifespan, influence the

  1. Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

    PubMed

    Huang, Zi-Ming; Kang, Jhi-Kai; Chen, Chih-Yu; Tseng, Tz-Hau; Chang, Chien-Wen; Chang, Yung-Chi; Tai, Shyh-Kuan; Hsieh, Shie-Liang; Leu, Chuen-Miin

    2012-06-15

    Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

  2. Tax-Independent Constitutive IκB Kinase Activation in Adult T-Cell Leukemia Cells1

    PubMed Central

    Hironaka, Noriko; Mochida, Kanako; Mori, Naoki; Maeda, Michiyuki; Yamamoto, Naoki; Yamaoka, Shoji

    2004-01-01

    Abstract Adult T-cell leukemia (ATL) is a fatal T-cell malignancy that arises long after infection with human T-cell leukemia virus type I (HTLV-I). We reported previously that nuclear factor-κB (NF-κB) was constitutively activated in ATL cells, although expression of the viral proteins was barely detectable, including Tax, which was known to persistently activate NF-κB. Here we demonstrate that ATL cells that do not express detectable Tax protein exhibit constitutive IκB kinase (IKK) activity. Transfection studies revealed that a dominant-negative form of IKK1, and not of IKK2 or NF-κB essential modulator (NEMO), suppressed constitutive NFκB activity in ATL cells. This IKK activity was accompanied by elevated expression of p52, suggesting that the recently described noncanonical pathway of NF-κB activation operates in ATL cells. We finally show that specific inhibition of NF-κB by a super-repressor form of IκBα (SR-IκBα) in HTLV-I-infected T cells results in cell death regardless of Tax expression, providing definitive evidence of an essential role for NF-κB in the survival of ATL cells. In conclusion, the IKK complex is constitutively activated in ATL cells through a cellular mechanism distinct from that of Tax-mediated IKK activation. Further elucidation of this cellular mechanism should contribute to establishing a rationale for treatment of ATL. PMID:15153339

  3. The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

    PubMed Central

    Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

    2010-01-01

    Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

  4. The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

    PubMed

    Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

    2016-06-28

    Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

    PubMed Central

    Wang, Jing; Tang, Shan; Wan, Zhengpeng; Gao, Yiren; Cao, Yiyun; Yi, Junyang; Si, Yanyan; Zhang, Haowen; Liu, Lei; Liu, Wanli

    2016-01-01

    Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens. PMID:26764382

  6. NFκBP65 transcription factor modulates resistance to doxorubicin through ABC transporters in breast cancer.

    PubMed

    Velaei, Kobra; Samadi, Nasser; Soltani, Sina; Barazvan, Balal; Soleimani Rad, Jafar

    2017-07-01

    Shedding light on chemoresistance biology of breast cancer could contribute to enhance the clinical outcome. Intrinsic or acquired resistance to chemotherapy is a major problem in breast cancer treatment. The NFκB pathway by siRNAP65 and JSH-23 as a translocational inhibitor of NFκBP65 in the doxorubicin-resistant MCF-7 (MCF-7/Dox) and MCF-7 cells was blocked. Then, the ABC transporter expression and function were assessed by real-time qRT-PCR and flow cytometry, respectively. Induction of apoptosis was evaluated after inhibition of the NFΚB pathway as well. Our study underlined the upregulation of NFκBP65 and anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax in the MCF-7/Dox cells compared with control MCF-7 cells. Here, we showed that interplay between nuclear factor kappa B P65 (NFkBP65) as a transcriptional regulator and ABC transporters in the MCF-7/Dox cancer cells. We found that inhibition of the elevated expression of NFκBP65 in the resistant breast cancer, whether translocational inhibition or silencing by siRNA, decreased the expression and function of MDR1 and MRP1 efflux pumps. Furthermore, the blockade of NFκBP65 promoted apoptosis via modulating Bcl-2 and BAX expression. After inhibition of the NFκBP65 signaling pathway, elevated baseline expression of survival Bcl-2 gene in the resistant breast cells significantly decreased. Suppression of the NFκB pathway has a profound dual impact on promoting the intrinsic apoptotic pathway and reducing ABC transporter function and expression, which are some of the chemoresistance features. It was speculated that the NFκB pathway directly acts on doxorubicin-induced MDR1 and MRP1 expression in MCF-7/Dox cells.

  7. Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

    PubMed Central

    Matsumura, Eitaro; Sakai, Masashi; Hayashi, Katsuaki; Murakami, Shuichiro; Takenaka, Shinji; Aoki, Kenji

    2005-01-01

    The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC. PMID:16156722

  8. A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

    NASA Astrophysics Data System (ADS)

    Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

    2006-06-01

    Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

  9. Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

    PubMed Central

    Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

    2012-01-01

    Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

  10. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

    Kärblane, Kairi; Gerassimenko, Jelena; Nigul, Lenne; Piirsoo, Alla; Smialowska, Agata; Vinkel, Kadri; Kylsten, Per; Ekwall, Karl; Swoboda, Peter; Truve, Erkki; Sarmiento, Cecilia

    2015-01-01

    ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference. PMID:25659154

  11. Alternative Basic Comprehension Program (Project A.B.C.). Final Evaluation Report, 1993-94. OER Report.

    ERIC Educational Resources Information Center

    Augustin, Marc A.; Yanping, Ann

    The Alternative Basic Comprehension Program (Project A.B.C.) was an Elementary and Secondary Education Act Title VII-funded special alternative instructional program in its fourth year at two high schools in the Bronx (New York). In 1993-94, the project served 264 students of limited English proficiency from many countries. Participating students…

  12. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury.

    PubMed

    Lee, Hyunjung; McKeon, Robert J; Bellamkonda, Ravi V

    2010-02-23

    Chondroitin sulfate proteoglycans (CSPGs) are a major class of axon growth inhibitors that are up-regulated after spinal cord injury (SCI) and contribute to regenerative failure. Chondroitinase ABC (chABC) digests glycosaminoglycan chains on CSPGs and can thereby overcome CSPG-mediated inhibition. But chABC loses its enzymatic activity rapidly at 37 degrees C, necessitating the use of repeated injections or local infusions for a period of days to weeks. These infusion systems are invasive, infection-prone, and clinically problematic. To overcome this limitation, we have thermostabilized chABC and developed a system for its sustained local delivery in vivo, obviating the need for chronically implanted catheters and pumps. Thermostabilized chABC remained active at 37 degrees C in vitro for up to 4 weeks. CSPG levels remained low in vivo up to 6 weeks post-SCI when thermostabilized chABC was delivered by a hydrogel-microtube scaffold system. Axonal growth and functional recovery following the sustained local release of thermostabilized chABC versus a single treatment of unstabilized chABC demonstrated significant differences in CSPG digestion. Animals treated with thermostabilized chABC in combination with sustained neurotrophin-3 delivery showed significant improvement in locomotor function and enhanced growth of cholera toxin B subunit-positive sensory axons and sprouting of serotonergic fibers. Therefore, improving chABC thermostability facilitates minimally invasive, sustained, local delivery of chABC that is potentially effective in overcoming CSPG-mediated regenerative failure. Combination therapy with thermostabilized chABC with neurotrophic factors enhances axonal regrowth, sprouting, and functional recovery after SCI.

  13. Expression, purification and thermostability of MBP-chondroitinase ABC I from Proteus vulgaris.

    PubMed

    Chen, Zhenya; Li, Ye; Yuan, Qipeng

    2015-01-01

    Chondroitinase ABC I (ChSase ABC I) which can degrade chondroitin sulfate (CS) and other glycosaminoglycan to oligosaccharide or unsaturated disaccharide, was fusionally expressed with maltose-binding protein (MBP) in Escherichia coli BL21(DE3) (E. coli BL21(DE3)) and purified for the first time in this study. The result showed that the productivity of recombinant MBP-ChSase ABC I was 3180 IU/(L fermentation liquor) with CS A as substrate, and the productivity might be the highest level when compared to the reported ones. The specific activity of recombinant MBP-ChSase ABC I was 76 IU/(mg protein) after purification. The Vmax, Km and kcat were 18.7 ± 0.3 μmol/Ls, 73.1 ± 4.1 μmol/L and 586.7 ± 10.8 s(-1), respectively. Enzyme activity of the purified enzyme remained about 78% after 210 min when the enzyme incubated at 30 °C. This study introduces a rapid method for highly expressing ChSase ABC I, and the method could be adopted in the process of industrial production. Furthermore the investigation of thermostability might lead to an important guide in clinical treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Syk Mediates BCR- and CD40-Signaling Intergration during B Cell Activation

    PubMed Central

    Ying, Haiyan; Li, Zhenping; Yang, Lifen; Zhang, Jian

    2010-01-01

    CD40 is essential for optimal B cell activation. It has been shown that CD40 stimulation can augment BCR-induced B cell responses, but the molecular mechanism(s) by which CD40 regulates BCR signaling is poorly understood. In this report, we attempted to characterize the signaling synergy between BCR- and CD40-mediated pathways during B cell activation. We found that spleen tyrosine kinase (Syk) is involved in CD40 signaling, and is synergistically activated in B cells in response to BCR/CD40 costimulation. CD40 stimulation alone also activates B cell linker (BLNK), Bruton tyrosine kinase (Btk), and Vav-2 downstream of Syk, and significantly enhances BCR-induced formation of complex consisting of, Vav-2, Btk, BLNK, and phospholipase C-gamma2 (PLC-γ2) leading to activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase, Akt, and NF-κB required for optimal B cell activation. Therefore, our data suggest that CD40 can strengthen BCR-signaling pathway and quantitatively modify BCR signaling during B cell activation. PMID:21074890

  15. COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells

    PubMed Central

    Litchfield, Lacey M.; Appana, Savitri N.; Datta, Susmita; Klinge, Carolyn M.

    2016-01-01

    Reduced COUP-TFII expression contributes to endocrine resistance in breast cancer cells. Endocrine-resistant breast cancer cells have higher NFkappa B (NFκB) activity and target gene expression. The goal of this study was to determine if COUP-TFII modulates NFκB activity. Endocrine-resistant LCC9 cells with low endogenous COUP-TFII displayed ~5-fold higher basal NFκB activity than parental endocrine-sensitive MCF-7 breast cancer cells. Transient transfection of LCC9 cells with COUP-TFII inhibited NFκB activation and reduced NFκB target gene expression. COUP-TFII and NFκB were inversely correlated in breast cancer patient samples. Endogenous COUP-TFII coimmunoprecipitated with NFκB subunits RelB and NFκB1 in MCF-7 cells. COUP-TFII inhibited NFκB-DNA binding in vitro and impaired coactivator induced NFκB transactivation. LCC9 cells were growth-inhibited by an NFκB inhibitor and 4-hydroxytamoxifen compared to MCF-7 cells. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity and explain, in part, why decreased COUP-TFII expression results in an endocrine-resistant phenotype. PMID:24141032

  16. Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

    PubMed

    Kubota, E; McKenzie, D T; Dutton, R W; Swain, S L

    1991-01-01

    Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.

  17. Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

    PubMed Central

    Lebedeva, Irina V.; Pande, Praveen; Patton, Wayne F.

    2011-01-01

    An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments. PMID:21799851

  18. Reproducibility of lung tumor position and reduction of lung mass within the planning target volume using active breathing control (ABC).

    PubMed

    Cheung, Patrick C F; Sixel, Katharina E; Tirona, Romeo; Ung, Yee C

    2003-12-01

    The active breathing control (ABC) device allows for temporary immobilization of respiratory motion by implementing a breath hold at a predefined relative lung volume and air flow direction. The purpose of this study was to quantitatively evaluate the ability of the ABC device to immobilize peripheral lung tumors at a reproducible position, increase total lung volume, and thereby reduce lung mass within the planning target volume (PTV). Ten patients with peripheral non-small-cell lung cancer tumors undergoing radiotherapy had CT scans of their thorax with and without ABC inspiration breath hold during the first 5 days of treatment. Total lung volumes were determined from the CT data sets. Each peripheral lung tumor was contoured by one physician on all CT scans to generate gross tumor volumes (GTVs). The lung density and mass contained within a 1.5-cm PTV margin around each peripheral tumor was calculated using CT numbers. Using the center of the GTV from the Day 1 ABC scan as the reference, the displacement of subsequent GTV centers on Days 2 to 5 for each patient with ABC applied was calculated in three dimensions. With the use of ABC inspiration breath hold, total lung volumes increased by an average of 42%. This resulted in an average decrease in lung mass of 18% within a standard 1.5-cm PTV margin around the GTV. The average (+/- standard deviation) displacement of GTV centers with ABC breath hold applied was 0.3 mm (+/- 1.8 mm), 1.2 mm (+/- 2.3 mm), and 1.1 mm (+/- 3.5 mm) in the lateral direction, anterior-posterior direction, and superior-inferior direction, respectively. Results from this study indicate that there remains some inter-breath hold variability in peripheral lung tumor position with the use of ABC inspiration breath hold, which prevents significant PTV margin reduction. However, lung volumes can significantly increase, thereby decreasing the mass of lung within a standard PTV.

  19. B Cell Activation by Outer Membrane Vesicles—A Novel Virulence Mechanism

    PubMed Central

    Perez Vidakovics, Maria Laura A.; Jendholm, Johan; Mörgelin, Matthias; Månsson, Anne; Larsson, Christer; Cardell, Lars-Olaf; Riesbeck, Kristian

    2010-01-01

    Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19+ IgD+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys. PMID:20090836

  20. Trastuzumab-Resistant Luminal B Breast Cancer Cells Show Basal-Like Cell Growth Features Through NF-κB-Activation

    PubMed Central

    Kanzaki, Hirotaka; Mukhopadhya, Nishit K.; Cui, Xiaojiang; Ramanujan, V. Krishnan

    2016-01-01

    A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(1) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed “progression through grade” or “evolution of resistance” hypothesis. Furthermore, we identified IKK-α/IKK-β and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation. PMID:26871511

  1. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

    PubMed

    Schmitz, Roland; Wright, George W; Huang, Da Wei; Johnson, Calvin A; Phelan, James D; Wang, James Q; Roulland, Sandrine; Kasbekar, Monica; Young, Ryan M; Shaffer, Arthur L; Hodson, Daniel J; Xiao, Wenming; Yu, Xin; Yang, Yandan; Zhao, Hong; Xu, Weihong; Liu, Xuelu; Zhou, Bin; Du, Wei; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Connors, Joseph M; Campo, Elias; Lopez-Guillermo, Armando; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M; Tay Kuang Wei, Kevin; Zelenetz, Andrew D; Leonard, John P; Bartlett, Nancy L; Tran, Bao; Shetty, Jyoti; Zhao, Yongmei; Soppet, Dan R; Pittaluga, Stefania; Wilson, Wyndham H; Staudt, Louis M

    2018-04-12

    Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88 L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).

  2. ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

    PubMed

    Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

    2018-03-08

    ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM

  3. Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease.

    PubMed

    Chapuy, Bjoern; Cheng, Hongwei; Watahiki, Akira; Ducar, Matthew D; Tan, Yuxiang; Chen, Linfeng; Roemer, Margaretha G M; Ouyang, Jing; Christie, Amanda L; Zhang, Liye; Gusenleitner, Daniel; Abo, Ryan P; Farinha, Pedro; von Bonin, Frederike; Thorner, Aaron R; Sun, Heather H; Gascoyne, Randy D; Pinkus, Geraldine S; van Hummelen, Paul; Wulf, Gerald G; Aster, Jon C; Weinstock, David M; Monti, Stefano; Rodig, Scott J; Wang, Yuzhuo; Shipp, Margaret A

    2016-05-05

    Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition. © 2016 by The American Society of Hematology.

  4. CVID-associated TACI mutations affect autoreactive B cell selection and activation

    PubMed Central

    Romberg, Neil; Chamberlain, Nicolas; Saadoun, David; Gentile, Maurizio; Kinnunen, Tuure; Ng, Yen Shing; Virdee, Manmeet; Menard, Laurence; Cantaert, Tineke; Morbach, Henner; Rachid, Rima; Martinez-Pomar, Natalia; Matamoros, Nuria; Geha, Raif; Grimbacher, Bodo; Cerutti, Andrea; Cunningham-Rundles, Charlotte; Meffre, Eric

    2013-01-01

    Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications. PMID:24051380

  5. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    PubMed Central

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  6. B-cell activating factor detected on both naïve and memory B cells in bullous pemphigoid.

    PubMed

    Qian, Hua; Kusuhara, Masahiro; Li, Xiaoguang; Tsuruta, Daisuke; Tsuchisaka, Atsunari; Ishii, Norito; Koga, Hiroshi; Hayakawa, Taihei; Ohara, Koji; Karashima, Tadashi; Ohyama, Bungo; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-08-01

    B-cell activating factor (BAFF), an important immune regulatory cytokine, is involved in development of autoimmune diseases. Although BAFF is expressed in various cells, including dendritic cells (DCs) and monocytes, BAFF expression on B cells has not been well documented. In the present study, BAFF molecules on DCs and naïve and memory B cells in autoimmune bullous diseases, including pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid (BP), were analysed by flow cytometry. Compared with healthy controls (HC), BAFF expression on naïve and memory B cells increased significantly in BP. No difference in BAFF receptor expression in naïve and memory B cells was shown among all study groups. Furthermore, BAFF expression in both naïve and memory B cells of BP, but not HC, was detected by confocal microscopic analysis. These results implied that BAFF expressed by B cells may play a pathogenic role in autoimmune bullous diseases, particularly BP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

    PubMed Central

    Yeung, A T; Mattes, W B; Grossman, L

    1986-01-01

    An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction. PMID:3960727

  8. Catalytic and transport cycles of ABC exporters.

    PubMed

    Al-Shawi, Marwan K

    2011-09-07

    ABC (ATP-binding cassette) transporters are arguably the most important family of ATP-driven transporters in biology. Despite considerable effort and advances in determining the structures and physiology of these transporters, their fundamental molecular mechanisms remain elusive and highly controversial. How does ATP hydrolysis by ABC transporters drive their transport function? Part of the problem in answering this question appears to be a perceived need to formulate a universal mechanism. Although it has been generally hoped and assumed that the whole superfamily of ABC transporters would exhibit similar conserved mechanisms, this is proving not to be the case. Structural considerations alone suggest that there are three overall types of coupling mechanisms related to ABC exporters, small ABC importers and large ABC importers. Biochemical and biophysical characterization leads us to the conclusion that, even within these three classes, the catalytic and transport mechanisms are not fully conserved, but continue to evolve. ABC transporters also exhibit unusual characteristics not observed in other primary transporters, such as uncoupled basal ATPase activity, that severely complicate mechanistic studies by established methods. In this chapter, I review these issues as related to ABC exporters in particular. A consensus view has emerged that ABC exporters follow alternating-access switch transport mechanisms. However, some biochemical data suggest that alternating catalytic site transport mechanisms are more appropriate for fully symmetrical ABC exporters. Heterodimeric and asymmetrical ABC exporters appear to conform to simple alternating-access-type mechanisms.

  9. Project A.B.C. (Bronx Academic Bilingual Career Program). O.E.E. Evaluation Report, 1981-1982.

    ERIC Educational Resources Information Center

    Collazo-Levy, Dora; And Others

    Project A.B.C. (Academic Bilingual Career Program) is a multisite project serving new immigrant students at three different high schools in the Bronx, New York: Vietnamese (Chinese ethnics) at Theodore Roosevelt, Italians at Christopher Columbus, and Cubans and Dominicans at John F. Kennedy high schools. Project students are incorporated into the…

  10. Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling

    PubMed Central

    Argyropoulos, K V; Vogel, R; Ziegler, C; Altan-Bonnet, G; Velardi, E; Calafiore, M; Dogan, A; Arcila, M; Patel, M; Knapp, K; Mallek, C; Hunter, Z R; Treon, S P; van den Brink, M R M; Palomba, M L

    2016-01-01

    Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a ‘high' and a ‘healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups. PMID:26867669

  11. Biological and Clinical Relevance of Associated Genomic Alterations in MYD88 L265P and non-L265P-Mutated Diffuse Large B-Cell Lymphoma: Analysis of 361 Cases.

    PubMed

    Dubois, Sydney; Viailly, Pierre-Julien; Bohers, Elodie; Bertrand, Philippe; Ruminy, Philippe; Marchand, Vinciane; Maingonnat, Catherine; Mareschal, Sylvain; Picquenot, Jean-Michel; Penther, Dominique; Jais, Jean-Philippe; Tesson, Bruno; Peyrouze, Pauline; Figeac, Martin; Desmots, Fabienne; Fest, Thierry; Haioun, Corinne; Lamy, Thierry; Copie-Bergman, Christiane; Fabiani, Bettina; Delarue, Richard; Peyrade, Frédéric; André, Marc; Ketterer, Nicolas; Leroy, Karen; Salles, Gilles; Molina, Thierry J; Tilly, Hervé; Jardin, Fabrice

    2017-05-01

    Purpose: MYD88 mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL), leading to constitutive NFκB pathway activation. The aim of this study was to examine the distinct genomic profiles of MYD88 -mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants. Experimental Design: A cohort of 361 DLBCL cases (94 MYD88 mutant and 267 MYD88 wild-type) was submitted to next-generation sequencing (NGS) focusing on 34 genes to analyze associated mutations and copy number variations, as well as gene expression profiling, and clinical and prognostic analyses. Results: Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P-mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis also segregated subgroups according to associated genetic alterations among patients with the same MYD88 mutation. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFκB pathway activation, although the majority of MYD88 L265P-mutant cases harbors downstream NFκB alterations, which can predict BTK inhibitor resistance. Finally, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated CD79B mutations significantly improved the survival of MYD88 L265P-mutant ABC DLBCL in our cohort. Conclusions: This study highlights the relative heterogeneity of MYD88 -mutant DLBCL, adding to the field's knowledge of the theranostic importance of MYD88 mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. Clin Cancer Res; 23(9); 2232-44. ©2016 AACR . ©2016 American Association for Cancer Research.

  12. Cellular cooperation in lymphocyte activation. III. B-cell helper effect in the enhancement of T-cell response.

    PubMed

    Kasahara, T; Kin, K; Itoh, Y; Kawai, T; Kano, Y; Shioiri-Nakano, K

    1979-01-01

    T and B cells were purified from human tonsil and peripheral blood by the removal of phagocytic cells, followed by filtration through a nylon fiber column (NC) and E-rosette formation. Purified T and B cells contained less than 1% of other cell types. The responses of T cells to concanavalin A (Con A) and soluble protein A were greatly enhanced in the presence of autologous B cells. Participation of B cells in T-cell enhancement was confirmed by the following observations: (a) purified B copulation, which was separated further from adherent B cells, retained its enhancing activity. (b) Another adherent cell-free B-cell preparation, which was purified from the NC-passed fraction, and (c) no T lymphoid but some B lymphoid cell lines, elicited strong T-cell enhancement. It was also found that the enhancing capacity of B cells required no metabolic activity, but rather an intact cell form and direct cell-to-cell contact with responding cells. The stimulatory determinants on B cells were resistant to trypsin and neuraminidase treatment. In this paper a hypothesis will be presented that at least two signals are prerequisite for the effective activation of T cells.

  13. Group B Strep Infection in Newborns

    MedlinePlus

    ... Active Bacterial Core surveillance (ABCs) CDC Streptococcus Laboratory Sepsis Group B Strep Disease in Newborns Language: English ( ... Active Bacterial Core surveillance (ABCs) CDC Streptococcus Laboratory Sepsis Language: English (US) Español (Spanish) File Formats Help: ...

  14. Cell-of-Origin in Diffuse Large B-Cell Lymphoma: Are the Assays Ready for the Clinic?

    PubMed

    Scott, David W

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide and consists of a heterogeneous group of cancers classified together on the basis of shared morphology, immunophenotype, and aggressive clinical behavior. It is now recognized that this malignancy comprises at least two distinct molecular subtypes identified by gene expression profiling: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) groups-the cell-of-origin (COO) classification. These two groups have different genetic mutation landscapes, pathobiology, and outcomes following treatment. Evidence is accumulating that novel agents have selective activity in one or the other COO group, making COO a predictive biomarker. Thus, there is now a pressing need for accurate and robust methods to assign COO, to support clinical trials, and ultimately guide treatment decisions for patients. The "gold standard" methods for COO are based on gene expression profiling (GEP) of RNA from fresh frozen tissue using microarray technology, which is an impractical solution when formalin-fixed paraffin-embedded tissue (FFPET) biopsies are the standard diagnostic material. This review outlines the history of the COO classification before examining the practical implementation of COO assays applicable to FFPET biopsies. The immunohistochemistry (IHC)-based algorithms and gene expression-based assays suitable for the highly degraded RNA from FFPET are discussed. Finally, the technical and practical challenges that still need to be addressed are outlined before robust gene expression-based assays are used in the routine management of patients with DLBCL.

  15. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

    NASA Astrophysics Data System (ADS)

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-12-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

  16. Effect of rituximab on B cell phenotype and serum B cell-activating factor levels in patients with thrombotic thrombocytopenic purpura

    PubMed Central

    Becerra, E; Scully, M A; Leandro, M J; Heelas, E O; Westwood, J-P; De La Torre, I; Cambridge, G

    2015-01-01

    Autoantibodies inhibiting the activity of the metalloproteinase, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), underlie the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Rituximab (RTX) combined with plasma-exchange (PEX) is an effective treatment in TTP. Patients can remain in remission for extended periods following PEX/RTX, and this is associated with continuing reduction in antibodies to ADAMTS13. Factors controlling B cell differentiation to autoantibody production, including stimulation through the B cell receptor and interactions with the B cell-activating factor (BAFF), may thus impact length of remission. In this cross-sectional study, we measured naive and memory B cell phenotypes [using CD19/immunoglobulin (Ig)D/CD27] following PEX/RTX treatment in TTP patients at B cell return (n = 6) and in 12 patients in remission 10–68 months post-RTX. We also investigated relationships among serum BAFF, soluble CD23 (sCD23– a surrogate measure of acquiring B memory (CD27+) phenotype) and BAFF receptor (BAFF-R) expression. At B cell return after PEX/RTX, naive B cells predominated and BAFF-R expression was reduced compared to healthy controls (P < 0·001). In the remission group, despite numbers of CD19+ B cells within normal limits in most patients, the percentage and absolute numbers of pre-switch and memory B cells remained low, with sCD23 levels at the lower end of the normal range. BAFF levels were correlated inversely with BAFF-R expression and time after therapy. In conclusion, the long-term effects of RTX therapy in patients with TTP included slow regeneration of memory B cell subsets and persistently reduced BAFF-R expression across all B cell subpopulations. This may reflect the delay in selection and differentiation of potentially autoreactive (ADAMTS13-specific) B cells, resulting in relatively long periods of low disease activity after therapy. PMID:25339550

  17. Implementation of activity-based costing (ABC) to drive cost reduction efforts in a semiconductor manufacturing operation

    NASA Astrophysics Data System (ADS)

    Naguib, Hussein; Bol, Igor I.; Lora, J.; Chowdhry, R.

    1994-09-01

    This paper presents a case study on the implementation of ABC to calculate the cost per wafer and to drive cost reduction efforts for a new IC product line. The cost reduction activities were conducted through the efforts of 11 cross-functional teams which included members of the finance, purchasing, technology development, process engineering, equipment engineering, production control, and facility groups. The activities of these cross functional teams were coordinated by a cost council. It will be shown that these activities have resulted in a 57% reduction in the wafer manufacturing cost of the new product line. Factors contributed to successful implementation of an ABC management system are discussed.

  18. Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling.

    PubMed

    Ackermann, Jochen A; Radtke, Daniel; Maurberger, Anna; Winkler, Thomas H; Nitschke, Lars

    2011-04-20

    Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

  19. IL-17B activated mesenchymal stem cells enhance proliferation and migration of gastric cancer cells.

    PubMed

    Bie, Qingli; Zhang, Bin; Sun, Caixia; Ji, Xiaoyun; Barnie, Prince Amoah; Qi, Chen; Peng, Jingjing; Zhang, Danyi; Zheng, Dong; Su, Zhaoliang; Wang, Shengjun; Xu, Huaxi

    2017-03-21

    Mesenchymal stem cells are important cells in tumor microenvironment. We have previously demonstrated that IL-17B/IL-17RB signal promoted progression of gastric cancer. In this study, we further explored the effect of IL-17B on mesenchymal stem cells in tumor microenvironment and its impact on the tumor progression. The results showed that IL-17B induced the expression of stemness-related genes Nanog, Sox2, and Oct4 in mesenchymal stem cells and enhanced its tumor-promoting effect. The supernatant from cultured mesenchymal stem cells after treating with exogenous rIL-17B promoted the proliferation and migration of MGC-803, therefor suggesting that rIL-17B might promote mesenchymal stem cells to produce soluble factors. In addition, rIL-17B also activated the NF-κΒ, STAT3, β-catenin pathway in mesenchymal stem cells. Our data revealed a new mechanism that IL-17B enhanced the progression of gastric cancer by activating mesenchymal stem cells.

  20. Suppression of hepatic stellate cell activation by microRNA-29b

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sekiya, Yumiko; Ogawa, Tomohiro; Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka

    Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has beenmore » previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.« less

  1. The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response

    PubMed Central

    Ronald, Pamela C.

    2014-01-01

    Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS. PMID:25386383

  2. The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response.

    PubMed

    Ronald, Pamela C

    2014-01-01

    Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS.

  3. Activation of B cells by non-canonical helper signals

    PubMed Central

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-01-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that—in addition to presenting antigens to T and B cells—macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry. PMID:22868664

  4. Involvement of Semaphorin (Sema4D) in T-Dependent Activation of B Cells.

    PubMed

    Kuklina, Е М; Nekrasova, I V; Valieva, Yu V

    2017-08-01

    The involvement of endogenous semaphorin (Sema4D) into the key stage of T-dependent differentiation of B cells, formation of plasmoblasts, was demonstrated in vitro in T/B cell co-culture under conditions of polyclonal activation of T cells. The effect of semaphorin was not associated with activation of high-affinity Sema4D receptor plexin B1, but involves lowaffinity receptor CD72. These data indicate that Sema4D-dependent signal regulates not only the initial stage of B-cell activation, proliferative response to the antigen, but also further differentiation of B cells into plasma cells.

  5. Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

    PubMed

    Kawano, M; Matsushima, K; Oppenheim, J J

    1987-08-01

    A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

  6. Glaucarubinone sensitizes KB cells to paclitaxel by inhibiting ABC transporters via ROS-dependent and p53-mediated activation of apoptotic signaling pathways

    PubMed Central

    Karthikeyan, Subburayan; Hoti, Sugeerappa Laxmanappa; Nazeer, Yasin; Hegde, Harsha Vasudev

    2016-01-01

    Multidrug resistance (MDR) is considered to be the major contributor to failure of chemotherapy in oral squamous cell carcinoma (SCC). This study was aimed to explore the effects and mechanisms of glaucarubinone (GLU), one of the major quassinoids from Simarouba glauca DC, in potentiating cytotoxicity of paclitaxel (PTX), an anticancer drug in KB cells. Our data showed that the administration of GLU pre-treatment significantly enhanced PTX anti-proliferative effect in ABCB1 over-expressing KB cells. The Rh 123 drug efflux studies revealed that there was a significant transport function inhibition by GLU-PTX treatment. Interestingly, it was also found that this enhanced anticancer efficacy of GLU was associated with PTX-induced cell arrest in the G2/M phase of cell cycle. Further, the combined treatment of GLU-PTX had significant decrease in the expression levels of P-gp, MRPs, and BCRP in resistant KB cells at both mRNA and protein levels. Furthermore, the combination treatments showed significant reactive oxygen species (ROS) production, chromatin condensation and reduced mitochondrial membrane potential in resistant KB cells. The results from DNA fragmentation analysis also demonstrated the GLU induced apoptosis in KB cells and its synergy with PTX. Importantly, GLU and/or PTX triggered apoptosis through the activation of pro-apoptotic proteins such as p53, Bax, and caspase-9. Our findings demonstrated for the first time that GLU causes cell death in human oral cancer cells via the ROS-dependent suppression of MDR transporters and p53-mediated activation of the intrinsic mitochondrial pathway of apoptosis. Additionally, the present study also focussed on investigation of the protective effect of GLU and combination drugs in human normal blood lymphocytes. Normal blood lymphocytes assay indicated that GLU is able to induce selective toxicity in cancer cells and in silico molecular docking studies support the choice of GLU as ABC inhibitor to enhance PTX efficacy

  7. Arginine methylation catalyzed by PRMT1 is required for B cell activation and differentiation.

    PubMed

    Infantino, Simona; Light, Amanda; O'Donnell, Kristy; Bryant, Vanessa; Avery, Danielle T; Elliott, Michael; Tangye, Stuart G; Belz, Gabrielle; Mackay, Fabienne; Richard, Stephane; Tarlinton, David

    2017-10-12

    Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in mammalian cells, regulating many important functions including cell signalling, proliferation and differentiation. Here we show the role of PRMT1 in B-cell activation and differentiation. PRMT1 expression and activity in human and mouse peripheral B cells increases in response to in vitro or in vivo activation. Deletion of the Prmt1 gene in mature B cells establishes that although the frequency and phenotype of peripheral B cell subsets seem unaffected, immune responses to T-cell-dependent and -independent antigens are substantially reduced. In vitro activation of Prmt1-deficient B cells with a variety of mitogens results in diminished proliferation, differentiation and survival, effects that are correlated with altered signal transduction from the B cell receptor. Thus PRMT1 activity in B cells is required for correct execution of multiple processes that in turn are necessary for humoral immunity.PRMT1 is an arginine methyltransferase involved in a variety of cell functions. Here the authors delete PRMT1 specifically in mature B cells to show the importance of arginine methylation for B cell proliferation, differentiation and survival, and thereby for humoral immunity.

  8. Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection

    PubMed Central

    Sciaranghella, Gaia; Tong, Neath; Mahan, Alison E.; Suscovich, Todd J.; Alter, Galit

    2013-01-01

    Objective To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection. Design Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction. Methods Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication. Results Although the emergence of exhausted, CD21low tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21low activated memory B cells was lower in spontaneous controllers. Conclusion Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment. PMID:23135171

  9. Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe-S cluster status and Mg²⁺: implication for ribosomal function.

    PubMed

    Sims, Lynn M; Igarashi, Robert Y

    2012-08-15

    Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe₄S₄ clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²⁺. Typically ATPases require Mg²⁺, as is true for PabABCE1, but Mg²⁺ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²⁺] inhibits the PabABCE1 ATPase (K(i) of ∼1 μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²⁺ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from ∼30 μM to over 1 mM with M²⁺. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²⁺]. These findings uncover intricate overlapping effects by both [Mg²⁺] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Zhou, Shu; Wheatley, Brittany; Song, Wenxia; Mochrie, Simon; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body for signs of infection in the form of antigen on the surface of antigen presenting cells. B cell receptor (BCR) binding to antigen induces a signaling cascade that leads to B cell activation and spreading. During activation, BCR form signaling microclusters that later coalesce as the cell contracts. We have studied the dynamics of BCRs on activated murine primary B cells using single particle tracking. The tracks are analyzed using perturbation expectation-maximization (pEM), a systems-level analysis, which allows identification of different short-time diffusive states from single molecule tracks. We identified four dominant diffusive states, two of which correspond to BCRs interacting with signaling molecules. For wild-type cells, the number of BCR in signaling states increases as the cell spreads and then decreases during cell contraction. In contrast, cells lacking the actin regulatory protein, N-WASP, are unable to contract and BCRs remain in the signaling states for longer times. These observations indicate that actin cytoskeleton dynamics modulate BCR diffusion and clustering. Our results provide novel information regarding the timescale of interaction between BCR and signaling molecules.

  11. Early activation of teleost B cells in response to rhabdovirus infection.

    PubMed

    Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J; Barreda, Daniel R; Tafalla, Carolina

    2015-02-01

    To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have

  12. The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin.

    PubMed

    Poudyal, Bandita; Sauer, Karin

    2018-02-01

    A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa , biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors. Copyright © 2018 American Society for Microbiology.

  13. ATP-binding cassette (ABC) proteins in aquatic invertebrates: Evolutionary significance and application in marine ecotoxicology.

    PubMed

    Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Jae-Seong

    2017-04-01

    The ATP-binding cassette (ABC) protein superfamily is known to play a fundamental role in biological processes and is highly conserved across animal taxa. The ABC proteins function as active transporters for multiple substrates across the cellular membrane by ATP hydrolysis. As this superfamily is derived from a common ancestor, ABC genes have evolved via lineage-specific duplications through the process of adaptation. In this review, we summarized information about the ABC gene families in aquatic invertebrates, considering their evolution and putative functions in defense mechanisms. Phylogenetic analysis was conducted to examine the evolutionary significance of ABC gene families in aquatic invertebrates. Particularly, a massive expansion of multixenobiotic resistance (MXR)-mediated efflux transporters was identified in the absence of the ABCG2 (BCRP) gene in Ecdysozoa and Platyzoa, suggesting that a loss of Abcg2 gene occurred sporadically in these species during divergence of Protostome to Lophotrochozoa. Furthermore, in aquatic invertebrates, the ecotoxicological significance of MXR is discussed while considering the role of MXR-mediated efflux transporters in response to various environmental pollutants. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. EGF receptor uses SOS1 to drive constitutive activation of NFκB in cancer cells

    PubMed Central

    De, Sarmishtha; Dermawan, Josephine Kam Tai; Stark, George R.

    2014-01-01

    Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation. PMID:25071181

  15. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

    PubMed Central

    Reilman, Ewoud; Mars, Ruben A. T.; van Dijl, Jan Maarten; Denham, Emma L.

    2014-01-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

  16. Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

    PubMed

    Kosowicz, John G; Lee, Jaeyeun; Peiffer, Brandon; Guo, Zufeng; Chen, Jianmeng; Liao, Gangling; Hayward, S Diane; Liu, Jun O; Ambinder, Richard F

    2017-08-15

    Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors. Copyright © 2017 American Society for Microbiology.

  17. Phase behavior of model ABC triblock copolymers

    NASA Astrophysics Data System (ADS)

    Chatterjee, Joon

    The phase behavior of poly(isoprene-b-styrene- b-ethylene oxide) (ISO), a model ABC triblock copolymer has been studied. This class of materials exhibit self-assembly, forming a large array of ordered morphologies at length scales of 5-100 nm. The formation of stable three-dimensionally continuous network morphologies is of special interest in this study. Since these nanostructures considerably impact the material properties, fundamental knowledge for designing ABC systems have high technological importance for realizing applications in the areas of nanofabrication, nanoporous media, separation membranes, drug delivery and high surface area catalysts. A comprehensive framework was developed to describe the phase behavior of the ISO triblock copolymers at weak to intermediate segregation strengths spanning a wide range of composition. Phases were characterized through a combination of characterization techniques, including small angle x-ray scattering, dynamic mechanical spectroscopy, transmission electron microscopy, and birefringence measurements. Combined with previous investigations on ISO, six different stable ordered state symmetries have been identified: lamellae (LAM), Fddd orthorhombic network (O70), double gyroid (Q230), alternating gyroid (Q214), hexagonal (HEX), and body-centered cubic (BCC). The phase map was found to be somewhat asymmetric around the fI = fO isopleth. This work provides a guide for theoretical studies and gives insight into the intricate effects of various parameters on the self-assembly of ABC triblock copolymers. Experimental SAXS data evaluated with a simple scattering intensity model show that local mixing varies continuously across the phase map between states of two- and three-domain segregation. Strategies of blending homopolymers with ISO triblock copolymer were employed for studying the swelling properties of a lamellar state. Results demonstrate that lamellar domains swell or shrink depending upon the type of homopolymer that

  18. Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism

    PubMed Central

    Farsang, Attila; Zádori, Zoltán; Hornyák, Ákos; Dencső, László; Almazán, Fernando; Enjuanes, Luis; Belák, Sándor

    2012-01-01

    The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log10 titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV. PMID:22438554

  19. Anti-cancer activity of withaferin A in B-cell lymphoma

    PubMed Central

    McKenna, MK; Gachuki, BW; Alhakeem, SS; Oben, KN; Rangnekar, VM; Gupta, RC; Bondada, S

    2015-01-01

    Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90. PMID:26020511

  20. Anti-cancer activity of withaferin A in B-cell lymphoma.

    PubMed

    McKenna, M K; Gachuki, B W; Alhakeem, S S; Oben, K N; Rangnekar, V M; Gupta, R C; Bondada, S

    2015-01-01

    Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90.

  1. Activation of NF-kappa B Signaling Promotes Growth of Prostate Cancer Cells in Bone

    PubMed Central

    Jin, Renjie; Sterling, Julie A.; Edwards, James R.; DeGraff, David J.; Lee, Changki; Park, Serk In; Matusik, Robert J.

    2013-01-01

    Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation. PMID:23577181

  2. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1)

    PubMed Central

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-01-01

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage. PMID:27827892

  3. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

    PubMed Central

    Honigberg, Lee A.; Smith, Ashley M.; Sirisawad, Mint; Verner, Erik; Loury, David; Chang, Betty; Li, Shyr; Pan, Zhengying; Thamm, Douglas H.; Miller, Richard A.; Buggy, Joseph J.

    2010-01-01

    Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway. PMID:20615965

  4. Target sequence accessibility limits activation-induced cytidine deaminase activity in primary mediastinal B-cell lymphoma.

    PubMed

    Popov, Sergey W; Moldenhauer, Gerhard; Wotschke, Beate; Brüderlein, Silke; Barth, Thomas F; Dorsch, Karola; Ritz, Olga; Möller, Peter; Leithäuser, Frank

    2007-07-15

    Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL.

  5. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

    2013-06-01

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

  6. Activation of B Cells by a Dendritic Cell-Targeted Oral Vaccine

    PubMed Central

    Sahay, Bikash; Owen, Jennifer L.; Yang, Tao; Zadeh, Mojgan; Lightfoot, Yaíma L.; Ge, Jun-Wei; Mohamadzadeh, Mansour

    2015-01-01

    Production of long-lived, high affinity humoral immunity is an essential characteristic of successful vaccination and requires cognate interactions between T and B cells in germinal centers. Within germinal centers, specialized T follicular helper cells assist B cells and regulate the antibody response by mediating the differentiation of B cells into memory or plasma cells after exposure to T cell-dependent antigens. It is now appreciated that local immune responses are also essential for protection against infectious diseases that gain entry to the host by the mucosal route; therefore, targeting the mucosal compartments is the optimum strategy to induce protective immunity. However, because the gastrointestinal mucosae are exposed to large amounts of environmental and dietary antigens on a daily basis, immune regulatory mechanisms exist to favor tolerance and discourage autoimmunity at these sites. Thus, mucosal vaccination strategies must ensure that the immunogen is efficiently taken up by the antigen presenting cells, and that the vaccine is capable of activating humoral and cellular immunity, while avoiding the induction of tolerance. Despite significant progress in mucosal vaccination, this potent platform for immunotherapy and disease prevention must be further explored and refined. Here we discuss recent progress in the understanding of the role of different phenotypes of B cells in the development of an efficacious mucosal vaccine against infectious disease. PMID:24372255

  7. Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression.

    PubMed

    Ding, Wei; Nowakowski, Grzegorz S; Knox, Traci R; Boysen, Justin C; Maas, Mary L; Schwager, Susan M; Wu, Wenting; Wellik, Linda E; Dietz, Allan B; Ghosh, Asish K; Secreto, Charla R; Medina, Kay L; Shanafelt, Tait D; Zent, Clive S; Call, Timothy G; Kay, Neil E

    2009-11-01

    It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.

  8. Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis.

    PubMed

    Touil, Hanane; Kobert, Antonia; Lebeurrier, Nathalie; Rieger, Aja; Saikali, Philippe; Lambert, Caroline; Fawaz, Lama; Moore, Craig S; Prat, Alexandre; Gommerman, Jennifer; Antel, Jack P; Itoyama, Yasuto; Nakashima, Ichiro; Bar-Or, Amit

    2018-04-19

    The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated. We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells. Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival

  9. CD45RO enriches for activated, highly mutated human germinal center B cells

    PubMed Central

    Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

    2007-01-01

    To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

  10. [German adaptation of the "Activities-Specific Balance Confidence (ABC) scale" for the assessment of falls-related self-efficacy].

    PubMed

    Schott, Nadja

    2008-12-01

    The present study investigates the psychometric properties and the factorial structure of the German adaptation of the Activities-Specific Balance Confidence (ABC) scale (Powell & Myers, 1995) for the evaluation of falls-related self-efficacy in community-dwelling older adults. The German adaptation of the ABC using a forward-backward procedure was administered to 113 older adults (age 68.9+/-8.5 years). The following internationally accepted instruments were used for validation: The Short Form Health Survey SF 36, the Geriatric Depression Scale (GDS), the Trail Making Test and the Letter Number Sequencing Test, and motor tests (balance, strength, mobility). The internal consistency (0.91-0.95) as well as the test-retest reliability of the subscales was excellent (0.94-0.98). The correlation coefficients with the validation instruments ranged between 0.33 and 0.58. Significant differences in the ABC-D scores were found in older adults with and without falls. Older adults with a recent fall history scored lower on the ABC-D than older adults without a recent fall history. To conclude, the German version of the ABC has properties analogous to the original English version and is apparently useful in assessing falls-related self-efficacy.

  11. B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

    PubMed

    Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

    2018-06-01

    Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

  12. SU-E-T-326: The Oxygen Saturation (SO2) and Breath-Holding Time Variation Applied Active Breathing Control (ABC)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gong, G; Yin, Y

    Purpose: To study the oxygen saturation (SO2) and breath-holding time variation applied active breathing control (ABC) in radiotherapy of tumor. Methods: 24 volunteers were involved in our trials, and they all did breath-holding motion assisted by ELEKTA Active Breathing Coordinator 2.0 for 10 times respectively. And the patient monitor was used to observe the oxygen saturation (SO2) variation. The variation of SO2, and length of breath-holding time and the time for recovering to the initial value of SO2 were recorded and analyzed. Results: (1) The volunteers were divided into two groups according to the SO2 variation in breath-holding: A group,more » 14 cases whose SO2 reduction were more than 2% (initial value was 97% to 99%, while termination value was 91% to 96%); B group, 10 cases were less than 2% in breath-holding without inhaling oxygen. (2) The interfraction breath holding time varied from 8 to 20s for A group compared to the first breath-holding time, and for B group varied from 4 to 14s. (3) The breathing holding time of B group prolonged mean 8s, compared to A group. (4) The time for restoring to the initial value of SO2 was from 10s to 30s. And the breath-holding time shortened obviously for patients whose SO2 did not recover to normal. Conclusion: It is very obvious that the SO2 reduction in breath-holding associated with ABC for partial people. It is necessary to check the SO2 variation in breath training, and enough time should be given to recover SO2.« less

  13. Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

    PubMed Central

    Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

    2015-01-01

    Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

  14. In vitro Reactivity to Implant Metals Demonstrates a Person Dependent Association with both T-Cell and B-Cell Activation

    PubMed Central

    Hallab, Nadim James; Caicedo, Marco; Epstein, Rachael; McAllister, Kyron; Jacobs, Joshua J

    2009-01-01

    Hypersensitivity to metallic implants remains relatively unpredictable and poorly understood. We initially hypothesized that metal-induced lymphocyte proliferation responses to soluble metal challenge (ions) are mediated exclusively by early T-cell activation (not B-cells), typical of a Delayed-Type-Hypersensitivity response. We tested this by comparing proliferation (6-days) of primary lymphocytes with early T-cell and B-cell activation (48-hours) in three groups of subjects likely to demonstrate elevated metal-reactivity: Group 1(n=12) history of metal-sensitivity with no implant; Group 2a(n=6) well performing metal-on-metal THRs, and Group 2b(n=20) subjects with poorly performing metal-on-polymer total joint arthroplasties (TJA). Group 1 showed 100%(12/12) metal reactivity (Stimulation Index>2) to Ni. Group 2a&2b were 83%(5/6) and 75%(15/22) metal reactive (to Co, Cr or Ni) respectively. Of the n=32 metal reactive subjects to Co, Cr or Ni (SI>2), n=22/32 demonstrated >2-fold elevations in % of T-cell or B-cell activation (CD25+,CD69+) to metal challenge compared to untreated control. 18/22 metal-activated subjects demonstrated an exclusively T-cell or B-cell activation response to metal challenge, where 6/18 demonstrated exclusively B-cell activation and 12/18 demonstrated a T-cell only response, as measured by surface activation markers CD25+ and CD69+. However, there was no direct correlation (R2<0.1) between lymphocyte proliferation and % T-cell or B-cell activation (CD25+:CD69+). Proliferation assays (LTT) showed greater ability to detect metal reactivity than did subject-dependent results of flow-cytometry analysis of T-cell or B-cell activation. The high incidence of lymphocyte reactivity and activation, indicate that more complex than initially hypothesized immune responses may contribute to the etiology of debris induced osteolysis in metal-sensitive individuals. PMID:19235773

  15. A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

    PubMed

    Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

    2009-02-01

    Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.

  16. Regulation of Expression of abcA and Its Response to Environmental Conditions

    PubMed Central

    Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

    2014-01-01

    The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

  17. Profiling of ABC transporters ABCB5, ABCF2 and nestin-positive stem cells in nevi, in situ and invasive melanoma.

    PubMed

    Setia, Namrata; Abbas, Ossama; Sousa, Yessica; Garb, Jane L; Mahalingam, Meera

    2012-08-01

    Distinct ABCB5 forms and ABCF2, members of the ATP-binding cassette (ABC) superfamily of transporters, are normally expressed in various tissues and cells, and enhanced expression of both has been demonstrated in select cancers. In melanoma cell lines, gene expression profiling of ABC transporters has revealed enhanced expression of melanocyte-specific ABCB5 and ABCF2 proteins. Given this, our primary aim was to ascertain immunohistochemical expression of the ABC transporters ABCB5 and ABCF2 and, the stem cell marker, nestin in a spectrum of benign and malignant nevomelanocytic proliferations, including nevi (n=30), in situ (n=31) and invasive (n=24) primary cutaneous melanomas to assess their role in the stepwise development of malignancy. In addition, their expression was compared with established melanoma prognosticators to ascertain their utility as independent prognosticators. A semiquantitative scoring system was utilized by deriving a cumulative score (based on percentage positivity cells and intensity of expression) and statistical analyses was carried out using analysis of variance with linear contrasts. Mean cumulative score in nevi, in situ and invasive melanoma were as follows: 3.8, 4.4 and 5.3 for ABCB5, respectively (P<0.005 for all), and 4.6, 4.6 and 5.3 for nestin, respectively (P=not significant for all). No appreciable expression of ABCF2 was noted in any of the groups. While ulcerated lesions of melanoma demonstrated lower levels of expression of ABCB5 and nestin than non-ulcerated lesions, and nestin expression was lower in lesions with mitoses >1, after controlling for the presence of ulceration and mitotic activity, the expression of both proteins did not significantly correlate with known melanoma prognosticators. The gradual increase in the expression of ABCB5 from benign nevus to in situ to invasive melanoma suggests that it plays a role in melanomagenesis. On the basis of our findings, a prospective study with follow-up data is required to

  18. Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation.

    PubMed

    Ramila, G; Studer, S; Kennedy, M; Sklenar, I; Erb, P

    1985-01-01

    Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.

  19. Polycystin-1 promotes PKC{alpha}-mediated NF-{kappa}B activation in kidney cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky

    2006-11-17

    Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-{kappa}B signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293{sup CTT}), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-{kappa}B nuclear levels and NF-{kappa}B-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-{kappa}B promoter activation was mediated by PKC{alpha}more » because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293{sup CTT} cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-{kappa}B inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKC{alpha}-mediated NF-{kappa}B signalling and cell survival.« less

  20. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

    PubMed

    Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

    2014-10-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

    PubMed Central

    Sarkar, Subhashis; Germeraad, Wilfred T. V.; Rouschop, Kasper M. A.; Steeghs, Elisabeth M. P.; van Gelder, Michel; Bos, Gerard M. J.; Wieten, Lotte

    2013-01-01

    Background Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses. Methods NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia. Results Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions. Conclusions Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions. PMID:23724099

  2. Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

    PubMed

    Bartlett, W C; McCann, J; Shepherd, D M; Roy, M; Noelle, R J

    1990-12-15

    After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

  3. An increase in circulating B cell-activating factor in childhood-onset ocular myasthenia gravis.

    PubMed

    Motobayashi, Mitsuo; Inaba, Yuji; Nishimura, Takafumi; Kobayashi, Norimoto; Nakazawa, Yozo; Koike, Kenichi

    2015-04-01

    Myasthenia gravis is a B cell-mediated autoimmune disorder. The pathophysiology of childhood-onset ocular myasthenia gravis remains unclear. We investigated serum B cell-activating factor levels and other immunological parameters in child patients with ocular myasthenia gravis. Blood samples were obtained from 9 children with ocular myasthenia gravis and 20 age-matched controls. We assayed serum concentrations of B cell-activating factor, anti-acetylcholine receptor antibody titers, 7 types of cytokines (interleukins-2, -4, -6, -10, and -17A; interferon-γ; tumor necrosis factor-α) as well as the percentages of peripheral blood CD4+, CD8+, and CD19+ cells. Serum B cell-activating factor levels were significantly higher before immunosuppressive therapy in patients with childhood-onset ocular myasthenia gravis than in controls and decreased after immunosuppressive therapy. A significant positive correlation was observed between serum B cell-activating factor levels and anti-acetylcholine receptor antibody titers in patients with myasthenia gravis. Serum B cell-activating factor concentrations did not correlate with the percentages of CD4+, CD8+, and CD19+ cells or the CD4+/CD8+ ratio. No significant differences were observed in the levels of the 7 different types of cytokines examined, including interleukin-17A, between preimmunosuppressive therapy myasthenia gravis patients and controls. Circulating B cell-activating factor may play a key role in the pathophysiology of childhood-onset ocular myasthenia gravis. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

    PubMed

    Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

    2016-04-01

    For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

  5. Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

    PubMed

    Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

    2009-02-24

    CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

  6. Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B.

    PubMed

    Paul, N L; Lenardo, M J; Novak, K D; Sarr, T; Tang, W L; Ruddle, N H

    1990-11-01

    Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia.

  7. B cells flying solo.

    PubMed

    Groom, Joanna; Mackay, Fabienne

    2008-01-01

    Systemic autoimmunity such as systemic lupus erythematosus (SLE) is associated with the loss of B-cell tolerance, B-cell dysregulation and autoantibody production. While some autoantibodies may contribute to the pathology seen with SLE, numerous studies have shown that dysregulation of T-cell function is another critical aspect driving disease. The positive results obtained in clinical trials using T-cell- or B-cell-specific treatments have suggested that cooperation between T and B cells probably underlies disease progression in many patients. A similar cooperative mechanism seemed to explain SLE developing in mice overexpressing the B-cell-activating factor from the tumor necrosis factor family (BAFF). However, surprisingly, T-cell-deficient BAFF transgenic (Tg) mice develop SLE similar to T-cell-sufficient BAFF Tg mice, and the disease was linked to innate activation of B cells and production of proinflammatory autoantibody isotypes. In conclusion, dysregulated innate activation of B cells alone can drive disease independently of T cells, and as such this aspect represents a new pathogenic mechanism in autoimmunity.

  8. ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

    PubMed Central

    Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

    2016-01-01

    Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617

  9. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niu, Mingshan; Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu; Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu

    Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has beenmore » reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.« less

  10. ATP-binding cassette transporters in tumor endothelial cells and resistance to metronomic chemotherapy.

    PubMed

    Hida, Kyoko; Kikuchi, Hiroshi; Maishi, Nako; Hida, Yasuhiro

    2017-08-01

    Drug resistance is a major problem in anticancer therapy. ATP-binding cassette (ABC) transporters have a role in the multidrug resistance. A new regimen of chemotherapy has been proposed, called "metronomic chemotherapy". Metronomic chemotherapy is the frequent, regular administration of drug doses designed to maintain low, but active, concentrations of chemotherapeutic drugs over prolonged periods of time, without causing serious toxicities. Metronomic chemotherapy regimens were developed to optimize the antitumor efficacy of agents that target the tumor vasculature instead of tumor cells, and to reduce toxicity of antineoplastic drugs [1]. Nevertheless, recent studies revealed that ABC transporters are expressed at a higher level in the endothelium in the tumor. To avoid resistance to metronomic anti-angiogenic chemotherapy, ABC transporter inhibition of tumor endothelial cells may be a promising strategy. In this mini-review, we discuss the possible mechanism of resistance to metronomic chemotherapy from the viewpoint of tumor endothelial cell biology, focusing on ABC transporters. Copyright © 2017. Published by Elsevier B.V.

  11. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    PubMed Central

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  12. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

    PubMed

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

    2016-03-29

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

  13. Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B.

    PubMed Central

    Paul, N L; Lenardo, M J; Novak, K D; Sarr, T; Tang, W L; Ruddle, N H

    1990-01-01

    Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia. Images PMID:1976820

  14. [Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

    PubMed

    Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

    2016-09-01

    Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

  15. UV light B-mediated inhibition of skin catalase activity promotes Gr-1+ CD11b+ myeloid cell expansion.

    PubMed

    Sullivan, Nicholas J; Tober, Kathleen L; Burns, Erin M; Schick, Jonathan S; Riggenbach, Judith A; Mace, Thomas A; Bill, Matthew A; Young, Gregory S; Oberyszyn, Tatiana M; Lesinski, Gregory B

    2012-03-01

    Skin cancer incidence and mortality are higher in men compared with women, but the causes of this sex discrepancy remain largely unknown. UV light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1(+)CD11b(+) myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present in the skin, has been associated with skin carcinogenesis. We used the outbred, immune-competent Skh-1 hairless mouse model of UVB-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1(+)CD11b(+) myeloid cell percentage. Acute UVB exposure induced Gr-1(+)CD11b(+) myeloid cell skin infiltration, which was inhibited to a greater extent in male mice by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1(+)CD11b(+) myeloid cells was 55% higher in male tumor-bearing mice compared with their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced generation of Gr-1(+)CD11b(+) myeloid cells and subsequent skin carcinogenesis.

  16. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    PubMed

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  17. NF-κB dynamics show digital activation and analog information processing in cells

    NASA Astrophysics Data System (ADS)

    Tay, Savas; Hughey, Jake; Lee, Timothy; Lipniacki, Tomasz; Covert, Markus; Quake, Stephen

    2010-03-01

    Cells operate in ever changing environments using extraordinary communication capabilities. Cell-to-cell communication is mediated by signaling molecules that form spatiotemporal concentration gradients, which requires cells to respond to a wide range of signal intensities. We used high-throughput microfluidic cell culture, quantitative gene expression analysis and mathematical modeling to investigate how single mammalian cells respond to different concentrations of the signaling molecule TNF-α via the transcription factor NF-κB. We measured NF-κB activity in thousands of live cells under TNF-α doses covering four orders of magnitude. In contrast to population studies, the activation is a stochastic, switch-like process at the single cell level with fewer cells responding at lower doses. The activated cells respond fully and express early genes independent of the TNF-α concentration, while only high dose stimulation results in the expression of late genes. Cells also encode a set of analog parameters such as the NF-κB peak intensity, response time and number of oscillations to modulate the outcome. We developed a stochastic model that reproduces both the digital and analog dynamics as well as the gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-α induced NF-κB signaling in various types of cells.

  18. Kalkipyrone B, a marine cyanobacterial γ-pyrone possessing cytotoxic and anti-fungal activities

    PubMed Central

    Bertin, Matthew J; Demirkiran, Ozlem; Navarro, Gabriel; Moss, Nathan A; Lee, John; Goldgof, Gregory M; Vigil, Edgar; Winzeler, Elizabeth A; Valeriote, Fred A; Gerwick, William H

    2015-01-01

    Bioassay-guided fractionation of two marine cyanobacterial extracts using the H-460 human lung cancer cell line and the OVC-5 human ovarian cancer cell line led to the isolation of three related α-methoxy-β, β’-dimethyl-γ-pyrones each containing a modified alkyl chain, one of which was identified as the previously reported kalkipyrone and designated kalkipyrone A. The second compound was an analog designated kalkipyrone B. The third was identified as the recently reported yoshipyrone A, also isolated from a marine cyanobacterium. Kalkipyrone A and B were obtained from a field-collection of the cyanobacterium Leptolyngbya sp. from Fagasa Bay, American Samoa, while yoshipyrone A was isolated from a field-collection of cyanobacteria (cf. Schizothrix sp.) from Panama. One-dimensional and two-dimensional NMR experiments were used to determine the overall structures and relative configurations of the kalkipyrones, and the absolute configuration of kalkipyrone B was determined by 1H NMR analysis of diastereomeric Mosher’s esters. Kalkipyrone A showed good cytotoxicity to H-460 human lung cancer cells (EC50 = 0.9 µM), w M), while kalkipyrone B and yoshipyrone A were less active (EC50 = 9.0 µM and > 10 µM, respectively). Both kalkipyrone A and B showed moderate toxicity to Saccharomyces cerevisiae ABC16-Monster strain (IC50 = 14.6 and 13.4 µM, respectively), whereas yoshipyrone A was of low toxicity to this yeast strain (IC50 = 63.8 µM). PMID:26632528

  19. Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation

    PubMed Central

    Jain, Ashish; Ma, Chi A.; Lopez-Granados, Eduardo; Means, Gary; Brady, William; Orange, Jordan S.; Liu, Shuying; Holland, Steven; Derry, Jonathan M.J.

    2004-01-01

    Hypomorphic mutations in the zinc finger domain of NF-κB essential modulator (NEMO) cause X-linked hyper-IgM syndrome with ectodermal dysplasia (XHM-ED). Here we report that patient B cells are characterized by an absence of Ig somatic hypermutation (SHM) and defective class switch recombination (CSR) despite normal induction of activation-induced cytidine deaminase (AID) and Iε-Cε transcripts. This indicates that AID expression alone is insufficient to support neutralizing antibody responses. Furthermore, we show that patient B cells stimulated with CD40 ligand are impaired in both p65 and c-Rel activation, and whereas addition of IL-4 can enhance p65 activity, c-Rel activity remains deficient. This suggests that these NF-κB components have different activation requirements and that IL-4 can augment some but not all NEMO-dependent NF-κB signaling. Finally, using microarray analysis of patient B cells we identified downstream effects of impaired NF-κB activation and candidate factors that may be necessary for CSR and SHM in B cells. PMID:15578091

  20. Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells

    PubMed Central

    Syrett, Camille M.; Sindhava, Vishal; Hodawadekar, Suchita; Myles, Arpita; Liang, Guanxiang; Zhang, Yue; Nandi, Satabdi; Cancro, Michael; Atchison, Michael

    2017-01-01

    X-chromosome inactivation (XCI) in female lymphocytes is uniquely regulated, as the inactive X (Xi) chromosome lacks localized Xist RNA and heterochromatin modifications. Epigenetic profiling reveals that Xist RNA is lost from the Xi at the pro-B cell stage and that additional heterochromatic modifications are gradually lost during B cell development. Activation of mature B cells restores Xist RNA and heterochromatin to the Xi in a dynamic two-step process that differs in timing and pattern, depending on the method of B cell stimulation. Finally, we find that DNA binding domain of YY1 is necessary for XCI in activated B cells, as ex-vivo YY1 deletion results in loss of Xi heterochromatin marks and up-regulation of X-linked genes. Ectopic expression of the YY1 zinc finger domain is sufficient to restore Xist RNA localization during B cell activation. Together, our results indicate that Xist RNA localization is critical for maintaining XCI in female lymphocytes, and that chromatin changes on the Xi during B cell development and the dynamic nature of YY1-dependent XCI maintenance in mature B cells predisposes X-linked immunity genes to reactivation. PMID:28991910

  1. EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas.

    PubMed

    Shair, Kathy H Y; Bendt, Katherine M; Edwards, Rachel H; Bedford, Elisabeth C; Nielsen, Judith N; Raab-Traub, Nancy

    2007-11-01

    Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.

  2. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yi, E-mail: wangyi2004a@126.com; Wang, Xiang; Sun, Minghui

    Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kBmore » (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB

  3. Relevance of P-glycoprotein on CXCR4+ B cells to organ manifestation in highly active rheumatoid arthritis.

    PubMed

    Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Kawabe, Akio; Tanaka, Yoshiya

    2018-03-01

    In rheumatoid arthritis (RA), P-glycoprotein (P-gp) expression on activated B cells is associated with active efflux of intracellular drugs, resulting in drug resistance. CXCR4 is associated with migration of B cells. This study was designed to elucidate the relevance of P-gp expression on CXCR4 + B cells to clinical manifestations in refractory RA. CD19 + B cells were analyzed using flow cytometry and immunohistochemistry. P-gp was highly expressed especially on CXCR4 + CD19 + B cells in RA. The proportion of P-gp-expressing CXCR4 + B cells correlated with disease activity, estimated by Simplified Disease Activity Index (SDAI), and showed marked expansion in RA patients with high SDAI and extra-articular involvement. In highly active RA, massive infiltration of P-gp + CXCR4 + CD19 + B cells was noted in CXCL12-expressing inflammatory lesions of RA synovitis and RA-associated interstitial pneumonitis. In RA patient with active extra-articular involvement, intracellular dexamethasone level (IDL) in lymphocytes diminished with expansion of P-gp + CXCR4 + CD19 + B cells. Adalimumab reduced P-gp + CXCR4 + CD19 + B cells, increased IDL in lymphocytes, and improved the clinical manifestation and allowed tapering of concomitant medications. Expansion of P-gp + CXCR4 + B cells seems to be associated with drug resistance, disease activity and progressive destructive arthritis with extra-articular involvement in RA.

  4. ABC transporters and immunity: mechanism of self-defense.

    PubMed

    Hinz, Andreas; Tampé, Robert

    2012-06-26

    The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.

  5. INTERFERON α ACTIVATES NF-κ B IN JAK1-DEFICIENT CELLS THROUGH A TYK2-DEPENDENT PATHWAY

    PubMed Central

    Yang, Chuan He; Murti, Aruna; Valentine, William J.; Du, Ziyun; Pfeffer, Lawrence M.

    2005-01-01

    In addition to activating members of the STAT transcription factor family, IFN α/β activates the NF-κ B transcription factor. To determine the role of the JAK-STAT pathway in NF-κ B activation by IFN, we examined NF-κ B activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH) IFN activates STAT1, STAT2 and STAT3, as well as NF-κB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells IFN induces NF-κB activation and NF-κB dependent gene transcription, but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically-inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-κB activation by IFN. Moreover, IFN does not promote NF-κB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-κB signaling pathway. In the IFN signaling pathway leading to STAT activation both JAK1 and TYK2 are essential, while NF-κB activation requires only TYK2. PMID:15883164

  6. Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter.

    PubMed

    Escudero, Leticia; Mariscal, Vicente; Flores, Enrique

    2015-08-01

    In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in

  7. Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

    PubMed

    Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

    2007-07-15

    (-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

  8. Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

    PubMed Central

    Oh, Yoon Sin; Shin, Seungjin; Lee, Youn-Jung; Kim, Eung Hwi; Jun, Hee-Sook

    2011-01-01

    Background Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation. Methodology/Principal Findings The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes. Conclusions/Significance These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells. PMID:21897861

  9. Evaluating the B-cell density with various activation functions using White Noise Path Integral Approach

    NASA Astrophysics Data System (ADS)

    Aban, C. J. G.; Bacolod, R. O.; Confesor, M. N. P.

    2015-06-01

    A The White Noise Path Integral Approach is used in evaluating the B-cell density or the number of B-cell per unit volume for a basic type of immune system response based on the modeling done by Perelson and Wiegel. From the scaling principles of Perelson [1], the B- cell density is obtained where antigens and antibodies mutates and activation function f(|S-SA|) is defined describing the interaction between a specific antigen and a B-cell. If the activation function f(|S-SA|) is held constant, the major form of the B-cell density evaluated using white noise analysis is similar to the form of the B-cell density obtained by Perelson and Wiegel using a differential approach.A piecewise linear functionis also used to describe the activation f(|S-SA|). If f(|S-SA|) is zero, the density decreases exponentially. If f(|S-SA|) = S-SA-SB, the B- cell density increases exponentially until it reaches a certain maximum value. For f(|S-SA|) = 2SA-SB-S, the behavior of B-cell density is oscillating and remains to be in small values.

  10. Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity[S

    PubMed Central

    Teague, Heather; Harris, Mitchel; Fenton, Jenifer; Lallemand, Perrine; Shewchuk, Brian M.; Shaikh, Saame Raza

    2014-01-01

    EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies. PMID:24837990

  11. Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mizukoshi, Terumi; Komori, Hideyuki; Mizuguchi, Mariko

    2013-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA bindingmore » was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection. - Highlights: • HTLV-1 Tax1 does not activate RelA of NF-κB in non-hematopoietic cell lines. • Tax1 activates the NF-κB non-canonical pathway in non-hematopoietic cell lines. • Tax1 does not induce RelA nuclear translocation in those cell lines, unlike TNFα. • The OX40L promoter κB site is activated by ectopic, but not endogenous, RelA.« less

  12. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  13. Oxaliplatin antagonizes HIV-1 latency by activating NF-κB without causing global T cell activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Xiaoli; Liu, Sijie; Wang, Pengfei

    Highlights: • The chemotherapeutic drug oxaliplatin reactivates latent HIV-1 in this cell line model of HIV-1 latency. • Reactivation is synergized when oxaliplatin is used in combination with valproic acid. • Oxaliplatin reactivates latent HIV-1 through activation of NF-kB and does not induce T cell activation. - Abstract: Reactivation of latent HIV-1 is a promising strategy for the clearance of the viral reservoirs. Because of the limitations of current agents, identification of new latency activators is urgently required. Using an established model of HIV-1 latency, we examined the effect of Oxaliplatin on latent HIV-1 reactivation. We showed that Oxaliplatin, alonemore » or in combination with valproic acid (VPA), was able to reactivate HIV-1 without inducing global T cell activation. We also provided evidence that Oxaliplatin reactivated HIV-1 expression by inducing nuclear factor kappa B (NF-κB) nuclear translocation. Our results indicated that Oxaliplatin could be a potential drug candidate for anti-latency therapies.« less

  14. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

    PubMed Central

    Rodgers, David T.; Mazagova, Magdalena; Hampton, Eric N.; Cao, Yu; Ramadoss, Nitya S.; Hardy, Ian R.; Schulman, Andrew; Du, Juanjuan; Wang, Feng; Singer, Oded; Ma, Jennifer; Nunez, Vanessa; Shen, Jiayin; Woods, Ashley K.; Wright, Timothy M.; Schultz, Peter G.; Kim, Chan Hyuk; Young, Travis S.

    2016-01-01

    Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR–T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens. PMID:26759369

  15. [Effect of NF-κB activation on the radiation response of esophageal cancer cells].

    PubMed

    Li, Baozhong; Chen, Zhaoli; Zhou, Fang; He, Jie

    2014-07-01

    To investigate the effect of NF-κB activation on radiation response of esophageal carcinoma. The expression of NF-κB was detected in pretreatment and posttreatment specimens of patients with ESCC by immunohistochemistry. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activation of NF-κB in esophageal cancer cell line KYSE150 cells. SN50, a specific NF-κB inhibitor, was applied to inhibit the activation of NF-κB. Clone formation test was used to detect the radiosensitivity of esophageal cancer cells. The median survival time of patients with activated and inactivated NF-κB in the pretreatment specimens were 16 and 19 months, respectively, with a non-significant difference between the two groups (P > 0.05). As to the patients with activated and inactivated NF-κB in posttreatment specimens, the median survival times were 13 and 35 months, respectively, with a significant difference (P < 0.01) between them. Western blot showed that the cytoplasmic expression of NF-κB was reduced with increasing radiation dose at 1.5 and 3 hours after radiation treatment. However, the expression of NF-κB in the cell nuclei was increased under the same condition, showing a trend of increased nucleus/cytoplasm ratio. The clone number in SN50 group was 96.66, 64.66, 76.66 and 10.00 under 0, 2, 4 and 12 Gy irradiation, which demonstrated a significant difference compared with the control groups (P < 0.001). Our results show that activation of NF-κB is induced by radiotherapy. Activation of NF-κB reduces the outcome of radiation treatment of esophageal cancer patients.

  16. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Lei; Nishihara, Hiroshi, E-mail: nisihara@patho2.med.hokudai.ac.jp; Kimura, Taichi

    2010-04-23

    DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines,more » Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.« less

  17. Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity

    PubMed Central

    Nakayama, Yumi; Kosek, Jolanta; Capone, Lori; Schafer, Peter H.

    2017-01-01

    BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27−IgD− double-negative (DN) B cells, but not CD27−IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines. PMID:28848067

  18. TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang Ling; Reinach, Peter; Lu, Luo

    2005-11-15

    Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less

  19. Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.

    PubMed

    Pasta, Saloni Yatin; Raman, Bakthisaran; Ramakrishna, Tangirala; Rao, Ch Mohan

    2002-11-29

    Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

  20. OCA-B regulation of B-cell development and function.

    PubMed

    Teitell, Michael A

    2003-10-01

    The transcriptional co-activator OCA-B [for Oct co-activator from B cells, also known as OBF-1 (OCT-binding factor-1) and Bob1] is not required for B-cell genesis but does regulate subsequent B-cell development and function. OCA-B deficient mice show strain-specific, partial blocks at multiple stages of B-cell maturation and a complete disruption of germinal center formation in all strains, causing humoral immune deficiency and susceptibility to infection. OCA-B probably exerts its effects through the regulation of octamer-motif controlled gene expression. The OCA-B gene encodes two proteins of distinct molecular weight, designated p34 and p35. The p34 isoform localizes in the nucleus, whereas the p35 isoform is myristoylated and is bound to the cytoplasmic membrane. p35 can traffic to the nucleus and probably activates octamer-dependent transcription, although this OCA-B isoform might regulate B cells through membrane-related signal transduction.

  1. The Preschool Activity, Technology, Health, Adiposity, Behaviour and Cognition (PATH-ABC) cohort study: rationale and design.

    PubMed

    Cliff, Dylan P; McNeill, Jade; Vella, Stewart; Howard, Steven J; Kelly, Megan A; Angus, Douglas J; Wright, Ian M; Santos, Rute; Batterham, Marijka; Melhuish, Edward; Okely, Anthony D; de Rosnay, Marc

    2017-04-04

    Prevalence estimates internationally suggest that many preschool-aged children (3-5 years) are insufficiently physically active and engage in high levels of screen-based entertainment. Early childhood is the developmental period for which we know the least about the effects of physical activity on development and health. Likewise, rapid technological advancements in mobile electronic media have made screen-based forms of entertainment for young children ubiquitous, and research demonstrating the impacts on cognition, psychosocial well-being, and health has lagged behind the rate of adoption of these technologies. The purpose of the Preschool Activity, Technology, Health, Adiposity, Behaviour and Cognition (PATH-ABC) study is to investigate if physical activity and screen-based entertainment are independently associated with cognitive and psychosocial development, and health outcomes in young children, and if so, how much and which types of these behaviours might be most influential. The PATH-ABC study is a prospective cohort, aiming to recruit 430 3-5 year-old children. Children are recruited through and complete initial assessments at their Early Childhood Education and Care (ECEC) centre, and then 12-months later at their centre or school. Direct assessments are made of children's habitual physical activity using accelerometry, cognitive (executive function) and language development (expressive vocabulary), psychosocial development (emotional understanding, Theory of Mind, empathy, and heart rate variability), adiposity (body mass index and waist circumference), and cardiovascular health (blood pressure and retinal micro- vasculature). Educators report on children's psychological strengths and difficulties and self-regulation. Parents report on children's habitual use of electronic media and other child, parent and household characteristics. The PATH-ABC study aims to provide evidence to enhance understanding of how much and which types of physical activity and

  2. Effect of body temperature on chondroitinase ABC's ability to cleave chondroitin sulfate glycosaminoglycans.

    PubMed

    Tester, Nicole J; Plaas, Anna H; Howland, Dena R

    2007-04-01

    Chondroitinase ABC (Ch'ase ABC) is a bacterial lyase that degrades chondroitin sulfate (CS), dermatan sulfate, and hyaluronan glycosaminoglycans (GAGs). This enzyme has received significant attention as a potential therapy for promoting central nervous system and peripheral nervous system repair based on its degradation of CS GAGs. Determination of the stability of Ch'ase ABC activity at temperatures equivalent to normal (37 degrees C) and elevated (39 degrees C) body temperatures is important for optimizing its clinical usage. We report here data obtained from examining enzymatic activity at these temperatures across nine lots of commercially available protease-free Ch'ase ABC. CS GAG degrading activity was assayed by using 1) immunohistochemical detection of unsaturated disaccharide stubs generated by digestion of proteoglycans in tissue sections and 2) fluorophore-assisted carbohydrate electrophoresis (FACE) and/or high-performance liquid chromatography (HPLC) to separate and quantify unsaturated disaccharide digestion products. Our results indicate that there is a significant effect of lot and time on enzymatic thermostability. Average enzymatic activity is significantly decreased at 1 and 3 days at 39 degrees C and 37 degrees C, respectively. Furthermore, the average activity seen after 1 day was significantly different between the two temperatures. Addition of bovine serum albumin as a stabilizer significantly preserved enzymatic activity at 1 day, but not 3 days, at 39 degrees C. These results show that the CS GAG degrading activity of Ch'ase ABC is significantly decreased with incubation at body temperature over time and that all lots do not show equal thermostability. These findings are important for the design and interpretation of experimental and potential clinical studies involving Ch'ase ABC. (c) 2007 Wiley-Liss, Inc.

  3. Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains.

    PubMed

    Hinrichs, John W J; Klappe, Karin; van Riezen, Manon; Kok, Jan W

    2005-11-01

    We have recently shown that two ATP binding cassette (ABC) transporters are enriched in Lubrol-resistant noncaveolar membrane domains in multidrug-resistant human cancer cells [Hinrichs, J. W. J., K. Klappe, I. Hummel, and J. W. Kok. 2004. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279: 5734-5738]. Here, we show that aminophospholipids are relatively enriched in Lubrol-resistant membrane domains compared with Triton X-100-resistant membrane domains, whereas sphingolipids are relatively enriched in the latter. Moreover, Lubrol-resistant membrane domains contain more protein and lipid mass. Based on these results, we postulate a model for detergent-insoluble glycosphingolipid-enriched membrane domains consisting of a Lubrol-insoluble/Triton X-100-insoluble region and a Lubrol-insoluble/Triton X-100-soluble region. The latter region contains most of the ABC transporters as well as lipids known to be necessary for their efflux activity. Compared with drug-sensitive cells, the detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in drug-resistant cells differ specifically in sphingolipid content and not in protein, phospholipid, or cholesterol content. In drug-resistant cells, sphingolipids with specific fatty acids (especially C24:1) are enriched in these membrane domains. Together, these data show that multidrug resistance-associated changes in both sphingolipids and ABC transporters occur in DIGs, but in different regions of these domains.

  4. Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Němcová-Fürstová, Vlasta, E-mail: vlasta.furstova@

    Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100 nM and 300 nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublinesmore » of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells. - Highlights: • Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed. • SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes. • Some taxanes are able to overcome developed resistance to

  5. Analysis of T cell-replacing factor-like activity: potent induction of T helper activity for human B cells by residual concanavalin A and interleukin 2.

    PubMed

    Sauerwein, R W; Van der Meer, W G; Aarden, L A

    1987-08-01

    At least two factors with the capacity to induce IgM synthesis in human B cells were found to be present in the 15-20-kDa fraction of the supernatant of mononuclear cells activated with concanavalin A (Con A) and phorbol ester. Previously, it has been shown (Sauerwein, R. W. et al., Eur. J. Immunol. 1985. 15: 611) that interleukin 2 (IL2) in this material is able to induce T cell-dependent IgM secretion in normal B cells. Evidence was obtained for the presence of another factor distinct from IL2 that could replace T cells in the induction of B cell differentiation. We have analyzed this factor with the use of a neoplastic B cell population of prolymphocytic origin that was functionally nonresponsive to IL2. T cell-replacing factor (TRF)-like activity and IL2 could be separated by ion-exchange chromatography, although a small amount of IL2 was recovered in the TRF fractions. This small amount of IL2 was found to be crucial for the observed TRF activity. Moreover, a substantial amount of monomeric Con A was detected in the TRF preparation. Our studies show that Con A in the presence of IL2 can act as a potent inducer of helper function in lower numbers of T cells for normal and neoplastic B cells. Functional assays for T cell contamination in B cell suspensions are therefore of limited value because they are determined by the efficiency of the stimulating signal. Particularly in those B cell factor preparations, obtained from mitogen-activated T cells with an obligatory or unidentified role of IL2, the possible effect of a contaminating mitogen must be considered.

  6. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo.

    PubMed

    Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

    2012-07-01

    The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me-/- mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me-/- erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me-/- erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me-/- erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo.

  7. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo

    PubMed Central

    Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

    2012-01-01

    The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me−/− mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me−/− erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me−/− erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me−/− erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo. PMID:22240895

  8. B-Cell Maturation Antigen, A Proliferation-Inducing Ligand, and B-Cell Activating Factor Are Candidate Mediators of Spinal Cord Injury-Induced Autoimmunity

    PubMed Central

    Saltzman, Jonah W.; Battaglino, Ricardo A.; Salles, Loise; Jha, Prateek; Sudhakar, Supreetha; Garshick, Eric; Stott, Helen L.; Zafonte, Ross

    2013-01-01

    Abstract Autoimmunity is thought to contribute to poor neurological outcomes after spinal cord injury (SCI). There are few mechanism-based therapies, however, designed to reduce tissue damage and neurotoxicity after SCI because the molecular and cellular bases for SCI-induced autoimmunity are not completely understood. Recent groundbreaking studies in rodents indicate that B cells are responsible for SCI-induced autoimmunity. This novel paradigm, if confirmed in humans, could aid in the design of neuroprotective immunotherapies. The aim of this study was to investigate the molecular signaling pathways and mechanisms by which autoimmunity is induced after SCI, with the goal of identifying potential targets in therapies designed to reduce tissue damage and inflammation in the chronic phase of SCI. To that end, we performed an exploratory microarray analysis of peripheral blood mononuclear cells to identify differentially expressed genes in chronic SCI. We identified a gene network associated with lymphoid tissue structure and development that was composed of 29 distinct molecules and five protein complexes, including two cytokines, a proliferation-inducing ligand (APRIL) and B-cell–activating factor (BAFF), and one receptor, B-cell maturation antigen (BMCA) involved in B cell development, proliferation, activation, and survival. Real-time polymerase chain reaction analysis from ribonucleic acid samples confirmed upregulation of these three genes in SCI. To our knowledge, this is the first report that peripheral blood mononuclear cells produce increased levels of BAFF and APRIL in chronic SCI. This finding provides evidence of systemic regulation of SCI-autoimmunity via APRIL and BAFF mediated activation of B cells through BMCA and points toward these molecules as potential targets of therapies designed to reduce neuroinflammation after SCI. PMID:23088438

  9. The ABC's of Learning in Infancy.

    ERIC Educational Resources Information Center

    Saunders, Minta M.

    Learning in infancy is based on activity, beginnings, and curiosity, the so-called ABC's. Earliest behavior consists of mass activity, the period from birth to 24 months of sensory-motor development which provides the foundation for all future learning. Adults must provide space, toys, and affectionate care to help infants proceed through…

  10. Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

    PubMed

    Nakayama, Yumi; Kosek, Jolanta; Capone, Lori; Hur, Eun Mi; Schafer, Peter H; Ringheim, Garth E

    2017-10-01

    BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27 + memory and memory-like CD27 - IgD - double-negative (DN) B cells, but not CD27 - IgD + naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27 + memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines. Copyright © 2017 by The American Association of Immunologists, Inc.

  11. Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

    PubMed

    Mędra, Aleksandra; Witkowska, Magdalena; Majchrzak, Agata; Cebula-Obrzut, Barbara; Bonner, Michael Y; Robak, Tadeusz; Arbiser, Jack L; Smolewski, Piotr

    2016-07-30

    Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended.

  12. Optimal Medical Equipment Maintenance Service Proposal Decision Support System combining Activity Based Costing (ABC) and the Analytic Hierarchy Process (AHP).

    PubMed

    da Rocha, Leticia; Sloane, Elliot; M Bassani, Jose

    2005-01-01

    This study describes a framework to support the choice of the maintenance service (in-house or third party contract) for each category of medical equipment based on: a) the real medical equipment maintenance management system currently used by the biomedical engineering group of the public health system of the Universidade Estadual de Campinas located in Brazil to control the medical equipment maintenance service, b) the Activity Based Costing (ABC) method, and c) the Analytic Hierarchy Process (AHP) method. Results show the cost and performance related to each type of maintenance service. Decision-makers can use these results to evaluate possible strategies for the categories of equipment.

  13. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Fei, E-mail: zhufei@zju.edu.cn; Yue, Wanfu; Wang, Yongxia

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at onemore » hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages. - Highlights: • NF-κB cascade genes such as Nfκb1 and Traf1 were up-regulated by heat-inactivated S. aureus. • Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus. • NF-κB activation is required for phagocytosis of S. aureus by macrophages.« less

  14. NF-κB activation impairs somatic cell reprogramming in ageing.

    PubMed

    Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

    2015-08-01

    Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

  15. Allelic Dependent Expression of an Activating Fc receptor on B cells Enhances Humoral Immune Responses

    PubMed Central

    Li, Xinrui; Wu, Jianming; Ptacek, Travis; Redden, David T; Brown, Elizabeth E; Alarcón, Graciela S; Ramsey-Goldman, Rosalind; Petri, Michelle A; Reveille, John D.; Kaslow, Richard A; Kimberly, Robert P; Edberg, Jeffrey C

    2014-01-01

    B cells are pivotal regulators of acquired immune responses and recent work in both experimental murine models and humans has demonstrated that subtle changes in the regulation of B cell function can significantly alter immunological responses. The balance of negative and positive signals in maintaining an appropriate B cell activation threshold is critical in B lymphocyte immune tolerance and autoreactivity. FcγRIIb (CD32B), the only recognized Fcγ receptor on B cells, provides IgG-mediated negative modulation through a tyrosine-based inhibition motif which down-regulates B cell receptor initiated signaling. These properties make FcγRIIb a promising target for antibody-based therapy. Here we report the discovery of allele-dependent expression of the activating FcγRIIc on B cells. Identical to FcγRIIb in the extracellular domain, FcγRIIc has a tyrosine-based activation motif in its cytoplasmic domain. In both human B cells and in B cells from mice transgenic for human FcγRIIc, FcγRIIc expression counterbalances the negative feedback of FcγRIIb and enhances humoral responses to immunization in mice and to BioThrax® vaccination in a human Anthrax vaccine trial. Moreover, the FCGR2C-ORF allele is associated with the risk of development of autoimmunity in humans. FcγRIIc expression on B cells challenges the prevailing paradigm of uni-directional negative feedback by IgG immune complexes via the inhibitory FcγRIIb, is a previously unrecognized determinant in human antibody/autoantibody responses, and opens the opportunity for more precise personalized use of B cell targeted antibody-based therapy. PMID:24353158

  16. Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

    PubMed

    Murota, Yoshitaka; Tabu, Kouichi; Taga, Tetsuya

    2016-11-04

    Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of

  17. Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection.

    PubMed

    Lokensgard, James R; Mutnal, Manohar B; Prasad, Sujata; Sheng, Wen; Hu, Shuxian

    2016-05-20

    persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact. CXCR3 is the primary chemokine receptor on CD19(+) B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

  18. Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

    PubMed

    Seelig, Davis M; Ito, Daisuke; Forster, Colleen L; Yoon, Una A; Breen, Matthew; Burns, Linda J; Bachanova, Veronika; Lindblad-Toh, Kerstin; O'Brien, Timothy D; Schmechel, Stephen C; Rizzardi, Anthony E; Modiano, Jaime F; Linden, Michael A

    2017-07-01

    Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

  19. Inhibition of the Human ABC Efflux Transporters P-gp and ...

    EPA Pesticide Factsheets

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

  20. The ABC and AUSSAT.

    ERIC Educational Resources Information Center

    McGarritty, Ian

    1985-01-01

    Discusses the Australian Broadcasting Corporation's (ABC) utilization of the AUSSAT telecommunications satellite to extend its television and radio transmission range to reach remote Australian audiences; the satellite's program gathering and interchange capabilities; and ABC's generation of other benefits to offset cost of satellite services.…

  1. eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

    PubMed Central

    Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

    2017-01-01

    The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

  2. DC-SIGN–expressing macrophages trigger activation of mannosylated IgM B-cell receptor in follicular lymphoma

    PubMed Central

    Amin, Rada; Mourcin, Frédéric; Uhel, Fabrice; Pangault, Céline; Ruminy, Philippe; Dupré, Loic; Guirriec, Marion; Marchand, Tony; Fest, Thierry; Lamy, Thierry

    2015-01-01

    Follicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM+ FL B cells activated a stronger BCR signaling network than IgG+ FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM+ FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN–dependent adhesion of highly mannosylated IgM+ FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN–expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets. PMID:26272216

  3. Towards depersonalized abacavir therapy: chemical modification eliminates HLA-B*57 : 01-restricted CD8+ T-cell activation.

    PubMed

    Naisbitt, Dean J; Yang, Emma L; Alhaidari, Mohammad; Berry, Neil G; Lawrenson, Alexandre S; Farrell, John; Martin, Philip; Strebel, Klaus; Owen, Andrew; Pye, Matthew; French, Neil S; Clarke, Stephen E; O'Neill, Paul M; Park, B Kevin

    2015-11-28

    Exposure to abacavir is associated with T-cell-mediated hypersensitivity reactions in individuals carrying human leukocyte antigen (HLA)-B57 : 01. To activate T cells, abacavir interacts directly with endogenous HLA-B57 : 01 and HLA-B57 : 01 expressed on the surface of antigen presenting cells. We have investigated whether chemical modification of abacavir can produce a molecule with antiviral activity that does not bind to HLA-B57 : 01 and activate T cells. An interdisciplinary laboratory study using samples from human donors expressing HLA-B57 : 01. Researchers were blinded to the analogue structures and modelling data. Sixteen 6-amino substituted abacavir analogues were synthesized. Computational docking studies were completed to predict capacity for analogue binding within HLA-B57 : 01. Abacavir-responsive CD8 clones were generated to study the association between HLA-B57 : 01 analogue binding and T-cell activation. Antiviral activity and the direct inhibitory effect of analogues on proliferation were assessed. Major histocompatibility complex class I-restricted CD8 clones proliferated and secreted IFNγ following abacavir binding to surface and endogenous HLA-B57 : 01. Several analogues retained antiviral activity and showed no overt inhibitory effect on proliferation, but displayed highly divergent antigen-driven T-cell responses. For example, abacavir and N-propyl abacavir were equally potent at activating clones, whereas the closely related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity. Docking abacavir analogues to HLA-B57 : 01 revealed a quantitative relationship between drug-protein binding and the T-cell response. These studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favourable antiviral profile. The in-silico model provides a tool to aid the design of safer antiviral agents that may not require a personalized medicines approach to therapy.

  4. Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

    PubMed

    Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

    2017-02-01

    The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells.

    PubMed

    Saulep-Easton, D; Vincent, F B; Quah, P S; Wei, A; Ting, S B; Croce, C M; Tam, C; Mackay, F

    2016-01-01

    Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory elements with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B-cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is critical for B-cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing regulatory B cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor transmembrane activator and cyclophilin ligand interactor. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL.

  6. Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.

    PubMed

    Hagn, Magdalena; Jahrsdörfer, Bernd

    2012-11-01

    B cells are generally believed to operate as producers of high affinity antibodies to defend the body against microorganisms, whereas cellular cytotoxicity is considered as an exclusive prerogative of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). In conflict with this dogma, recent studies have demonstrated that the combination of interleukin-21 (IL-21) and B-cell receptor (BCR) stimulation enables B cells to produce and secrete the active form of the cytotoxic serine protease granzyme B (GrB). Although the production of GrB by B cells is not accompanied by that of perforin as in the case of many other GrB-secreting cells, recent findings suggest GrB secretion by B cells may play a significant role in early antiviral immune responses, in the regulation of autoimmune responses, and in cancer immunosurveillance. Here, we discuss in detail how GrB-secreting B cells may influence a variety of immune processes. A better understanding of the role that GrB-secreting B cells are playing in the immune system may allow for the development and improvement of novel immunotherapeutic approaches against infectious, autoimmune and malignant diseases.

  7. Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

    PubMed

    Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

    2010-09-01

    Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

  8. SU-F-P-14: Oxygen Inhalation Should Be the Conventional Approach in the Treatment of Thoracic and Abdominal Cancer by Radiotherapy with Active Breathing Control (ABC)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gong, G; Guo, Y; Yin, Y

    Purpose: To investigate the feasibility and potential benefit of oxygen inhalation (OI) during radiotherapy applying an active breathing control (ABC) device, by analyzing the blood oxygen saturation (SpO2) and the instantaneous heart rate (IHR) variation in breath holding with OI and oxygen non-inhalation (ONI). Methods: The 27 healthy volunteers (16 males, 11 females) who were involved in this trial were all required to hold their breath for 10 times, non-inhaling and inhaling oxygen successively. The breath-holding time (BHT), rest time (RT), SpO2 and IHR under different oxygen status were recorded and compared. Results: The volunteers were divided into two groupsmore » according to SpO2 variations in breath-holding: group A (12 cases), with less than2% decline of SpO2; group B (15 cases), with a decline that surpassed 2%, and which could reach 3–6%. The BHT of group A, without inhaling oxygen, was significantly longer than that of group B (mean 33.77s Vs 30.51s, p<0.05); and was extended by 26.6% and 27.85%, after inhaling oxygen, in groups A and B, respectively. The SpO2 decreased in all volunteers during RT with ONI, to an extent that could reach up to 6%. The IHR of all volunteers showed the fast-slow-fast variation rule, and the oxygen had little effect. More than 70% of the volunteers stated that oxygen made them feel more comfortable and were more cooperative when ABC was used. Conclusion: The SpO2 declines during breath holding and RT could not be ignored while applying ABC, oxygen inhalation should become a conventional method with lengthening BHT and shortening RT, which yielded the benefit of improving the stability and reproducibility.« less

  9. Knowing the ABCs: A Comparative Effectiveness Study of Two Methods of Diabetes Education

    PubMed Central

    Naik, Aanand D.; Teal, Cayla R.; Rodriguez, Elisa; Haidet, Paul

    2011-01-01

    Objective To test an active-learning, empowerment approach to teaching patients about the “diabetes ABCs” (hemoglobin A1C, systolic blood pressure, and low density lipoprotein cholesterol). Methods 84 (97%) diabetic patients who participated in a randomized effectiveness trial of two clinic-based group educational methods and completed a post-intervention assessment. The empowerment arm participated in a group session that incorporated two educational innovations (a conceptual metaphor to foster understanding, and team-based learning methods to foster active learning). The traditional diabetes education arm received a didactic group session focused on self-management and educational materials about the diabetes ABCs. Participants in both arms received individual review of their current ABC values. Results A questionnaire evaluated knowledge, understanding, and recall of the diabetes ABCs was administered three months after enrollment in the study. At three months, participants in the empowerment group demonstrated greater understanding of the diabetes ABCs (P<.0001), greater knowledge of their own values (P<.0001), and greater knowledge of guideline-derived target goals for the ABCs compared with participants in the traditional arm (P<.0001). Conclusion An active-learning, empowerment-based approach applied to diabetes education can lead to greater understanding and knowledge retention. Practice Implications An empowerment approach to education can facilitate informed, activated patients and increase performance of self-management behaviors. PMID:21300516

  10. Poor concordance among nine immunohistochemistry classifiers of cell-of-origin for Diffuse Large B-cell Lymphoma: implications for therapeutic strategies

    PubMed Central

    Coutinho, Rita; Clear, Andrew James; Owen, Andrew; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; da Silva, Maria Gomes; Cabeçadas, José; Calaminici, Maria; Gribben, John G.

    2014-01-01

    Purpose The opportunity to improve therapeutic choices on the basis of molecular features of the tumour cells is on the horizon in Diffuse Large B-cell Lymphoma (DLBCL). Agents such as bortezomib exhibit selective activity against the poor outcome activated B-cell type DLBCL. In order for targeted therapies to succeed in this disease, robust strategies that segregate patients into molecular groups with high reliability are needed. While molecular studies are considered gold standard, several immunohistochemistry (IHC) algorithms have been published that claim to be able to stratify patients according to their cell-of-origin and to be relevant for patient outcome. However results are poorly reproducible by independent groups. Experimental design We investigated nine IHC algorithms for molecular classification in a dataset of DLBCL diagnostic biopsies, incorporating immunostaining for CD10, BCL6, BCL2, MUM1, FOXP1, GCET1 and LMO2. IHC profiles were assessed and agreed among three expert observers. A consensus matrix based on all scoring combinations and the number of subjects for each combination allowed to assess reliability. The survival impact of individual markers and classifiers was evaluated using Kaplan-Meier curves and the log-rank test. Results The concordance in patient’s classification across the different algorithms was low. Only 4% the tumors have been classified as GCB and 21% as ABC/non-GCB by all methods. None of the algorithms provided prognostic information in the R-CHOP treated cohort. Conclusion Further work is required to standardize IHC algorithms for DLBCL cell-of-origin classification for these to be considered reliable alternatives to molecular-based methods to be used for clinical decisions. PMID:24122791

  11. The ABC Model and its Applicability to Basal Angiosperms

    PubMed Central

    Soltis, Douglas E.; Chanderbali, André S.; Kim, Sangtae; Buzgo, Matyas; Soltis, Pamela S.

    2007-01-01

    Background Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular. Scope and Conclusions Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer ‘fading borders’ as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms. PMID:17616563

  12. Polyclonal activation of human lymphocytes in vitro-II. Reappraisal of T and B cell-specific mitogens.

    PubMed

    Dosch, H M; Schuurman, R K; Gelfand, E W

    1980-08-01

    The capacity of the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcus protein A (SpA) to induce B cell proliferation and differentiation was compared with the B cell mitogen, formalinized Staphylococcus aureus (STA). Lymphocyte subpopulations from normal donors and patients with various immunodeficiency diseases were studied. In the presence of the T cell mitogens, irradiated T cells were capable of providing a helper cell activity that enabled co-cultured B lymphocytes to proliferate in response to these mitogens and to differentiate into IgM-secreting (direct) hemolytic plaque-forming cells (PFC). In the PFC response, radioresistant T-helper and radiosensitive T-suppressor cell activities could be demonstrated. T-suppressor cell activity outweighed helper activity only in nonirradiated co-cultures stimulated with Con A. Patients with severe combined immunodeficiency lacked mitogen-induced helper T cells, whereas patients with various forms of humoral immune deficiency were normal in this respect. These findings and the tissue distribution of the helper activity is aquired early in post-thymic T cell differentiation. The data suggest that experiments with cell lineage-specific lymphocyte mitogens should be considered in the context of more complex cell-cell interactions.

  13. DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells.

    PubMed

    Camponeschi, Alessandro; Todi, Laura; Cristofoletti, Cristina; Lazzeri, Cristina; Carbonari, Maurizio; Mitrevski, Milica; Marrapodi, Ramona; Del Padre, Martina; Fiorilli, Massimo; Casato, Milvia; Visentini, Marcella

    2018-06-01

    The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21 low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  14. HLA-B*57:01 allele prevalence in HIV-infected North American subjects and the impact of allele testing on the incidence of abacavir-associated hypersensitivity reaction in HLA-B*57:01-negative subjects.

    PubMed

    Small, Catherine Butkus; Margolis, David A; Shaefer, Mark S; Ross, Lisa L

    2017-04-11

    The presence of the HLA-B*57:01 allele in HIV-infected subjects is associated with a higher risk of abacavir-associated hypersensitivity reaction (ABC HSR). HLA-B*57:01 allele prevalence varies in different populations, but HLA-B*57:01 testing with immunological confirmation has had a negative predictive value for ABC HSR between 97 and 100%. In the ASSURE study (EPZ113734), the HLA-B*57:01 prevalence in virologically suppressed, antiretroviral treatment-experienced, HIV-infected subjects from the United States, including Puerto Rico, was assessed. Three hundred eighty-five subjects were screened; 13 were HLA-B*57:01 positive and 372 were negative. Only HLA-B*57:01-negative, abacavir-naive subjects were eligible to enroll into the ASSURE trial. Eleven of the 13 subjects who possessed the HLA-B*57:01 allele were white, the other 2 were African-American. There was no geographic clustering of HLA-B*57:01-positive subjects, and the incidence correlated roughly with those states with the greatest numbers of subjects screened. Similarly, there was no statistically significant correlation between subjects who possessed or lacked the allele and age, gender, ethnicity or CD4+ T-cell numbers. The incidence of ABC HSR following abacavir initiation was also evaluated. Only 1 of 199 HLA-B*57:01-negative subjects (an African-American male) randomized to receive abacavir-containing treatment developed symptoms consistent with suspected ABC HSR; ABC HSR was not immunologically confirmed. These findings confirm the utility of HLA-B*57:01 allele testing to reduce the frequency of ABC HSR. The prevalence of HLA-B*57:01 positivity was higher in white than in African-American subjects. In HLA-B*57:01-negative subjects, suspected ABC HSR is very rare, but should lead to discontinuation of abacavir when ABC HSR cannot be definitively excluded from the differential diagnosis. The ASSURE (EPZ113734) study was registered on ClinicalTrials.gov registration on April 8th 2010 and the

  15. The interaction of gut microbes with host ABC transporters

    PubMed Central

    Mercado-Lubo, Regino

    2010-01-01

    ATP binding cassette (ABC) transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, secretion and toxicity of xenobiotics. In addition to their essential function in drug resistance, there is also emerging evidence documenting the important role ABC transporters play in tissue defense. In this respect, the gastrointestinal tract represents a critical vanguard of defense against oral exposure of drugs while at the same time functions as a physical barrier between the lumenal contents (including bacteria) and the intestinal epithelium. Given emerging evidence suggesting that multidrug resistance protein (MDR) plays an important role in host-bacterial interactions in the gastrointestinal tract, this review will discuss the interplay between MDR of the intestinal epithelial cell barrier and gut microbes in health and disease. In particular, we will explore host-microbe interactions involving three apically restricted ABC transporters of the intestinal epithelium; P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cystic fibrosis transmembrane regulator (CFTR). PMID:21327038

  16. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

    PubMed Central

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

  17. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

    PubMed

    Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

    2014-07-29

    ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

  18. Substrate stiffness governs the initiation of B cell activation by the concerted signaling of PKCβ and focal adhesion kinase

    PubMed Central

    Shaheen, Samina; Wan, Zhengpeng; Li, Zongyu; Chau, Alicia; Li, Xinxin; Zhang, Shaosen; Liu, Yang; Yi, Junyang; Zeng, Yingyue; Wang, Jing; Chen, Xiangjun; Xu, Liling; Chen, Wei; Wang, Fei; Lu, Yun; Zheng, Wenjie; Shi, Yan; Sun, Xiaolin; Li, Zhanguo; Xiong, Chunyang; Liu, Wanli

    2017-01-01

    The mechanosensing ability of lymphocytes regulates their activation in response to antigen stimulation, but the underlying mechanism remains unexplored. Here, we report that B cell mechanosensing-governed activation requires BCR signaling molecules. PMA-induced activation of PKCβ can bypass the Btk and PLC-γ2 signaling molecules that are usually required for B cells to discriminate substrate stiffness. Instead, PKCβ-dependent activation of FAK is required, leading to FAK-mediated potentiation of B cell spreading and adhesion responses. FAK inactivation or deficiency impaired B cell discrimination of substrate stiffness. Conversely, adhesion molecules greatly enhanced this capability of B cells. Lastly, B cells derived from rheumatoid arthritis (RA) patients exhibited an altered BCR response to substrate stiffness in comparison with healthy controls. These results provide a molecular explanation of how initiation of B cell activation discriminates substrate stiffness through a PKCβ-mediated FAK activation dependent manner. DOI: http://dx.doi.org/10.7554/eLife.23060.001 PMID:28755662

  19. RED CELL STROMA PROTEIN RICH IN VITAMIN B12 DURING ACTIVE REGENERATION

    PubMed Central

    Whipple, G. H.; Robscheit-Robbins, F. S.; Bale, W. F.

    1955-01-01

    During active blood regeneration in anemia in dogs an increase occurs in the stroma protein of the red cells. When vitamin B12 with radioactive cobalt is given at the start of this blood regeneration one finds concentration of labeled B 12 in the stroma protein but not in the hemoglobin. After the acute phase of red cell regeneration is ended the concentration of B12 in stroma protein falls rapidly to very low levels within 2 weeks. Subsequent episodes of red blood cell regeneration seems not to cause remobilization of radioactive cobalt into red cells from other body stores. It appears that the vitamin B12 is a factor of importance in the first steps of stroma protein formation in the first few days of the life of the red cell in the dog. This response in dogs and the response in pernicious anemia to vitamin B12 may have some points in common. Distribution of the B12-radioactive cobalt in the organs and tissues at autopsy has been recorded. Some very suggestive localizations were noted and some variation 1 week and 7 weeks after B12 injections. Radioactive cobalt escapes in the urine during the weeks following B12 injections. PMID:13271685

  20. HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib.

    PubMed

    Yang, Guang; Buhrlage, Sara J; Tan, Li; Liu, Xia; Chen, Jie; Xu, Lian; Tsakmaklis, Nicholas; Chen, Jiaji G; Patterson, Christopher J; Brown, Jennifer R; Castillo, Jorge J; Zhang, Wei; Zhang, Xiaofeng; Liu, Shuai; Cohen, Philip; Hunter, Zachary R; Gray, Nathanael; Treon, Steven P

    2016-06-23

    Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. © 2016 by The American Society of Hematology.

  1. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

    PubMed

    Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi

    2017-12-01

    Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    PubMed

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  3. Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

    PubMed

    Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  4. Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

    PubMed

    Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

    2017-06-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. Mechanisms of B cell activation in patients with acquired immunodeficiency syndrome and related disorders. Contribution of antibody-producing B cells, of Epstein-Barr virus-infected B cells, and of immunoglobulin production induced by human T cell lymphotropic virus, type III/lymphadenopathy-associated virus.

    PubMed Central

    Yarchoan, R; Redfield, R R; Broder, S

    1986-01-01

    Patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) have hyperimmunoglobulinemia and increased numbers of circulating immunoglobulin-secreting cells. In this paper, we studied the basis for this B cell hyperactivity. Limiting dilution studies of B cells from seven patients with ARC and four with AIDS revealed that some B cells spontaneously produced antibodies to human T cell lymphotropic virus, type III/lymphadenopathy-associated virus (HTLV-III/LAV) (39:10(6) and 7:10(6) B cells, respectively), suggesting that chronic antigenic stimulation by HTLV-III/LAV was one contributing factor. The patients also had an increased number of spontaneously outgrowing B cells than did normals (6:10(6) vs. less than 2:10(6) B cells), suggesting that they had an increased number of Epstein-Barr virus (EBV)-infected B cells. However, fewer B cells from patients were immortalized by exogenously added EBV than were B cells from normals. In additional studies, HTLV-III/LAV induced immunoglobulin secretion (mean 2,860 ng/ml) by peripheral blood mononuclear cells from normals; this HTLV-III/LAV-induced immunoglobulin secretion required the presence of both B and T cells. Thus, antigenic stimulation by HTLV-III/LAV, increased numbers of EBV-infected B cells, and HTLV-III/LAV-induced T cell-dependent B cell activation all contribute to the B cell hyperactivity in patients with HTLV-III/LAV disease. PMID:3016028

  6. The Plasma Concentration of the B Cell Activating Factor Is Increased in Children With Acute Malaria

    PubMed Central

    Nduati, Eunice; Gwela, Agnes; Karanja, Henry; Mugyenyi, Cleopatra; Langhorne, Jean; Marsh, Kevin

    2011-01-01

    Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells. PMID:21849293

  7. Gold nanoparticles induce transcriptional activity of NF-κB in a B-lymphocyte cell line

    NASA Astrophysics Data System (ADS)

    Sharma, Monita; Salisbury, Richard L.; Maurer, Elizabeth I.; Hussain, Saber M.; Sulentic, Courtney E. W.

    2013-04-01

    Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKβ were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects

  8. Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

    PubMed Central

    Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

    2005-01-01

    Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

  9. Downregulation of STAT3/NF-κB potentiates gemcitabine activity in pancreatic cancer cells.

    PubMed

    Gong, Jingjing; Muñoz, Amanda R; Pingali, Subramanya; Payton-Stewart, Florastina; Chan, Daniel E; Freeman, James W; Ghosh, Rita; Kumar, Addanki P

    2017-02-01

    There is an unmet need to develop new agents or strategies against therapy resistant pancreatic cancer (PanCA). Recent studies from our laboratory showed that STAT3 negatively regulates NF-κB and that inhibition of this crosstalk using Nexrutine® (Nx) reduces transcriptional activity of COX-2. Inhibition of these molecular interactions impedes pancreatic cancer cell growth as well as reduces fibrosis in a preclinical animal model. Nx is an extract derived from the bark of Phellodendron amurense and has been utilized in traditional Chinese medicine as antidiarrheal, astringent, and anti-inflammatory agent for centuries. We hypothesized that "Nx-mediated inhibition of survival molecules like STAT3 and NF-κB in pancreatic cancer cells will improve the efficacy of the conventional chemotherapeutic agent, gemcitabine (GEM)." Therefore, we explored the utility of Nx, one of its active constituents berberine and its derivatives, to enhance the effects of GEM. Using multiple human pancreatic cancer cells we found that combination treatment with Nx and GEM resulted in significant alterations of proteins in the STAT3/NF-κB signaling axis culminating in growth inhibition in a synergistic manner. Furthermore, GEM resistant cells were more sensitive to Nx treatment than their parental GEM-sensitive cells. Interestingly, although berberine, the Nx active component used, and its derivatives were biologically active in GEM sensitive cells they did not potentiate GEM activity when used in combination. Taken together, these results suggest that the natural extract, Nx, but not its active component, berberine, has the potential to improve GEM sensitivity, perhaps by down regulating STAT3/NF-κB signaling. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

    PubMed Central

    Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

    2016-01-01

    The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

  11. EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

    PubMed

    Takada, Honami; Imadome, Ken-Ichi; Shibayama, Haruna; Yoshimori, Mayumi; Wang, Ludan; Saitoh, Yasunori; Uota, Shin; Yamaoka, Shoji; Koyama, Takatoshi; Shimizu, Norio; Yamamoto, Kouhei; Fujiwara, Shigeyoshi; Miura, Osamu; Arai, Ayako

    2017-01-01

    Epstein-Barr virus (EBV) has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV). However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

  12. EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells

    PubMed Central

    Shibayama, Haruna; Yoshimori, Mayumi; Wang, Ludan; Saitoh, Yasunori; Uota, Shin; Yamaoka, Shoji; Koyama, Takatoshi; Shimizu, Norio; Yamamoto, Kouhei; Fujiwara, Shigeyoshi; Miura, Osamu

    2017-01-01

    Epstein–Barr virus (EBV) has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV). However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells. PMID:28346502

  13. Exosomes derived from gastric cancer cells activate NF-κB pathway in macrophages to promote cancer progression.

    PubMed

    Wu, Lijun; Zhang, Xu; Zhang, Bin; Shi, Hui; Yuan, Xiao; Sun, Yaoxiang; Pan, Zhaoji; Qian, Hui; Xu, Wenrong

    2016-09-01

    Exosomes are nano-sized membrane vesicles secreted by both normal and cancer cells. Emerging evidence indicates that cancer cells derived exosomes contribute to cancer progression through the modulation of tumor microenvironment. However, the effects of exosomes derived from gastric cancer cells on macrophages are not well understood. In this study, we investigated the biological role of gastric cancer cells derived exosomes in the activation of macrophages. We demonstrated that gastric cancer cells derived exosomes activated macrophages to express increased levels of proinflammatory factors, which in turn promoted tumor cell proliferation and migration. In addition, gastric cancer cells derived exosomes remarkably upregulated the phosphorylation of NF-κB in macrophages. Inhibiting the activation of NF-κB reversed the upregulation of proinflammatory factors in macrophages and blocked their promoting effects on gastric cancer cells. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. In conclusion, our results suggest that gastric cancer cells derived exosomes stimulate the activation of NF-κB pathway in macrophages to promote cancer progression, which provides a potential therapeutic approach for gastric cancer by interfering with the interaction between exosomes and macrophages in tumor microenvironment.

  14. Activation of the Endoplasmic Reticulum Stress-Associated Transcription Factor X Box-Binding Protein-1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive B-Cell Lymphomas with Prognostic Implications

    PubMed Central

    Balague, Olga; Mozos, Ana; Martinez, Daniel; Hernandez, Luis; Colomo, Lluis; Mate, Jose Luis; Teruya-Feldstein, Julie; Lin, Oscar; Campo, Elias; Lopez-Guillermo, Armando; Martinez, Antonio

    2009-01-01

    X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4+/Bcl-6−/Pax-5− B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas. PMID:19389935

  15. EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

    PubMed Central

    Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

    2013-01-01

    The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

  16. Discovery of a Brown Dwarf Companion to Gliese 570ABC: A 2MASS T Dwarf Significantly Cooler than Gliese 229B.

    PubMed

    Burgasser; Kirkpatrick; Cutri; McCallon; Kopan; Gizis; Liebert; Reid; Brown; Monet; Dahn; Beichman; Skrutskie

    2000-03-01

    We report the discovery of a widely separated (258&farcs;3+/-0&farcs;4) T dwarf companion to the Gl 570ABC system. This new component, Gl 570D, was initially identified from the Two Micron All-Sky Survey. Its near-infrared spectrum shows the 1.6 and 2.2 µm CH4 absorption bands characteristic of T dwarfs, while its common proper motion with the Gl 570ABC system confirms companionship. Gl 570D (MJ=16.47+/-0.07) is nearly a full magnitude dimmer than the only other known T dwarf companion, Gl 229B, and estimates of L=&parl0;2.8+/-0.3&parr0;x10-6 L middle dot in circle and Teff=750+/-50 K make it significantly cooler and less luminous than any other known brown dwarf companion. Using evolutionary models by Burrows et al. and an adopted age of 2-10 Gyr, we derive a mass estimate of 50+/-20 MJup for this object.

  17. ABC estimation of unit costs for emergency department services.

    PubMed

    Holmes, R L; Schroeder, R E

    1996-04-01

    Rapid evolution of the health care industry forces managers to make cost-effective decisions. Typical hospital cost accounting systems do not provide emergency department managers with the information needed, but emergency department settings are so complex and dynamic as to make the more accurate activity-based costing (ABC) system prohibitively expensive. Through judicious use of the available traditional cost accounting information and simple computer spreadsheets. managers may approximate the decision-guiding information that would result from the much more costly and time-consuming implementation of ABC.

  18. B cell-stimulatory cytokines and markers of immune activation are elevated several years prior to the diagnosis of systemic AIDS-associated non-Hodgkin B cell lymphoma

    PubMed Central

    Breen, Elizabeth Crabb; Hussain, Shehnaz K.; Magpantay, Larry; Jacobson, Lisa P.; Detels, Roger; Rabkin, Charles S.; Kaslow, Richard A.; Variakojis, Daina; Bream, Jay H.; Rinaldo, Charles R.; Ambinder, Richard F.; Martínez-Maza, Otoniel

    2011-01-01

    Background The risk of developing non-Hodgkin lymphoma (NHL) is greatly increased in HIV infection. The aim of this study was to determine if elevated serum levels of molecules associated with B cell activation precede the diagnosis of AIDS-associated NHL. Methods Serum levels of B cell activation-associated molecules, interleukin-6 (IL6), interleukin-10 (IL10), soluble CD23 (sCD23), soluble CD27 (sCD27), soluble CD30 (sCD30), C-reactive protein (CRP), and IgE were determined in 179 NHL cases and HIV+ controls in the Multicenter AIDS Cohort Study, collected at up to three time points per subject, 0–5 years prior to AIDS-NHL diagnosis. Results Serum IL6, IL10, CRP, sCD23, sCD27, and sCD30 levels were all significantly elevated in the AIDS-NHL group, when compared to HIV+ controls or to AIDS controls, after adjusting for CD4 T cell number. Elevated serum levels of B cell activation-associated molecules were seen to be associated with the development of systemic (non-CNS) NHL, but not with the development of primary CNS lymphoma. Conclusions Levels of certain B cell stimulatory cytokines and molecules associated with immune activation are elevated for several years preceding the diagnosis of systemic AIDS-NHL. This observation is consistent with the hypothesis that chronic B cell activation contributes to the development of these hematologic malignancies. Impact Marked differences in serum levels of several molecules are seen for several years pre-diagnosis in those who eventually develop AIDS-NHL. Some of these molecules may serve as candidate biomarkers and provide valuable information to better define the etiology of NHL. PMID:21527584

  19. Similar disturbances in B cell activity and regulatory T cell function in Henoch-Schonlein purpura and systemic lupus erythematosus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beale, M.G.; Nash, G.S.; Bertovich, M.J.

    1982-01-01

    The immunoglobulin synthesizing activities of peripheral mononuclear cells (MNC) from five patients with Henoch-Schonlein purpura (HSP) and eight patients with active systemic lupus erythematosus (SLE) were compared. Cumulative amounts of IgM, IgG, and IgA synthesized and secreted by unstimulated and PWM-stimulated patient cells over a 12-day period were determied in a solid-phase radioimmunoassay. In unstimulated control cultures mean rates of IgM, IgG, and IgA synthesis were less than 250 ng/ml. The synthetic activities of patient MNC were markedly increased. In HSP cultures IgA was the major immunoglobulin class produced (2810 x/divide 1.33 ng/ml) followed by IgG (1754 x/divide 1.32 ng/ml)more » and IgM (404 x/divide 1.16 ng/ml). In SLE cultures IgA and IgG syntheses were equally elevated (4427 x/divide 1.20 and 4438 x/divide 1.49 ng/ml, respectively) whereas IgM synthesis averaged 967 x/divide 1.66 ng/ml. PWM stimulation of pateient MNC caused a sharp decline in the synthesis of all three immunoglobulin classes. After T cell depletion B cell-enriched fractions from HSP and SLE patients maintained high levels of IgA and IgG synthesis that were inhibited by PWM and by normal allogeneic but not autologous T cells. In PWM-stimulted co-cultures, patient T cells nonspecifically suppressed the synthetic activities of autologous and control B cells. in contrast patient B cells achieved normal levels of immunoglobulin synthesis when cultured with control T cells plus PWM. In longitudinal studies patient B and T cell disturbances persisted despite clinical improvement.« less

  20. Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

    PubMed

    Chung, Inyoung; Hah, Young-Sool; Ju, SunMi; Kim, Ji-Hye; Yoo, Woong-Sun; Cho, Hee-Young; Yoo, Ji-Myong; Seo, Seong-Wook; Choi, Wan-Sung; Kim, Seong-Jae

    2017-07-01

    Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm 2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm 2 ) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

  1. B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis.

    PubMed

    Al Tabaa, Yassine; Tuaillon, Edouard; Jeziorski, Eric; Ouedraogo, David Eric; Bolloré, Karine; Rubbo, Pierre-Alain; Foulongne, Vincent; Rodière, Michel; Vendrell, Jean-Pierre

    2011-09-01

    Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. In vitro B-cell polyclonal activation induced 8000-24,000 BZLF1- and 1000-3000gp350-SCs/10(6) B cells, respectively. These data suggest that only 11.1-19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20-120 and 10-30/10(6) B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1-10.2% of CD19(+) B cells). The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1.

    PubMed

    Jeffery, Hannah C; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R; Adams, David H; Klenerman, Paul; Oo, Ye H

    2016-05-01

    Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  3. Anti-Campylobacter activity of resveratrol and an extract from waste Pinot noir grape skins and seeds, and resistance of Camp. jejuni planktonic and biofilm cells, mediated via the CmeABC efflux pump.

    PubMed

    Klančnik, A; Šikić Pogačar, M; Trošt, K; Tušek Žnidarič, M; Mozetič Vodopivec, B; Smole Možina, S

    2017-01-01

    To define anti-Campylobacter jejuni activity of an extract from waste skins and seeds of Pinot noir grapes (GSS), resveratrol and possible resistance mechanisms, and the influence of these on Camp. jejuni morphology. Using gene-specific knock-out Camp. jejuni mutants and an efflux pump inhibitor, we showed CmeABC as the most active efflux pump for extrusion across the outer membrane of GSS extract and resveratrol. Using polystyrene surface and pig small intestine epithelial (PSI) and human foetal small intestine (H4) cell lines, GSS extract shows an efficient inhibition of adhesion of Camp. jejuni to these abiotic and biotic surfaces. Low doses of GSS extract can inhibit Camp. jejuni adhesion to polystyrene surfaces and to PSI and H4 cells, and can thus modulate Camp. jejuni invasion and intracellular survival. An understanding of the activities of GSS extract and resveratrol as bacterial growth inhibitors and the specific mechanisms of cell accumulation is crucial for our understanding of Camp. jejuni resistance. GSS extract inhibition of Camp. jejuni adhesion to abiotic and biotic surfaces provides a further step towards the application of new innovative strategies to control Campylobacter contamination and infection via the food chain. © 2016 The Society for Applied Microbiology.

  4. INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia.

    PubMed

    Jin, Hongjun; Yang, Liyuan; Wang, Lu; Yang, Zailin; Zhan, Qian; Tao, Yao; Zou, Qin; Tang, Yuting; Xian, Jingrong; Zhang, Shuaishuai; Jing, Yipei; Zhang, Ling

    2018-01-17

    Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P 2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan-Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells

  5. Neutrophil mediated IFN activation in the bone marrow alters B cell development in human and murine SLE1

    PubMed Central

    Palanichamy, Arumugam; Bauer, Jason W; Yalavarthi, Srilakshmi; Meednu, Nida; Barnard, Jennifer; Owen, Teresa; Cistrone, Christopher; Bird, Anna; Rabinovich, Alfred; Nevarez, Sarah; Knight, Jason S.; Dedrick, Russell; Rosenberg, Alexander; Wei, Chungwen; Rangel-Moreno, Javier; Liesveld, Jane; Sanz, Inaki; Baechler, Emily; Kaplan, Mariana J.; Anolik, Jennifer H

    2014-01-01

    Inappropriate activation of type I interferon (IFN) plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). Here we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood (PB) and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre B cells suggesting an inhibition in early B cell development and an expansion of B cells at the transitional (T2) stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type I IFN receptor. BM neutrophils were abundant, responsive to and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil mediated IFN activation and alterations in B cell ontogeny and selection. PMID:24379124

  6. Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses.

    PubMed

    Jahnmatz, Maja; Kesa, Gun; Netterlid, Eva; Buisman, Anne-Marie; Thorstensson, Rigmor; Ahlborg, Niklas

    2013-05-31

    B-cell responses after infection or vaccination are often measured as serum titers of antigen-specific antibodies. Since this does not address the aspect of memory B-cell activity, it may not give a complete picture of the B-cell response. Analysis of memory B cells by ELISpot is therefore an important complement to conventional serology. B-cell ELISpot was developed more than 25 years ago and many assay protocols/reagents would benefit from optimization. We therefore aimed at developing an optimized B-cell ELISpot for the analysis of vaccine-induced human IgG-secreting memory B cells. A protocol was developed based on new monoclonal antibodies to human IgG and biotin-avidin amplification to increase the sensitivity. After comparison of various compounds commonly used to in vitro-activate memory B cells for ELISpot analysis, the TLR agonist R848 plus interleukin (IL)-2 was selected as the most efficient activator combination. The new protocol was subsequently compared to an established protocol, previously used in vaccine studies, based on polyclonal antibodies without biotin avidin amplification and activation of memory B-cells using a mix of antigen, CpG, IL-2 and IL-10. The new protocol displayed significantly better detection sensitivity, shortened the incubation time needed for the activation of memory B cells and reduced the amount of antigen required for the assay. The functionality of the new protocol was confirmed by analyzing specific memory B cells to five different antigens, induced in a limited number of subjects vaccinated against tetanus, diphtheria and pertussis. The limited number of subjects did not allow for a direct comparison with other vaccine studies. Optimization of the B-cell ELISpot will facilitate an improved analysis of IgG-secreting B cells in vaccine studies. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

    PubMed

    Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S; McMurray, John S; Gandhi, Varsha

    2016-01-19

    The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

  8. Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

    PubMed

    Wagner, Tristan; Koch, Jürgen; Ermler, Ulrich; Shima, Seigo

    2017-08-18

    In methanogenic archaea, the carbon dioxide (CO 2 ) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

    PubMed Central

    Foubert, Philippe; Silvestre, Jean-Sébastien; Souttou, Boussad; Barateau, Véronique; Martin, Coralie; Ebrahimian, Téni G.; Leré-Déan, Carole; Contreres, Jean Olivier; Sulpice, Eric; Levy, Bernard I.; Plouët, Jean; Tobelem, Gérard; Le Ricousse-Roussanne, Sophie

    2007-01-01

    Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy. PMID:17510705

  10. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

    PubMed Central

    Tzeng, E; Billiar, T R; Robbins, P D; Loftus, M; Stuehr, D J

    1995-01-01

    Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8524846

  11. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  12. The short version of the Activities-specific Balance Confidence (ABC) scale: its validity, reliability, and relationship to balance impairment and falls in older adults.

    PubMed

    Schepens, Stacey; Goldberg, Allon; Wallace, Melissa

    2010-01-01

    A shortened version of the ABC 16-item scale (ABC-16), the ABC-6, has been proposed as an alternative balance confidence measure. We investigated whether the ABC-6 is a valid and reliable measure of balance confidence and examined its relationship to balance impairment and falls in older adults. Thirty-five community-dwelling older adults completed the ABC-16, including the 6 questions of the ABC-6. They also completed the following clinical balance tests: unipedal stance time (UST), functional reach (FR), Timed Up and Go (TUG), and maximum step length (MSL). Participants reported 12-month falls history. Balance confidence on the ABC-6 was significantly lower than on the ABC-16, however scores were highly correlated. Fallers reported lower balance confidence than non-fallers as measured by the ABC-6 scale, but confidence did not differ between the groups with the ABC-16. The ABC-6 significantly correlated with all balance tests assessed and number of falls. The ABC-16 significantly correlated with all balance tests assessed, but not with number of falls. Test-retest reliability for the ABC-16 and ABC-6 was good to excellent. The ABC-6 is a valid and reliable measure of balance confidence in community-dwelling older adults, and shows stronger relationships to falls than does the ABC-16. The ABC-6 may be a more useful balance confidence assessment tool than the ABC-16. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  13. The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

    PubMed

    Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

    2011-10-28

    B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

  14. Invariant NKT cells provide innate and adaptive help for B cells

    PubMed Central

    Vomhof-DeKrey, Emilie E.; Yates, Jennifer; Leadbetter, Elizabeth A.

    2014-01-01

    B cells rely on CD4+ T cells helper signals to optimize their responses to T-dependent antigens. Recently another subset of T cells has been identified which provides help for B cells, invariant natural killer T (iNKT) cells. INKT cells are unique because they provide both innate and adaptive forms of help to B cells, with divergent outcomes. iNKT cells are widely distributed throughout the spleen at rest, consolidate in the marginal zone of the spleen early after activation, and are later found in germinal centers. Understanding the activation requirements for iNKT cells has led to the development of glycolipid containing nanoparticles which efficiently activate iNKT cells, enhance their cooperation with B cells, and which hold promise for vaccine development. PMID:24514004

  15. Social Experiences of Beginning Braille Readers in Literacy Activities: Qualitative and Quantitative Findings of the ABC Braille Study

    ERIC Educational Resources Information Center

    Sacks, Sharon Z.; Kamei-Hannan, Cheryl; Erin, Jane N.; Barclay, Lizbeth; Sitar, Debbie

    2009-01-01

    This mixed-design investigation examined the social experiences of beginning braille readers who were initially taught contracted or alphabetic braille in literacy activities as part of the ABC Braille Study. No differences in the quality or quantity of social experiences were found between the two groups over time. (Contains 4 tables.)

  16. Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

    PubMed

    Kihira, T; Kawanishi, H

    1995-08-01

    The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

  17. Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

    PubMed Central

    Stengel, Kristy R.; Barnett, Kelly R.; Wang, Jing; Liu, Qi; Hodges, Emily; Hiebert, Scott W.; Bhaskara, Srividya

    2017-01-01

    Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/−Mb1-Cre+/− mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/− bone marrow. For Hdac3Δ/− B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/− progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells. PMID:28739911

  18. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Sang-Myeong; Kleiboeker, Steven B.

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradationmore » of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.« less

  19. CD22 Promotes B-1b Cell Responses to T Cell-Independent Type 2 Antigens.

    PubMed

    Haas, Karen M; Johnson, Kristen L; Phipps, James P; Do, Cardinal

    2018-03-01

    CD22 (Siglec-2) is a critical regulator of B cell activation and survival. CD22 -/- mice generate significantly impaired Ab responses to T cell-independent type 2 (TI-2) Ags, including haptenated Ficoll and pneumococcal polysaccharides, Ags that elicit poor T cell help and activate BCR signaling via multivalent epitope crosslinking. This has been proposed to be due to impaired marginal zone (MZ) B cell development/maintenance in CD22 -/- mice. However, mice expressing a mutant form of CD22 unable to bind sialic acid ligands generated normal TI-2 Ab responses, despite significantly reduced MZ B cells. Moreover, mice treated with CD22 ligand-binding blocking mAbs, which deplete MZ B cells, had little effect on TI-2 Ab responses. We therefore investigated the effects of CD22 deficiency on B-1b cells, an innate-like B cell population that plays a key role in TI-2 Ab responses. B-1b cells from CD22 -/- mice had impaired BCR-induced proliferation and significantly increased intracellular Ca 2+ concentration responses following BCR crosslinking. Ag-specific B-1b cell expansion and plasmablast differentiation following TI-2 Ag immunization was significantly impaired in CD22 -/- mice, consistent with reduced TI-2 Ab responses. We generated CD22 -/- mice with reduced CD19 levels (CD22 -/- CD19 +/- ) to test the hypothesis that augmented B-1b cell BCR signaling in CD22 -/- mice contributes to impaired TI-2 Ab responses. BCR-induced proliferation and intracellular Ca 2+ concentration responses were normalized in CD22 -/- CD19 +/- B-1b cells. Consistent with this, TI-2 Ag-specific B-1b cell expansion, plasmablast differentiation, survival, and Ab responses were rescued in CD22 -/- CD19 +/- mice. Thus, CD22 plays a critical role in regulating TI-2 Ab responses through regulating B-1b cell signaling thresholds. Copyright © 2018 by The American Association of Immunologists, Inc.

  20. Early Observations of the Type Ia Supernova iPTF 16abc

    NASA Astrophysics Data System (ADS)

    Miller, Adam; iPTF Collaboration

    2018-01-01

    Early observations of Type Ia supernovae (SNe) provide a unique probe of their progenitor systems and explosion physics. Here, we report the intermediate Palomar Transient Factory (iPTF) discovery of an extraordinarily young SN Ia, iPTF 16abc. By fitting a power law to our early light curve, we infer that first light for the SN only occurred 0.15 +0.15-0.07 d before our first detection. In the ~24 hr after discovery, iPTF 16abc rose by ~2 mag, following a near-linear rise in flux for ~3 d. Strong C II absorption is detected in the early spectra of iPTF 16abc, before disappearing after ~7 d. Unlike the extensively-observed Type Ia SN 2011fe, the (B-V)_0 colors of iPTF 16abc are blue and nearly constant in the days after explosion. We show that our early observations of iPTF 16abc cannot be explained by either SN shock breakout and the associated, subsequent cooling, or the SN ejecta colliding with a stellar companion. Instead, we argue that the early characteristics of iPTF 16abc, including: (i) the rapid, near-linear rise, (ii) the non-evolving blue colors, and (iii) the strong absorption from ionized carbon, are the result of either vigorous mixing of radioactive-Ni in the SN ejecta, or ejecta interaction with diffuse material, or a combination of the two. In the next few years, dozens of very young normal SNe Ia will be discovered, and observations similar to those presented here will constrain the white dwarf explosion mechanism.

  1. ABC's of Construction. Final Report.

    ERIC Educational Resources Information Center

    Greater Baton Rouge Chamber of Commerce, LA.

    The ABC's of Construction project was a demonstration project designed to integrate basic skills training with an industry-developed vocational-craft training program. The program was located at the central training facility of the Pelican Chapter of Associated Builders and Contractors (ABC), an organization made up of nearly 300 member companies…

  2. Statistical Hypothesis Testing in Intraspecific Phylogeography: NCPA versus ABC

    PubMed Central

    Templeton, Alan R.

    2009-01-01

    Nested clade phylogeographic analysis (NCPA) and approximate Bayesian computation (ABC) have been used to test phylogeographic hypotheses. Multilocus NCPA tests null hypotheses, whereas ABC discriminates among a finite set of alternatives. The interpretive criteria of NCPA are explicit and allow complex models to be built from simple components. The interpretive criteria of ABC are ad hoc and require the specification of a complete phylogeographic model. The conclusions from ABC are often influenced by implicit assumptions arising from the many parameters needed to specify a complex model. These complex models confound many assumptions so that biological interpretations are difficult. Sampling error is accounted for in NCPA, but ABC ignores important sources of sampling error that creates pseudo-statistical power. NCPA generates the full sampling distribution of its statistics, but ABC only yields local probabilities, which in turn make it impossible to distinguish between a good fitting model, a non-informative model, and an over-determined model. Both NCPA and ABC use approximations, but convergences of the approximations used in NCPA are well defined whereas those in ABC are not. NCPA can analyze a large number of locations, but ABC cannot. Finally, the dimensionality of tested hypothesis is known in NCPA, but not for ABC. As a consequence, the “probabilities” generated by ABC are not true probabilities and are statistically non-interpretable. Accordingly, ABC should not be used for hypothesis testing, but simulation approaches are valuable when used in conjunction with NCPA or other methods that do not rely on highly parameterized models. PMID:19192182

  3. Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

    PubMed Central

    Martorana, Alessandra M.; Benedet, Mattia; Maccagni, Elisa A.; Sperandeo, Paola; Villa, Riccardo; Dehò, Gianni

    2016-01-01

    ABSTRACT The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCE The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a

  4. IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity

    PubMed Central

    Simonetti, Giorgia; Carette, Amanda; Silva, Kathryn; Wang, Haowei; De Silva, Nilushi S.; Heise, Nicole; Siebel, Christian W.; Shlomchik, Mark J.

    2013-01-01

    The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity. PMID:24323359

  5. Murine Lupus Susceptibility Locus Sle2 Activates DNA-Reactive B Cells through Two Sub-Loci with Distinct Phenotypes

    PubMed Central

    Zeumer, Leilani; Sang, Allison; Niu, Haitao; Morel, Laurence

    2010-01-01

    The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B cell differentiation which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b, and Sle2c. Sle2 also enhances the breach of B cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that play a critical role in B cell tolerance. PMID:21270826

  6. Do You Know Your ABC?

    ERIC Educational Resources Information Center

    Neale, Claire

    2013-01-01

    Within primary schools, the core subjects of literacy and numeracy are highly regarded, and rightly so, as children need to learn to read, write and be numerically literate. This means that all children learn their ABCs at an early age, But, what about the "other ABC"--"Airway, Breathing and Circulation?" Accidents and medical…

  7. Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death*

    PubMed Central

    Chowdhury, Sayan; Mukhopadhyay, Rupkatha; Saha, Sourav; Mishra, Amartya; Sengupta, Souvik; Roy, Syamal; Majumder, Hemanta K.

    2014-01-01

    In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB25R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance. PMID:24706751

  8. B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

    PubMed

    Nakaya, K; Mizuno, R; Ohhashi, T

    2001-12-01

    We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of <1,000 molecular weight (MW), producing complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating or digestion with protease had no significant effect on supernatant-mediated response. These findings suggest that B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

  9. Vaccinium bracteatum Thunb. Exerts Anti-Inflammatory Activity by Inhibiting NF-κB Activation in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Ko, Yong-Hyun; Seo, Jee-Yeon; Lee, Bo-Ram; Lee, Taek Hwan; Kim, Sun Yeou; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-κB p65). VBME significantly inhibited LPS-induced production of NO and PGE2 and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-κB p65 translocation by blocking IκB-α phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-κB signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells. PMID:27169820

  10. Vaccinium bracteatum Thunb. Exerts Anti-Inflammatory Activity by Inhibiting NF-κB Activation in BV-2 Microglial Cells.

    PubMed

    Kwon, Seung-Hwan; Ma, Shi-Xun; Ko, Yong-Hyun; Seo, Jee-Yeon; Lee, Bo-Ram; Lee, Taek Hwan; Kim, Sun Yeou; Lee, Seok-Yong; Jang, Choon-Gon

    2016-09-01

    This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E₂ (PGE₂), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-κB p65). VBME significantly inhibited LPS-induced production of NO and PGE2 and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-κB p65 translocation by blocking IκB-α phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-κB signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells.

  11. The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.

    PubMed

    Paul, N L; Millet, I; Ruddle, N H

    1993-07-01

    The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.

  12. Loss of calreticulin function decreases NFκB activity by stabilizing IκB protein.

    PubMed

    Massaeli, Hamid; Jalali, Shahrzad; Viswanathan, Divya; Mesaeli, Nasrin

    2014-11-01

    Transcription factor NFκB is activated by several processes including inflammation, endoplasmic-reticulum (ER) stress, increase in Akt signaling and enhanced proteasomal degradation. Calreticulin (CRT) is an ER Ca(2+)-binding chaperone that regulates many cellular processes. Gene-targeted deletion of CRT has been shown to induce ER stress that is accompanied with a significant increase in the proteasome activity. Loss of CRT function increases the resistance of CRT-deficient (crt-/-) cells to UV- and drug-induced apoptosis. Based on these reports we hypothesized that loss of CRT will activate NFκB signaling thus contributing to enhanced resistance to apoptosis. In contrast to our hypothesis, we observed a significant decrease in the basal transcriptional activity of NFκB in CRT-deficient cells. Treatment with lipopolysaccharide failed to increase the transcriptional activity of NFκB in the crt-/- cells to the same level as in the wt cells. Our data illustrate that the mechanism of decreased NFκB activity in CRT-deficient cells is mediated by a significant increase in IκB protein expression. Furthermore, we showed a significant increase in protein phosphatase 2A activity inhibition which resulted in decreased IκBα protein level in CRT-deficient cells. Based on our data we concluded that loss of CRT increases the stability of IκB protein thus reducing NFκB activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

    PubMed

    Jia, Qingmei; Bowen, Richard; Lee, Bai-Yu; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

    2016-09-22

    A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC

  14. Understanding B-cell activation and autoantibody repertoire selection in systemic lupus erythematosus: A B-cell immunomics approach.

    PubMed

    Tipton, Christopher M; Hom, Jennifer R; Fucile, Christopher F; Rosenberg, Alexander F; Sanz, Inaki

    2018-07-01

    Understanding antibody repertoires and in particular, the properties and fates of B cells expressing potentially pathogenic antibodies is critical to define the mechanisms underlying multiple immunological diseases including autoimmune and allergic conditions as well as transplant rejection. Moreover, an integrated knowledge of the antibody repertoires expressed by B cells and plasma cells (PC) of different functional properties and longevity is essential to develop new therapeutic strategies, better biomarkers for disease segmentation, and new assays to measure restoration of B-cell tolerance or, at least, of normal B-cell homeostasis. Reaching these goals, however, will require a more precise phenotypic, functional and molecular definition of B-cell and PC populations, and a comprehensive analysis of the antigenic reactivity of the antibodies they express. While traditionally hampered by technical and ethical limitations in human experimentation, new technological advances currently enable investigators to address these questions in a comprehensive fashion. In this review, we shall discuss these concepts as they apply to the study of Systemic Lupus Erythematosus. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

    PubMed

    Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

    2003-01-01

    Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

  16. Eupatilin inhibits T-cell activation by modulation of intracellular calcium flux and NF-kappaB and NF-AT activity.

    PubMed

    Kim, Young-Dae; Choi, Suck-Chei; Oh, Tae-Young; Chun, Jang-Soo; Jun, Chang-Duk

    2009-09-01

    Eupatilin, one of the pharmacologically active ingredients of Artemisia princeps, exhibits a potent anti-ulcer activity, but its effects on T-cell immunity have not been investigated. Here, we show that eupatilin has a profound inhibitory effect on IL-2 production in Jurkat T cells as well as in human peripheral blood leukocytes. Eupatilin neither influenced clustering of CD3 and LFA-1 to the immunological synapse nor inhibited conjugate formation between T cells and B cells in the presence or absence of superantigen (SEE). Eupatilin also failed to inhibit T-cell receptor (TCR) internalization, thereby, suggesting that eupatilin does not interfere with TCR-mediated signals on the membrane proximal region. In unstimulated T cells, eupatilin significantly induced apoptotic cell death, as evidenced by an increased population of annexin V(+)/PI(+) cells and cleavage of caspase-3 and PARP. To our surprise, however, once cells were activated, eupatilin had little effect on apoptosis, and instead slightly protected cells from activation-induced cell death, suggesting that apoptosis also is not a mechanism for eupatilin-induced T-cell suppression. On the contrary, eupatilin dramatically inhibited I-kappaBalpha degradation and NF-AT dephosphorylation and, consequently, inhibited NF-kappaB and NF-AT promoter activities in PMA/A23187-stimulated T cells. Interestingly, intracellular calcium flux was significantly perturbed in cells pre-treated with eupatilin, suggesting that calcium-dependent cascades might be targets for eupatilin action. Collectively, our results provide evidence for dual regulatory functions of eupatilin: (1) a pro-apoptotic effect on resting T cells and (2) an immunosuppressive effect on activated T cells, presumably through modulation of Ca(2+) flux. (c) 2009 Wiley-Liss, Inc.

  17. Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling.

    PubMed

    Jones, P M; George, A M

    2005-04-30

    Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.

  18. Protein Kinase Cδ Promotes Transitional B Cell-Negative Selection and Limits Proximal B Cell Receptor Signaling To Enforce Tolerance

    PubMed Central

    Zikherman, Julie; Lau, Tannia; Leitges, Michael; Weiss, Arthur

    2014-01-01

    Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca2+-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation. PMID:24515435

  19. Altered B cell signalling in autoimmunity

    PubMed Central

    Rawlings, David J.; Metzler, Genita; Wray-Dutra, Michelle; Jackson, Shaun W.

    2017-01-01

    Recent work has provided new insights into how altered B cell-intrinsic signals — through the B cell receptor (BCR) and key co-receptors — function together to promote the pathogenesis of autoimmunity. These combined signals affect B cells at two distinct stages: first, in the selection of the naive repertoire; and second, during extrafollicular or germinal centre activation responses. Thus, dysregulated signalling can lead to both an altered naive BCR repertoire and the generation of autoantibody-producing B cells. Strikingly, high-affinity autoantibodies predate and predict disease in several autoimmune disorders, including type 1 diabetes and systemic lupus erythematosus. This Review summarizes how, rather than being a downstream consequence of autoreactive T cell activation, dysregulated B cell signalling can function as a primary driver of many human autoimmune diseases. PMID:28393923

  20. Iowa ABC connections : [tech transfer summary].

    DOT National Transportation Integrated Search

    2015-06-01

    The Iowa Department of Transportation (DOT) and other organizations have : been developing accelerated bridge construction (ABC) concepts, details, and : processes, and Iowa has come to be viewed as a national leader in the area of : ABC. However, th...

  1. Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

    PubMed

    Allan, Lenka L; Mann, Koren K; Matulka, Raymond A; Ryu, Heui-Young; Schlezinger, Jennifer J; Sherr, David H

    2003-12-01

    Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.

  2. Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue

    2016-01-29

    The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-basedmore » cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.« less

  3. Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-κB pathway in sertoli TM4 cells.

    PubMed

    Liu, Xiaozhen; Nie, Shaoping; Huang, Danfei; Xie, Mingyong

    2015-09-01

    The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)-2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX-2 protein expression and interleukin-6 (IL)-6 and prostaglandin E2 (PGE2) secretion in a dose-dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), attenuated NP-induced ROS production, COX-2 expression, and IL-6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF-κB, whereas the NF-κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP-enhanced COX-2 expression and IL-6 and PGE2 release in TM4 cells in a dose-dependent manner. Furthermore, NAC blocked NP-induced activation of NF-κB. In addition, inhibition of COX-2 mitigated NP-induced IL-6 release. In conclusion, NP induced ROS generation, activation of NF-κB pathway, COX-2 upregulation, and IL-6 and PGE2 secretion in TM4 cells. NP may regulate COX-2 expression via ROS-activated NF-κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc.

  4. Non-specific factor enhancement of human in vitro antigen-dependent antibody synthesis: role of B cell activation and T cell help.

    PubMed Central

    Brenner, M K; North, M E; Chadda, H R; Farrant, J

    1984-01-01

    Lectin-free supernatants obtained from PWM-stimulated lymphocytes, enable B cells to proliferate and secrete immunoglobulin. Both functions are augmented by the addition of irradiated T cells. In the presence of antigen, these supernatants also enhance specific anti-tetanus toxoid antibody production. The components of the supernatant responsible for these activities have a molecular weight between 30,000 and 60,000, and have the characteristics of non-specific factors: they are genetically unrestricted, and do not bind to either antigen or anti-DR affinity columns. There is no evidence that the partial T dependency of these factors is an indication that their target is a T cell. Instead, T cells appear necessary to move the B cell into a state of activation in which it becomes responsive to the factor. Alternative activation signals such as Staph. A. Cowan can substitute for T cell help in the proliferative response, but not for immunoglobulin or antibody synthesis. The implications of these results for the approaches used to detect and classify B cell growth factors are discussed. PMID:6608488

  5. Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

    PubMed

    Nagata, Keiko; Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

    2017-04-01

    Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

  6. Epstein–Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease

    PubMed Central

    Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

    2017-01-01

    Abstract Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein–Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases. PMID:28333576

  7. Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

    USDA-ARS?s Scientific Manuscript database

    Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

  8. Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

    PubMed

    Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

    2013-01-01

    Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

  9. Interaction of the B cell-specific transcriptional coactivator OCA-B and galectin-1 and a possible role in regulating BCR-mediated B cell proliferation.

    PubMed

    Yu, Xin; Siegel, Rachael; Roeder, Robert G

    2006-06-02

    OCA-B is a B cell-specific transcriptional coactivator for OCT factors during the activation of immunoglobulin genes. In addition, OCA-B is crucial for B cell activation and germinal center formation. However, the molecular mechanisms for OCA-B function in these processes are not clear. Our previous studies documented two OCA-B isoforms and suggested a novel mechanism for the function of the myristoylated, membrane-bound form of OCA-B/p35 as a signaling molecule. Here, we report the identification of galectin-1, and related galectins, as a novel OCA-B-interacting protein. The interaction of OCA-B and galectin-1 can be detected both in vivo and in vitro. The galectin-1 binding domain in OCA-B has been localized to the N terminus of OCA-B. In B cells lacking OCA-B expression, increased galectin-1 expression, secretion, and cell surface association are observed. Consistent with these observations, and a reported inhibitory interaction of galectin-1 with CD45, the phosphatase activity of CD45 is reduced modestly, but significantly, in OCA-B-deficient B cells. Finally, galectin-1 is shown to negatively regulate B cell proliferation and tyrosine phosphorylation upon BCR stimulation. Together, these results raise the possibility that OCA-B may regulate BCR signaling through an association with galectin-1.

  10. Pim-2 activates API-5 to inhibit the apoptosis of hepatocellular carcinoma cells through NF-kappaB pathway.

    PubMed

    Ren, Ke; Zhang, Wei; Shi, Yujun; Gong, Jianping

    2010-06-01

    Pim-2 is proved to be relevant to the tumorigenesis of hepatocellular carcinoma (HCC), but the mechanism is unclear. We studied the relationship among Pim-2, NF-kappaB and API-5. In our experiment, expression level of the three factors and phosphorylation level of API-5, as well as NF-kappaB activity, were detected in HCC tissues and the nontumorous controls. Then Pim-2 gene was transfected into nontumorous liver cells L02, and Pim-2 SiRNA was transfected into hepatoblastoma cell line HepG2. Parthenolide was added as NF-kappaB inhibitor. The same detections as above were repeated in the cells, along with the apoptosis analysis. We found the levels of Pim-2, NF-kappaB and API-5, as well as NF-kappaB activity, were significantly higher in HCC tissues. Pim-2 level was increased in L02 cells after the transfection of Pim-2 gene, but decreased in HepG2 cells after the transfection of Pim-2 SiRNA. The levels of NF-kappaB and API-5, as well as NF-kappaB activity and API-5 phosphorylation level, were in accordance with Pim-2 level, but could be reversed by Parthenolide. Cell apoptosis rates were negatively correlated with API-5 phosphorylation level. Therefore, we infer that Pim-2 could activate API-5 to inhibit the apoptosis of liver cells, and NF-kappaB is the key regulator.

  11. CD22 ligation inhibits downstream B cell receptor signaling and Ca(2+) flux upon activation.

    PubMed

    Sieger, N; Fleischer, S J; Mei, H E; Reiter, K; Shock, A; Burmester, G R; Daridon, C; Dörner, T

    2013-03-01

    CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases. Copyright © 2013 by the American College of Rheumatology.

  12. B kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-κB in the central nervous system

    PubMed Central

    Raasch, Jenni; Zeller, Nicolas; van Loo, Geert; Merkler, Doron; Mildner, Alexander; Erny, Daniel; Knobeloch, Klaus-Peter; Bethea, John R.; Waisman, Ari; Knust, Markus; Del Turco, Domenico; Deller, Thomas; Blank, Thomas; Priller, Josef; Brück, Wolfgang

    2011-01-01

    The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system diseases. PMID:21310728

  13. Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

    PubMed

    Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff

    2013-09-01

    Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  14. Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

    PubMed

    Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

    2016-04-15

    B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

  15. Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin

    PubMed Central

    Kotlo, Kumar; Bhattacharyya, Sumit; Yang, Bo; Feferman, Leonid; Tejaskumar, Shah; Linhardt, Robert; Danziger, Robert

    2013-01-01

    N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation. PMID:23385884

  16. Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin.

    PubMed

    Kotlo, Kumar; Bhattacharyya, Sumit; Yang, Bo; Feferman, Leonid; Tejaskumar, Shah; Linhardt, Robert; Danziger, Robert; Tobacman, Joanne K

    2013-10-01

    N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.

  17. Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong

    2014-05-15

    Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1more » expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.« less

  18. Evolutionary Trajectories of Entomopathogenic Fungi ABC Transporters.

    PubMed

    Baral, Bikash

    2017-01-01

    The ABC protein superfamily-also called traffic ATPases-are energy-dependent ubiquitous proteins, representing one of the crucial and the largest family in the fungal genomes. The ATP-binding cassette endows a characteristic 200-250 amino acids and is omnipresent in all organisms ranging from prokaryotes to eukaryotes. Unlike in bacteria with nutrient import functions, ABC transporters in fungal entomopathogens serve as effective efflux pumps that are largely involved in the shuttle of metabolites across the biological membranes. Thus, the search for ABC proteins may prove of immense importance in elucidating the functional and molecular mechanism at the host-pathogen (insect-fungus) interface. Their sequence homology, domain topology, and functional traits led to the actual identification of nine different families in fungal entomopathogens. Evolutionary relationships within the ABC superfamily are discussed, concentrating on computational approaches for comparative identification of ABC transporters in insect-pathogenic fungi (entomopathogens) with those of animals, plants, and their bacterial orthologs. Ancestors of some fungal candidates have duplicated extensively in some phyla, while others were lost in one lineage or the other, and predictions for the cause of their duplications and/or loss in some phyla are made. ABC transporters of fungal insect-pathogens serve both defensive and offensive functions effective against land-dwelling and ground foraging voracious insects. This study may help to unravel the molecular cascades of ABC proteins to illuminate the means through which insects cope with fungal infection and fungal-related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

    PubMed

    Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

    2017-11-01

    imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Automated brainstem co-registration (ABC) for MRI.

    PubMed

    Napadow, Vitaly; Dhond, Rupali; Kennedy, David; Hui, Kathleen K S; Makris, Nikos

    2006-09-01

    Group data analysis in brainstem neuroimaging is predicated on accurate co-registration of anatomy. As the brainstem is comprised of many functionally heterogeneous nuclei densely situated adjacent to one another, relatively small errors in co-registration can manifest in increased variance or decreased sensitivity (or significance) in detecting activations. We have devised a 2-stage automated, reference mask guided registration technique (Automated Brainstem Co-registration, or ABC) for improved brainstem co-registration. Our approach utilized a brainstem mask dataset to weight an automated co-registration cost function. Our method was validated through measurement of RMS error at 12 manually defined landmarks. These landmarks were also used as guides for a secondary manual co-registration option, intended for outlier individuals that may not adequately co-register with our automated method. Our methodology was tested on 10 healthy human subjects and compared to traditional co-registration techniques (Talairach transform and automated affine transform to the MNI-152 template). We found that ABC had a significantly lower mean RMS error (1.22 +/- 0.39 mm) than Talairach transform (2.88 +/- 1.22 mm, mu +/- sigma) and the global affine (3.26 +/- 0.81 mm) method. Improved accuracy was also found for our manual-landmark-guided option (1.51 +/- 0.43 mm). Visualizing individual brainstem borders demonstrated more consistent and uniform overlap for ABC compared to traditional global co-registration techniques. Improved robustness (lower susceptibility to outliers) was demonstrated with ABC through lower inter-subject RMS error variance compared with traditional co-registration methods. The use of easily available and validated tools (AFNI and FSL) for this method should ease adoption by other investigators interested in brainstem data group analysis.

  1. Cross Talk in HEK293 Cells Between Nrf2, HIF, and NF-κB Activities upon Challenges with Redox Therapeutics Characterized with Single-Cell Resolution.

    PubMed

    Johansson, Katarina; Cebula, Marcus; Rengby, Olle; Dreij, Kristian; Carlström, Karl E; Sigmundsson, Kristmundur; Piehl, Fredrik; Arnér, Elias S J

    2017-02-20

    Many transcription factors with importance in health and disease are redox regulated. However, how their activities may be intertwined in responses to redox-perturbing stimuli is poorly understood. To enable in-depth characterization of this aspect, we here developed a methodology for simultaneous determination of nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation at single-cell resolution, using a new tool named pTRAF (plasmid for transcription factor reporter activation based upon fluorescence). The pTRAF allowed determination of Nrf2, HIF, and NF-κB activities in a high-resolution and high-throughput manner, and we here assessed how redox therapeutics affected the activities of these transcription factors in human embryonic kidney cells (HEK293). Cross talk was detected between the three signaling pathways upon some types of redox therapeutics, also by using inducers typically considered specific for Nrf2, such as sulforaphane or auranofin, hypoxia for HIF activation, or tumor necrosis factor alpha (TNFα) for NF-κB stimulation. Doxorubicin, at low nontoxic doses, potentiated TNFα-induced activation of NF-κB and HIF, without effects in stand-alone treatment. Stochastic activation patterns in cell cultures were also considerable upon challenges with several redox stimuli. A novel strategy was here used to study simultaneous activation of Nrf2, HIF, and NF-κB in single cells. The method can also be adapted for studies of other transcription factors. The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-κB are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells. Antioxid. Redox Signal. 26, 229-246.

  2. Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells.

    PubMed

    Van De Walle, Jacqueline; Romier, Béatrice; Larondelle, Yvan; Schneider, Yves-Jacques

    2008-04-01

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. In human intestinal Caco-2 cells, DON activates the mitogen-activated protein kinases (MAPKs). We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion. In addition, Caco-2 cells were co-exposed to pro-inflammatory stimuli in order to mimic an inflamed intestinal epithelium. Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed, reaching 1.4- and 7.6-fold, respectively using DON at 10 microg/ml. Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml. Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion. Synergistic interactions between these stimuli and DON were observed. These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.

  3. Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells.

    PubMed

    Ou-Yang, P; Chiang, B L; Hwang, L H; Chen, Y G; Yang, P M; Chi, W K; Chen, P J; Chen, D S

    1999-04-01

    The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.

  4. TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.

    2011-09-30

    Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases.more » Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.« less

  5. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

    PubMed

    Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

    2015-06-01

    Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

  6. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells

    PubMed Central

    JEONG, KWON; KIM, KIYOON; KIM, HUNSUNG; OH, YOOJUNG; KIM, SEONG-JIN; JO, YUNHEE; CHOE, WONCHAE

    2015-01-01

    Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions. PMID:26137159

  7. Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

    PubMed

    Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

    2007-01-01

    Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

  8. The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

    PubMed

    Martin, Audrey; Daniel, Jaiyanth

    2018-02-05

    Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. 4th International Consensus Conference on Advanced Breast Cancer (ABC4), Lisbon, November 4, 2017 : ABC4 Consensus: Assessment by a Panel of German Experts.

    PubMed

    Untch, Michael; Würstlein, Rachel; Marschner, Norbert; Lüftner, Diana; Augustin, Doris; Briest, Susanne; Ettl, Johannes; Haidinger, Renate; Müller, Lothar; Müller, Volkmar; Ruckhäberle, Eugen; Harbeck, Nadia; Thomssen, Christoph

    2018-05-01

    The fourth international advanced breast cancer consensus conference (ABC4) on the diagnosis and treatment of advanced breast cancer (ABC) headed by Professor Fatima Cardoso was once again held in Lisbon on November 2 - 4, 2017. To simplify matters, the abbreviation ABC will be used hereinafter in the text. In clinical practice, the abbreviation corresponds to metastatic breast cancer or locally far-advanced disease. This year the focus was on new developments in the treatment of ABC. Topics discussed included the importance of CDK4/6 inhibition in hormone receptor (HR)-positive ABC, the use of dual antibody blockade to treat HER2-positive ABC, PARP inhibition in triple-negative ABC and the potential therapeutic outcomes. Another major area discussed at the conference was BRCA-associated breast cancer, the treatment of cerebral metastasis, and individualized treatment decisions based on molecular testing (so-called precision medicine). As in previous years, close cooperation with representatives from patient organizations from around the world is an important aspect of the ABC conference. This cooperation was reinforced and expanded at the ABC4 conference. A global alliance was founded at the conclusion of the consensus conference, which aims to promote and coordinate the measures considered necessary by patient advocates worldwide. Because the panel of experts was composed of specialists from all over the world, it was inevitable that the ABC consensus also reflected country-specific features. As in previous years, a team of German breast cancer specialists who closely followed the consensus voting of the ABC panelists in Lisbon and intensively discussed the votes has therefore commented on the consensus in the context of the current German guidelines on the diagnosis and treatment of breast cancer 1 ,  2 used in clinical practice in Germany. The ABC consensus is based on the votes of the ABC panelists in Lisbon.

  10. Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity

    PubMed Central

    Campos, Regis A; Szczepanik, Marian; Itakura, Atsuko; Lisbonne, Mariette; Dey, Neelendu; Leite-de-Moraes, Maria C; Askenase, Philip W

    2006-01-01

    We showed that hepatic Vα14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)-4 production, activate B-1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL-4–/– and signal transducer and activator of transcription (STAT)-6–/– mice and was inhibited by anti-IL-4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream-acting 1-day immune B-1 cells from wild-type mice. Responses were not reconstituted with B-1 cells from IL-4 receptor-α–/– or STAT-6–/– mice, nor by IL-4 treatment of B cell-deficient mice at immunization. Finally, IL-4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate-like iNKT cells and the B-1 B cells that participate in the recruitment of effector T cells in vivo. PMID:16556268

  11. Decoy receptor 3 suppresses B cell functions and has a negative correlation with disease activity in rheumatoid arthritis.

    PubMed

    Chen, Ming-Han; Liu, Po-Chun; Chang, Chien-Wen; Chen, Yi-Ann; Chen, Ming-Huang; Liu, Chun-Yu; Leu, Chuen-Miin; Lin, Hsiao-Yi

    2014-01-01

    The decoy receptor 3 (DcR3) is a member of the tumour necrosis factor (TNF) receptor superfamily and may regulate inflammation. The aim of this study was to investigate the role of DcR3 in B cell functions and its correlation to disease activity in patients with rheumatoid arthritis (RA). The concentrations of DcR3 and TNF-α were measured by ELISA. B cell proliferation was assessed by quantification of 3H-thymidine uptake. Staphylococcus aureus Cowan (SAC) strain were used to stimulate B cell proliferation and TNF-α production. Compared to the osteoarthritis (OA) patients, the RA group had higher synovial DcR3 levels (3273.6±1623.2 vs. 1594.8±1190.0 pg/ml, p=0.003), which were negatively correlated with the serum erythrocyte sedimentation rate and Disease Activity Score using 28 joint counts (DAS28) scores (r=-0.560, p=0.002; r=-0.579, p<0.001, respectively). Although the RA B cells have more active characteristics, B cell proliferation induced by SAC was successfully suppressed by recombinant DcR3.Fc fusion protein with an average inhibition of 44.8%. Moreover, DcR3.Fc fusion protein was found to suppress SAC-induced TNF-α production by B cells in 8 RA patients (average inhibition 47.0%). The results of our study indicated that the inhibition of B cell functions by DcR3 may partially explain the negative correlation between DcR3 level and disease activity in RA patients. Our findings imply that DcR3 may be used as a biomarker for disease activity and a potential therapeutic agent in the treatment of RA.

  12. Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis.

    PubMed

    Qiu, Jingfan; Zhuo, Ying; Zhu, Dongqing; Zhou, Xiufen; Zhang, Lixin; Bai, Linquan; Deng, Zixin

    2011-10-01

    Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

  13. T-dependent activation of resting B cells mediated by concanavalin A.

    PubMed

    Ratcliffe, M J; Julius, M H

    1984-03-01

    In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

  14. Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

    PubMed

    Kim, Pan Soo; Shin, Ji Hyun; Jo, Doo Sin; Shin, Dong Woon; Choi, Dong-Hwa; Kim, Woo Jung; Park, Kyuhee; Kim, Jin Kyu; Joo, Chul Gue; Lee, Jong Suk; Choi, Yongmun; Shin, Yong Won; Shin, Joong Jin; Jeon, Hong Bae; Seo, Jin-Ho; Cho, Dong-Hyung

    2018-06-08

    Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy. Copyright © 2018. Published by Elsevier Inc.

  15. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis*

    PubMed Central

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F. Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M.

    2016-01-01

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

  16. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

    PubMed

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

    2016-07-08

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

    PubMed Central

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-01-01

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

  18. Yang-Mills theory and the ABC conjecture

    NASA Astrophysics Data System (ADS)

    He, Yang-Hui; Hu, Zhi; Probst, Malte; Read, James

    2018-05-01

    We establish a precise correspondence between the ABC Conjecture and 𝒩 = 4 super-Yang-Mills theory. This is achieved by combining three ingredients: (i) Elkies’ method of mapping ABC-triples to elliptic curves in his demonstration that ABC implies Mordell/Faltings; (ii) an explicit pair of elliptic curve and associated Belyi map given by Khadjavi-Scharaschkin; and (iii) the fact that the bipartite brane-tiling/dimer model for a gauge theory with toric moduli space is a particular dessin d’enfant in the sense of Grothendieck. We explore this correspondence for the highest quality ABC-triples as well as large samples of random triples. The conjecture itself is mapped to a statement about the fundamental domain of the toroidal compactification of the string realization of 𝒩 = 4 SYM.

  19. Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

    PubMed Central

    Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

    2014-01-01

    The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

  20. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    PubMed

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-07-09

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

  1. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

    PubMed Central

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  2. Quasiparticle renormalization in ABC graphene trilayers

    NASA Astrophysics Data System (ADS)

    Dou, Xu; Jaefari, Akbar; Barlas, Yafis; Uchoa, Bruno

    2015-03-01

    We investigate the effect of electron-electron interactions in ABC stacked graphene trilayers. In the gapless regime, we show that the self-energy corrections lead to the renormalization of the dynamical exponent z = 3 +α1 / N , with α1 ~ 0 . 52 and N is the number of fermionic species. Although the quasiparticle residue is suppressed near the neutrality point, the lifetime has a sublinear scaling with the energy and the quasiparticles are well defined even at zero energy. We calculate the renormalization of a variety of physical observables, which can be directly measured in experiments. X.D., A.J., and B.U. acknowledge University of Oklahoma for support. B.U. acknowledges NSF Career Grant No. DMR-1352604 for partial support.

  3. Polymethoxylated flavones potentiate the cytolytic activity of NK leukemia cell line KHYG-1 via enhanced expression of granzyme B.

    PubMed

    Saito, Takeshi; Abe, Daigo; Nogata, Yoichi

    2015-01-16

    Polymethoxylated flavones (PMFs) are found in the peel tissues of some citrus species. Here, we report that PMFs, such as nobiletin, potentiate the cytolytic activity of KHYG-1 natural killer (NK) leukemia cells. Nobiletin markedly enhanced the expression of granzyme B, a serine protease that plays critical roles in the cytolytic activity of NK cells. The potentiated cytolytic activity induced by nobiletin was canceled by the granzyme B inhibitor Z-AAD-CMK. Nobiletin also increased the levels of phosphorylated CREB, ERK1/2, and p38 MAPK in KHYG-1 cells, which are known to participate in NK cell function. Inhibition of an upstream kinase of ERK1/2 failed to reduce the granzyme B expression and KHYG-1 cytolytic activity. Meanwhile, inhibition of p38 MAPK attenuated both granzyme B expression and KHYG-1 cytolytic activity. These results suggest that the primary role of nobiletin in KHYG-1 cytolytic activity lies in upregulation of granzyme B expression, at least in part, mediated through p38 MAPK function. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Curcumin Regulates Low-Linear Energy Transfer {gamma}-Radiation-Induced NF{kappa}B-Dependent Telomerase Activity in Human Neuroblastoma Cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aravindan, Natarajan, E-mail: naravind@ouhsc.ed; Veeraraghavan, Jamunarani; Madhusoodhanan, Rakhesh

    2011-03-15

    Purpose: We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NF{kappa}B regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NF{kappa}B-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. Methods and Materials: SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NF{kappa}B-dependent regulation was investigated either by luciferase reporter assays using pNF{kappa}B-, pGL3-354-, pGL3-347-, or pUSE-I{kappa}B{alpha}-Luc, p50/p65, or RelA siRNA-transfectedmore » cells. NF{kappa}B activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. Results: Curcumin profoundly inhibited IR-induced NF{kappa}B. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NF{kappa}B becomes functionally activated after IR and mediates TA upregulation by binding to the {kappa}B-binding region in the promoter region of the TERT gene. Consistently, elimination of the NF{kappa}B-recognition site on the telomerase promoter or inhibition of NF{kappa}B by the I{kappa}B{alpha} mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NF{kappa}B overexpressed cells. Furthermore, curcumin

  5. Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers

    PubMed Central

    Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A.; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

    2016-01-01

    B-cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL+ MHC class-IHi CD86Hi B cells of unknown origin. Here we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. The 4BL cells induce expression of 4-1BBL and IFNγR1 on B1a cells resulting in subsequent up regulation of membrane TNFα (mTNFα) and CD86. As a result, B1a cells induce expression of granzyme B in CD8+T cells by targeting TNFR2 via mTNFα while providing co-stimulation with CD86. Thus, for the first time, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8+T cells. PMID:26983789

  6. [Role of let-7 in maintaining characteristics of breast cancer stem cells].

    PubMed

    Sun, Xin; Fan, Chong; Hu, Li-juan; Du, Ning; Xu, Chong-wen; Ren, Hong

    2012-08-01

    To observe the expression of let-7 in breast cancer stem cells and explore the role of let-7 in maintaining the characteristics of breast cancer stem cells. We separated breast cancer stem cells (SP and NSP) from MCF-7 cell line using SP sorting, and observed the expression of let-7a/b/c on SP and NSP cells using quantitative real-time PCR and the expressions of Ras and ERK using Western blotting to study the mechanism by which let-7 maintains the characteristics of breast cancer stem cells. The SP cells accounted for 3.3% in MCF-7 cells, however, the rate dropped to 0.4% when verapamil was added into the process of seperation. The level of Let-7a/b/c in SP cells were lower than that in NSP cells, and among let-7 miRNAs, let-7b/c showed the most obvious difference. The expressions of t-Ras and t-ERK showed no difference between SP and NSP cells, nevertheless, the expressions of p-Ras, p-ERK were higher in SP cells than in NSP cells. SP sorting is an effective method to separate cancer stem cells. There do exist cancer stem cells in MCF-7 breast cancer cell line. Let-7 is down-regulated in SP cells, and the down-regulation makes let-7 lose the opportunity to restrain Ras mRNA, finally, p-Ras and p-ERK are activated. They play an important role in maintaining the characteristics of breast cancer stem cells.

  7. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

    PubMed Central

    2017-01-01

    ABSTRACT Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8+ T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a−/− CD8+ T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a−/− CD8+ T cells responded equivalently to wild-type CD8+ T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8+ T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8+ T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8+ T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas. PMID:28344876

  8. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells.

    PubMed

    Huang, Yu-Hsuan; Tsai, Kevin; Tan, Sara Y; Kang, Sohyeong; Ford, Mandy L; Harder, Kenneth W; Priatel, John J

    2017-01-01

    Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8 + T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a - / - CD8 + T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a - / - CD8 + T cells responded equivalently to wild-type CD8 + T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8 + T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8 + T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8 + T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

  9. Hydroquinone stimulates inflammatory functions in microvascular endothelial cells via NF-κB nuclear activation.

    PubMed

    Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Farsky, Sandra Helena Poliselli

    2011-11-01

    Hydroquinone impairs several leucocyte cell functions, which alter the immune response. Although endothelial cell functions are important for the development of immune responses, hydroquinone actions on endothelial cell have not been shown. Therefore, the effect of hydroquinone exposure (10 or 100 μM for 2 hr) on primary culture of microvascular endothelial cells (PMECs) obtained from the cremaster muscle of Wistar rats incubated in the presence or absence of lipopolysaccharide (LPS, 2 μg/mL) was investigated. Hydroquinone treatment induced the membrane expression of cell adhesion molecules (CAMs) from the immunoglobulin superfamilies ICAM-1 (intercellular), VCAM-1(vascular) and PECAM-1 (platelet endothelial) and induced the secretion of cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The effects were dependent on transcriptional modifications because enhanced CAM mRNA expression as well as both cytokines and nuclear factor κB (NF-κB) nuclear activation was found. These effects may be due to the direct action of hydroquinone rather than its quinone metabolites, because endothelial cells do not present myeloperoxidase enzyme and hydroquinone incubation did not induce the expression of cytochrome P450 2E1 (CYP2E1) or prostaglandin H synthase 1. In addition, the incubation of endothelial cells with benzoquinone (10 μM, 2 hr) impaired PECAM-1 expression and did not modify NF-κB nuclear activation. Taken together, the data herein presented reveal that hydroquinone evokes pro-inflammatory properties in endothelial cells that are triggered by the enhancement of NF-κB nuclear translocation-dependent gene transcription. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

  10. Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

    PubMed Central

    Tze, Lina E; Schram, Brian R; Lam, Kong-Peng; Hogquist, Kristin A; Hippen, Keli L; Liu, Jiabin; Shinton, Susan A; Otipoby, Kevin L; Rodine, Peter R; Vegoe, Amanda L; Kraus, Manfred; Hardy, Richard R; Schlissel, Mark S; Rajewsky, Klaus

    2005-01-01

    In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms. PMID:15752064

  11. Global Phosphoproteomics of Activated B Cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers

    PubMed Central

    2015-01-01

    Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration. PMID:24905233

  12. The ABC of non-inferiority margin setting from indirect comparisons.

    PubMed

    Julious, Steven A

    2011-01-01

    In a non-inferiority trial to assess a new investigative treatment, there may need to be consideration of an indirect comparison with placebo using the active control in the current trial. We can, therefore, use the fact that there is a common active control in the comparisons of the investigative treatment and placebo. In analysing a non-inferiority trial, the ABC of: Assay sensitivity, Bias minimisation and Constancy assumption needs to be considered. It is highlighted how the ABC assumptions can potentially fail when there is placebo creep or a patient population shift. In this situation, the belief about the placebo response expressed in terms of a prior probability in Bayesian formulation could be used with the observed treatment effects to set the non-inferiority limit. Copyright © 2011 John Wiley & Sons, Ltd.

  13. [Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

    PubMed

    Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

    2013-05-04

    To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

  14. The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells.

    PubMed

    Lee, Jinmin; Sengupta, Prabuddha; Brzostowski, Joseph; Lippincott-Schwartz, Jennifer; Pierce, Susan K

    2017-02-15

    B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen--activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high -frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions. © 2017 Lee, Sengupta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

    PubMed

    Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

    2015-03-01

    This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Interaction of ABC multidrug transporters with anticancer protein kinase inhibitors: substrates and/or inhibitors?

    PubMed

    Hegedus, Csilla; Ozvegy-Laczka, Csilla; Szakács, Gergely; Sarkadi, Balázs

    2009-05-01

    Protein kinase inhibitors (PKI) are becoming key agents in modern cancer chemotherapy, and combination of PKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. Since chemotherapy resistance is a recurrent problem, mechanisms of resistance should be clarified in order to help further drug development. Here we suggest that in addition to PKI resistance based on altered target structures, the active removal of these therapeutic agents by the MDR-ABC transporters should also be considered as a major cause of clinical resistance. We discuss the occurring systemic and cellular mechanisms, which may hamper PKI efficiency, and document the role of selected MDR-ABC transporters in these phenomena through their interactions with these anticancer agents. Moreover, we suggest that PKI interactions with ABC transporters may modulate overall drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interaction with PKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. We propose that these interactions should be carefully considered in clinical application, and a combined MDR-ABC transporter and PKI effect may bring a major advantage in future drug development.

  17. Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout.

    PubMed

    Abos, Beatriz; Estensoro, Itziar; Perdiguero, Pedro; Faber, Marc; Hu, Yehfang; Díaz Rosales, Patricia; Granja, Aitor G; Secombes, Christopher J; Holland, Jason W; Tafalla, Carolina

    2018-01-01

    Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout ( Oncorhynchus mykiss ) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae . We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT + B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD

  18. Iowa ABC connections.

    DOT National Transportation Integrated Search

    2015-06-01

    For several years the Iowa Department of Transportation (DOT), Iowa State University, the Federal Highway Administration, : and several Iowa counties have been working to develop accelerated bridge construction (ABC) concepts, details, and processes....

  19. The effect of organic anion-transporting polypeptides 1B1, 1B3 and 2B1 on the antitumor activity of flavopiridol in breast cancer cells.

    PubMed

    Brenner, Stefan; Riha, Juliane; Giessrigl, Benedikt; Thalhammer, Theresia; Grusch, Michael; Krupitza, Georg; Stieger, Bruno; Jäger, Walter

    2015-01-01

    The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.

  20. HSV-1-induced activation of NF-κB protects U937 monocytic cells against both virus replication and apoptosis

    PubMed Central

    Marino-Merlo, Francesca; Papaianni, Emanuela; Medici, Maria Antonietta; Macchi, Beatrice; Grelli, Sandro; Mosca, Claudia; Borner, Christoph; Mastino, Antonio

    2016-01-01

    The transcription factor nuclear factor-kappa B (NF-κB) is a crucial player of the antiviral innate response. Intriguingly, however, NF-κB activation is assumed to favour herpes simplex virus (HSV) infection rather than restrict it. Apoptosis, a form of innate response to viruses, is completely inhibited by HSV in fully permissive cells, but not in cells incapable to fully sustain HSV replication, such as immunocompetent cells. To resolve the intricate interplay among NF-κB signalling, apoptosis and permissiveness to HSV-1 in monocytic cells, we utilized U937 monocytic cells in which NF-κB activation was inhibited by expressing a dominant-negative IκBα. Surprisingly, viral production was increased in monocytic cells in which NF-κB was inhibited. Moreover, inhibition of NF-κB led to increased apoptosis following HSV-1 infection, associated with lysosomal membrane permeabilization. High expression of late viral proteins and induction of apoptosis occurred in distinct cells. Transcriptional analysis of known innate response genes by real-time quantitative reverse transcription-PCR excluded a contribution of the assayed genes to the observed phenomena. Thus, in monocytic cells NF-κB activation simultaneously serves as an innate process to restrict viral replication as well as a mechanism to limit the damage of an excessive apoptotic response to HSV-1 infection. This finding may clarify mechanisms controlling HSV-1 infection in monocytic cells. PMID:27584793

  1. CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis.

    PubMed

    Gambi, D; Porrini, A M; Giampietro, A; Macor, S

    1991-08-01

    Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper-inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS.

  2. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

    PubMed Central

    Yao, Cheng; Oh, Jang-hee; Oh, Inn Gyung; Park, Chi-hyun; Chung, Jin Ho

    2013-01-01

    Aim: To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. Methods: B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Results: Treatment of the cells with [6]-shogaol (1, 5, 10 μmol/L) reduced the melanin content in a concentration-dependent manner. [6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity, and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells. Furthermore, [6]-shogaol (10 μmol/L) activated ERK, which was known to negatively regulate melanin synthesis in these cells. Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L). Conclusion: The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway. PMID:23123645

  3. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway.

    PubMed

    Yao, Cheng; Oh, Jang-hee; Oh, Inn Gyung; Park, Chi-hyun; Chung, Jin Ho

    2013-02-01

    To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Treatment of the cells with [6]-shogaol (1, 5, 10 μmol/L) reduced the melanin content in a concentration-dependent manner. [6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity, and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells. Furthermore, [6]-shogaol (10 μmol/L) activated ERK, which was known to negatively regulate melanin synthesis in these cells. Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L). The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway.

  4. PHF21B overexpression promotes cancer stem cell-like traits in prostate cancer cells by activating the Wnt/β-catenin signaling pathway.

    PubMed

    Li, Qiji; Ye, Liping; Guo, Wei; Wang, Min; Huang, Shuai; Peng, Xinsheng

    2017-06-23

    PHF21B is newly identified to be involved in the tumor progression; however, its biological role and molecular mechanism in prostate cancer have not been defined. This study is aimed to study the role of PHF21B in the progression of prostate cancer. Real-time PCR, immunohistochemistry and western blotting analysis were used to determine PHF21B expression in prostate cancer cell lines and clinical specimens. The role of PHF21B in maintaining prostate cancer stem cell-like phenotype was examined by tumor-sphere formation assay and expression levels of stem cell markers. Luciferase reporter assay, western blot analysis, enzyme-linked immunosorbent assay and ChIP assay were used to determine whether PHF21B activates the Wnt/β-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2. Our results revealed that PHF21B was markedly upregulated in prostate cancer cell lines and tissues. High PHF21B levels predicted poorer recurrence-free survival in prostate cancer patients. Gain-of-function and loss-of-function studies showed that overexpression of PHF21B enhanced, while downregulation suppressed, the cancer stem cell-like phenotype in prostate cancer cells. Xenograft tumor model showed that silencing PHF21B decreased the ability of tumorigenicity in vivo. Notably, Wnt/β-catenin signaling was hyperactivated in prostate cancer cells overexpressing PHF21B, and mediated PHF21B-induced cancer stem cell-like phenotype. Furthermore, PHF21B suppressed repressors of the Wnt/β-catenin signaling cascade, including SFRP1 and SFRP2. These results demonstrated that PHF21B constitutively activated wnt/β-catenin signaling by transcriptionally downregulating SFRP1 and SFRP2, which promotes prostate cancer stem cell-like phenotype. Our results revealed that PHF21B functions as an oncogene in prostate cancer, and may represent a promising prognostic biomarker and an attractive candidate for target therapy of prostate cancer.

  5. Inhibitory activity of synthesized acetylated Procyanidin B1 analogs against HeLa S3 cells proliferation.

    PubMed

    Okamoto, Syuhei; Ishihara, Sayaka; Okamoto, Taisuke; Doi, Syoma; Harui, Kota; Higashino, Yusuke; Kawasaki, Takashi; Nakajima, Noriyuki; Saito, Akiko

    2014-02-04

    Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.

  6. B-cell acquisition of antigen: Sensing the surface.

    PubMed

    Knight, Andrew M

    2015-06-01

    B-cell antigen receptor (BCR) recognition and acquisition of antigen by B cells is the essential first step in the generation of effective antibody responses. As B-cell-mediated antigen presentation is also believed to play a significant role in the activation of CD4(+) Th-cell responses, considerable effort has focused on clarifying the nature of antigen/BCR interactions. Following earlier descriptions of interactions of soluble antigens with the BCR, it is now clear that B cells also recognize, physically extract and present antigens that are tethered to, or integral components of, the surfaces or extracellular matrix of other cells. In this issue of the European Journal of Immunology, Zeng et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] examine how the physical property or "stiffness" of the surface displaying antigens to B cells influences the B-cell response. This commentary reports that antigen tethered on "less stiff" surfaces induces increased B-cell activation and antibody responses. I then infer how "sensing the surface" by B cells may represent a new component of the immune system's ability to detect "damage," and how this understanding may influence approaches to clinical therapies where immune activity is either unwanted or desired. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

    PubMed Central

    Xia, Wenle; Gooden, David; Liu, Leihua; Zhao, Sumin; Soderblom, Erik J.; Toone, Eric J.; Beyer, Wayne F.; Walder, Harold; Spector, Neil L.

    2014-01-01

    Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies. PMID:24551203

  8. Regulation of VH Replacement by B Cell Receptor (BCR)-mediated Signaling in Human Immature B Cells

    PubMed Central

    Liu, Jing; Lange, Miles D.; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R. A.; Zemlin, Michael; Burrows, Peter D.; Su, Kaihong; Carter, Robert H.; Zhang, Zhixin

    2013-01-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit non-functional IgH genes or IgH genes encoding self reactive B cell receptors (BCRs) and contributes to the diversification of antibody repertoire in mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. Here we show that crosslinking BCRs induces VH replacement in human EU12 μHC+ cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases; but is inhibited by CD19 co-stimulation, presumably through activation of the PI3 kinase pathway. These results show for the first time that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments. PMID:23630348

  9. The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

    PubMed Central

    Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

    2009-01-01

    Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

  10. Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4 + T cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shu, Guangwen; Yang, Tianming; Wang, Chaoyuan

    2013-06-15

    Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearingmore » mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4 + T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8 + T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4 + T cells but not in CD8 + T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4 + T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4 + T cells is implicated in its immunomodulatory activity. - Highlights: • Gastrodin stimulates anticancer immune response. • Gastrodin represses tumor transplantation-induced CD4 + T cell apoptosis. • Gastrodin activates NF-κB activity in CD4 + T cells.« less

  11. Continuous infusion of amphotericin B: preliminary experience at Faculdade de Medicina da Fundação ABC.

    PubMed

    Uehara, Roberto Palermo; Sá, Victor Hugo Lara de; Koshimura, Erika Tae; Prudente, Fernanda Vilas Boas; Tucunduva, Luciana Tomanik Cardozo de Mello; Gonçalves, Marina Sahade; Samano, Eliana Sueco Tibana; del Giglio, Auro

    2005-09-01

    Intravenous amphotericin B deoxycholate (AmB-D) infusions, usually given over 4 hours, frequently induce nephrotoxicity and undesirable infusion-related side effects such as rigors and chills. There is evidence in the literature that the use of AmB-D in the form of continuous 24-hour infusion is less toxic than the usual four-hour infusion of this drug. Our objective was to evaluate the efficacy and safety of continuous infusion of AmB-D for the treatment of persistent fever in neutropenic patients with hematological malignancies after chemotherapy. Observational retrospective analysis of our experience with continuous infusion of AmB-D, at Faculdade de Medicina da Fundação ABC and Hospital Estadual Mário Covas in Santo André. From October 2003 to May 2004, 12 patients with hematological malignancies and chemotherapy-induced neutropenia received 13 cycles of continuous infusion of AmB-D. The median dose of AmB-D was 0.84 mg/kg/day (0.33 to 2.30 mg/kg/day). Concomitant use of nephrotoxic medications occurred in 92% of the cycles. Nephrotoxicity occurred in 30.76% of the cycles, hypokalemia in 16.67%, hepatotoxicity in 30% and adverse infusion-related events in 23%. All patients survived for at least seven days after starting continuous infusion of AmB-D, and clinical resolution occurred in 76% of the cycles. Continuous infusion of AmB-D can be used in our Institution as an alternative to the more toxic four-hour infusion of AmB-D and possibly also as an alternative to the more expensive liposomal formulations of the drug.

  12. ABC1 Consensus Conference - a German Perspective: First International Consensus Conference on Advanced Breast Cancer (ABC1), Lisbon, November 5, 2011.

    PubMed

    Thomssen, Christoph; Marschner, Norbert; Untch, Michael; Decker, Thomas; Hegewisch-Becker, Susanna; Jackisch, Christian; Janni, Wolfgang; Hans-Joachim, Lück; von Minckwitz, Gunter; Scharl, Anton; Schneeweiss, Andreas; Tesch, Hans; Welt, Anja; Harbeck, Nadia

    2012-02-01

    A group of German breast cancer experts (medical oncologists and gynaecologists) reviewed and commented on the results of the first international 'Advanced Breast Cancer First Consensus Conference' (ABC1) for the diagnosis and treatment of advanced breast cancer. The ABC1 Conference is an initiative of the European School of Oncology (ESO) Metastatic Breast Cancer Task Force in cooperation with the EBCC (European Breast Cancer Conference), ESMO (European Society of Medical Oncology) and the American JNCI (Journal of the National Cancer Institute). The main focus of the ABC1 Conference was metastatic breast cancer (stage IV). The ABC1 consensus is based on the vote of 33 breast cancer experts from different countries and has been specified as a guideline for therapeutic practice by the German expert group. It is the objective of the ABC1 consensus as well as of the German comments to provide an internationally standardized and evidence-based foundation for qualified decision-making in the treatment of metastatic breast cancer.

  13. How Follicular Dendritic Cells Shape the B-Cell Antigenome

    PubMed Central

    Kranich, Jan; Krautler, Nike Julia

    2016-01-01

    Follicular dendritic cells (FDCs) are stromal cells residing in primary follicles and in germinal centers of secondary and tertiary lymphoid organs (SLOs and TLOs). There, they play a crucial role in B-cell activation and affinity maturation of antibodies. FDCs have the unique capacity to bind and retain native antigen in B-cell follicles for long periods of time. Therefore, FDCs shape the B-cell antigenome (the sum of all B-cell antigens) in SLOs and TLOs. In this review, we discuss recent findings that explain how this stromal cell type can arise in almost any tissue during TLO formation and, furthermore, focus on the mechanisms of antigen capture and retention involved in the generation of long-lasting antigen depots displayed on FDCs. PMID:27446069

  14. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

    PubMed

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-02-20

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases

    PubMed Central

    Joseph, Raji E.; Kleino, Iivari; Wales, Thomas E.; Xie, Qian; Fulton, D. Bruce; Engen, John R.; Berg, Leslie J.; Andreotti, Amy H.

    2014-01-01

    Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses. PMID:23982207

  16. Neuroprotective effect of chondroitinase ABC on primary and secondary brain injury after stroke in hypertensive rats.

    PubMed

    Chen, Xin-ran; Liao, Song-jie; Ye, Lan-xiang; Gong, Qiong; Ding, Qiao; Zeng, Jin-sheng; Yu, Jian

    2014-01-16

    Focal cerebral infarction causes secondary damage in the ipsilateral ventroposterior thalamic nucleus (VPN). Chondroitin sulfate proteoglycans (CSPGs) are a family of putative inhibitory components, and its degradation by chondroitinase ABC (ChABC) promotes post-injury neurogenesis. This study investigated the role of ChABC in the primary and secondary injury post stroke in hypertension. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), and were subjected to continuous intra-infarct infusion of ChABC (0.12 U/d for 7 days) 24 h later. Neurological function was evaluated by a modified neurologic severity score. Neurons were counted in the peri-infarct region and the ipsilateral VPN 8 and 14 days after MCAO by Nissl staining and NeuN labeling. The expressions of CSPGs, growth-associated protein-43 (GAP-43) and synaptophysin (SYN) were detected with immunofluorescence or Western blotting. The intra-infarct infusion of ChABC, by degrading accumulated CSPGs, rescued neuronal loss and increased the levels of GAP-43 and SYN in both the ipsilateral cortex and VPN, indicating enhancd neuron survival as well as augmented axonal growth and synaptic plasticity, eventually improving overall neurological function. The study demonstrated that intra-infarct ChABC infusion could salvage the brain from both primary and secondary injury by the intervention on the neuroinhibitory environment post focal cerebral infarction. © 2013 Published by Elsevier B.V.

  17. HIF-1α regulates epithelial inflammation by cell autonomous NFκB activation and paracrine stromal remodeling

    PubMed Central

    Scortegagna, Marzia; Cataisson, Christophe; Martin, Rebecca J.; Hicklin, Daniel J.; Schreiber, Robert D.; Yuspa, Stuart H.

    2008-01-01

    Hypoxia inducible factor-1 (HIF-1) is a master regulatory transcription factor controlling multiple cell-autonomous and non–cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal keratinocytes and demonstrated a signaling pathway from HIF-1 to nuclear factor κ B (NFκB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 12-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1–induced NFκB activation was composed of 2 elements, IκB hyperphosphorylation and phosphorylation of Ser276 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFκB transcriptional targets macrophage inflammatory protein-2 (MIP-2/CXCL2/3), keratinocyte chemokine (KC/CXCL1), and tumor necrosis factor [alfa] (TNFα) were constitutively up-regulated and further increased after TPA challenge both in cultured keratinocytes and in transgenic mice. Whole animal KC, MIP-2, or TNFα immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta growth factor (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFκB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression. PMID:18199827

  18. Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function

    PubMed Central

    Woltman, Andrea M.; Shi, Cui C.; Janssen, Harry L. A.

    2011-01-01

    Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNα production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNα induction in animal models and patients and chronic HBV patients display impaired IFNα production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTOR-mediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNα gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFα, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNα production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV. PMID:21246041

  19. Fungal-Induced Cell Cycle Impairment, Chromosome Instability and Apoptosis via Differential Activation of NF-κB

    PubMed Central

    Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

    2012-01-01

    Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a

  20. Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-κB.

    PubMed

    Ben-Abdallah, Mariem; Sturny-Leclère, Aude; Avé, Patrick; Louise, Anne; Moyrand, Frédérique; Weih, Falk; Janbon, Guilhem; Mémet, Sylvie

    2012-01-01

    Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a

  1. Bacterial Pathogens Induce Abscess Formation by CD4+ T-Cell Activation via the CD28–B7-2 Costimulatory Pathway

    PubMed Central

    Tzianabos, Arthur O.; Chandraker, Anil; Kalka-Moll, Wiltrud; Stingele, Francesca; Dong, Victor M.; Finberg, Robert W.; Peach, Robert; Sayegh, Mohamed H.

    2000-01-01

    Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naïve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, including Staphylococcus aureus, Bacteroides fragilis, and a combination of Enterococcus faecium and Bacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28–B7-2 pathway. PMID:11083777

  2. Noncanonical NF-κB Signaling Is Limited by Classical NF-κB Activity

    PubMed Central

    Gray, Carolyn M.; Remouchamps, Caroline; McCorkell, Kelly A.; Solt, Laura A.; Dejardin, Emmanuel; Orange, Jordan S.; May, Michael J.

    2014-01-01

    Precise regulation of nuclear factor κB (NF-κB) signaling is crucial for normal immune responses, and defective NF-κB activity underlies a range of immunodeficiencies. NF-κB is activated through two signaling cascades: the classical and noncanonical pathways. The classical pathway requires inhibitor of κB kinase β (IKKβ) and NF-κB essential modulator (NEMO), and hypomorphic mutations in the gene encoding NEMO (ikbkg) lead to inherited immunodeficiencies, collectively termed NEMO-ID. Noncanonical NF-κB activation requires NF-κB–inducing kinase (NIK) and IKKα, but not NEMO. We found that noncanonical NF-κB was basally active in peripheral blood mononuclear cells from NEMO-ID patients, and that noncanonical NF-κB signaling was similarly enhanced in cell lines lacking functional NEMO. NIK, which normally undergoes constitutive degradation, was aberrantly present in resting NEMO-deficient cells, and regulation of its abundance was rescued by reconstitution with full-length NEMO, but not a mutant NEMO protein unable to physically associate with IKKα or IKKβ. Binding of NEMO to IKKα was not required for ligand-dependent stabilization of NIK or noncanonical NF-κB signaling. Rather, an intact and functional IKK complex was essential to suppress basal NIK activity in unstimulated cells. Despite interacting with IKKα and IKKβ to form an IKK complex, NEMO mutants associated with immunodeficiency failed to rescue classical NF-κB signaling or reverse the accumulation of NIK. Together, these findings identify a crucial role for classical NF-κB activity in the suppression of basal noncanonical NF-κB signaling. PMID:24497610

  3. Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

    PubMed

    Hoogendoorn, M; Wolbers, J Olde; Smit, W M; Schaafsma, M R; Barge, R M Y; Willemze, R; Falkenburg, J H F

    2004-07-01

    Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

  4. Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner.

    PubMed

    Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M; Strasser, Andreas; Heierhorst, Jörg

    2018-01-01

    The proapoptotic BH3-only protein BIM ( Bcl2l11 ) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre ) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre ) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim -deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro , conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc -driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions.

  5. Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner

    PubMed Central

    Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M.; Strasser, Andreas; Heierhorst, Jörg

    2018-01-01

    The proapoptotic BH3-only protein BIM (Bcl2l11) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim-deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro, conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc-driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions. PMID:29623080

  6. The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells.

    PubMed

    Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

    2017-03-01

    Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immunomodulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17‑34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17‑34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17‑34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17‑34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17‑34-mediated pigmentation. Taken together, these results suggest that LfB17‑34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17‑34 could be further developed for the treatment of hypopigmentation disorders.

  7. The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells

    PubMed Central

    Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

    2017-01-01

    Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immuno-modulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17-34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17-34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17-34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17-34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17-34-mediated pigmentation. Taken together, these results suggest that LfB17-34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17-34 could be further developed for the treatment of hypopigmentation disorders. PMID:28204812

  8. Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

    PubMed Central

    Harris, Deshea L.

    2007-01-01

    Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

  9. Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

    PubMed

    Ridley, Anna; Hatano, Hiroko; Wong-Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K; Al-Mossawi, Hussein; Ladell, Kristin; Price, David A; Bowness, Paul; Kollnberger, Simon

    2016-04-01

    In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA. © 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

  10. Our bodies are our own: resistance to ABC-based HIV-prevention programmes in northern Tanzanian conservation organisations.

    PubMed

    Reid-Hresko, John

    2014-01-01

    ABC-based HIV-prevention programmes have been widely employed in northern Tanzanian wildlife conservation settings in an attempt to (re)shape the sexual behaviours of conservation actors. Utilising findings from 66 semi-structured interviews conducted in 2009-2010, this paper examines ABC prevention as a form of Foucauldian governmentality--circulating technologies of power that mobilise disciplinary technologies and attempt to transform such efforts into technologies of the self--and explores how individuals understand and respond to attempts to govern their behaviour. ABC regimes attempt to rework subjectivity, positioning HIV-related behaviours within a risk-based neoliberal rationality. However, efforts to use ABC as a technology to govern populations and individual bodies are largely incommensurate with existing Tanzanian sociocultural formations, including economic and gendered inequalities, and local understandings of sexuality. The language research participants used to talk about ABC and the justifications they offered for non-compliance illuminate this discrepancy. Data reveal that the recipients of ABC campaigns are active producers of understandings that work for them in their lives, but may not produce the behavioural shifts envisioned by programme goals. These findings corroborate previous research, which questions the continued plausibility of ABC as a stand-alone HIV- prevention framework.

  11. Lipoic Acid Decreases the Viability of Breast Cancer Cells and Activity of PTP1B and SHP2.

    PubMed

    Kuban-Jankowska, Alicja; Gorska-Ponikowska, Magdalena; Wozniak, Michal

    2017-06-01

    Protein tyrosine phosphatases PTP1B and SHP2 are potential targets for anticancer therapy, because of the essential role they play in the development of tumors. PTP1B and SHP2 are overexpressed in breast cancer cells, thus inhibition of their activity can be potentially effective in breast cancer therapy. Lipoic acid has been previously reported to inhibit the proliferation of colon, breast and thyroid cancer cells. We investigated the effect of alpha-lipoic acid (ALA) and its reduced form of dihydrolipoic acid (DHLA) on the viability of MCF-7 cancer cells and on the enzymatic activity of PTP1B and SHP2 phosphatases. ALA and DHLA decrease the activity of PTP1B and SHP2, and have inhibitory effects on the viability and proliferation of breast cancer cells. ALA and DHLA can be considered as potential agents for the adjunctive treatment of breast cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  12. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    PubMed

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-05-01

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  13. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    PubMed

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  14. The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells.

    PubMed

    Byrd, Matthew S; Pang, Bing; Mishra, Meenu; Swords, W Edward; Wozniak, Daniel J

    2010-06-29

    In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213-8221, 2006). Using an NF-kappaB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-kappaB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-kappaB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-kappaB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

  15. The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

    PubMed Central

    Byrd, Matthew S.; Pang, Bing; Mishra, Meenu; Swords, W. Edward; Wozniak, Daniel J.

    2010-01-01

    In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells. PMID:20802825

  16. β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

    PubMed

    Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

    2017-01-01

    The aim of this study was to explore the effects of β 3 -adrenoceptor (β 3 -AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β 3 -AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β 3 -AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining 3 H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β 3 -AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β 3 -AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β 3 -AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

  17. Human T-Cell Leukemia Virus Type 1 Tax Induction of NF-κB Involves Activation of the IκB Kinase α (IKKα) and IKKβ Cellular Kinases

    PubMed Central

    Geleziunas, Romas; Ferrell, Sharon; Lin, Xin; Mu, Yajun; Cunningham, Emmett T.; Grant, Mark; Connelly, Margery A.; Hambor, John E.; Marcu, Kenneth B.; Greene, Warner C.

    1998-01-01

    Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-κB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IκBα, a principal cytoplasmic inhibitor of NF-κB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IκB kinase α (IKKα) and IKKβ, which normally phosphorylate IκBα on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-κB, fails to activate either IKKα or IKKβ. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKα and IKKβ into either human Jurkat T or 293 cells also inhibits NF-κB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-κB-inducing kinase), which represents an upstream kinase in the TNF-α and IL-1 signaling pathways leading to IKKα and IKKβ activation, blocks Tax induction of NF-κB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-α and IL-1 receptors, respectively, do not inhibit Tax induction of NF-κB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-κB. The pathological alteration of this cytokine pathway leading to NF-κB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia. PMID

  18. Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB, uPA activator and MMP9.

    PubMed

    Tong, Weihua; Wang, Quan; Sun, Donghui; Suo, Jian

    2016-11-01

    Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

  19. Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells

    PubMed Central

    Rakhmanov, Mirzokhid; Keller, Baerbel; Gutenberger, Sylvia; Foerster, Christian; Hoenig, Manfred; Driessen, Gertjan; van der Burg, Mirjam; van Dongen, Jacques J.; Wiech, Elisabeth; Visentini, Marcella; Quinti, Isabella; Prasse, Antje; Voelxen, Nadine; Salzer, Ulrich; Goldacker, Sigune; Fisch, Paul; Eibel, Hermann; Schwarz, Klaus; Peter, Hans-Hartmut; Warnatz, Klaus

    2009-01-01

    The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population. PMID:19666505

  20. Spatio-temporal modelling of the NF-κB intracellular signalling pathway: the roles of diffusion, active transport, and cell geometry.

    PubMed

    Terry, Alan J; Chaplain, Mark A J

    2011-12-07

    The nuclear factor kappa B (NF-κB) intracellular signalling pathway is central to many stressful, inflammatory, and innate immune responses. NF-κB proteins themselves are transcription factors for hundreds of genes. Experiments have shown that the NF-κB pathway can exhibit oscillatory dynamics-a negative feedback loop causes oscillatory nuclear-cytoplasmic translocation of NF-κB. Given that cell size and shape are known to influence intracellular signal transduction, we consider a spatio-temporal model of partial differential equations for the NF-κB pathway, where we model molecular movement by diffusion and, for several key species including NF-κB, by active transport as well. Through numerical simulations we find values for model parameters such that sustained oscillatory dynamics occur. Our spatial profiles and animations bear a striking resemblance to experimental images and movie clips employing fluorescent fusion proteins. We discover that oscillations in nuclear NF-κB may occur when active transport is across the nuclear membrane only, or when no species are subject to active transport. However, when active transport is across the nuclear membrane and NF-κB is additionally actively transported through the cytoplasm, oscillations are lost. Hence transport mechanisms in a cell will influence its response to activation of its NF-κB pathway. We also demonstrate that sustained oscillations in nuclear NF-κB are somewhat robust to changes in the shape of the cell, or the shape, location, and size of its nucleus, or the location of ribosomes. Yet if the cell is particularly flat or the nucleus sufficiently small, then oscillations are lost. Thus the geometry of a cell may partly determine its response to NF-κB activation. The NF-κB pathway is known to be constitutively active in several human cancers. Our spatially explicit modelling approach will allow us, in future work, to investigate targeted drug therapy of tumours. Copyright © 2011 Elsevier Ltd

  1. CD24(hi)CD27(+) B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide.

    PubMed

    van der Vlugt, L E P M; Mlejnek, E; Ozir-Fazalalikhan, A; Janssen Bonas, M; Dijksman, T R; Labuda, L A; Schot, R; Guigas, B; Möller, G M; Hiemstra, P S; Yazdanbakhsh, M; Smits, H H

    2014-04-01

    Regulatory B cells have been identified that strongly reduce allergic and auto-immune inflammation in experimental models by producing IL-10. Recently, several human regulatory B-cell subsets with an impaired function in auto-immunity have been described, but there is no information on regulatory B cells in allergic asthma. In this study, the frequency and function of IL-10 producing B-cell subsets in allergic asthma were investigated. Isolated peripheral blood B cells from 13 patients with allergic asthma and matched healthy controls were analyzed for the expression of different regulatory B-cell markers. Next, the B cells were activated by lipopolysaccharide (LPS), CpG or through the B-cell receptor, followed by co-culture with endogenous memory CD4(+) T cells and house dust mite allergen DerP1. Lower number of IL-10 producing B cells were found in patients in response to LPS, however, this was not the case when B cells were activated through the B-cell receptor or by CpG. Further dissection showed that only the CD24(hi)CD27(+) B-cell subset was reduced in number and IL-10 production to LPS. In response to DerP1, CD4(+) T cells from patients co-cultured with LPS-primed total B cells produced less IL-10 compared to similar cultures from controls. These results are in line with the finding that sorted CD24(hi)CD27(+) B cells are responsible for the induction of IL-10(+) CD4(+) T cells. Taken together, these data indicate that CD24(hi)CD27(+) B cells from allergic asthma patients produce less IL-10 in response to LPS leading to a weaker IL-10 induction in T cells in response to DerP1, which may play a role in allergic asthma. © 2013 John Wiley & Sons Ltd.

  2. The Structure of Urease Activation Complexes Examined by Flexibility Analysis, Mutagenesis, and Small-Angle X-Ray Scattering

    PubMed Central

    Quiroz-Valenzuela, Soledad; Sukuru, Sai Chetan K.; Hausinger, Robert P.; Kuhn, Leslie A.; Heller, William T.

    2008-01-01

    Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC)3 induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle x-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD)3, and (UreABC-UreDF)3 confirm that UreD and UreF bind near UreB at the periphery of the (UreAC)3 structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF)3 allows CO2 and nickel ions to gain access to the nascent active site. PMID:18823937

  3. T cell-dependent antibody production by Ly-1 B cells.

    PubMed

    Taki, S; Schmitt, M; Tarlinton, D; Förster, I; Rajewsky, K

    1992-05-04

    Through the use of a SCID transfer system, we have demonstrated that under certain conditions, the production of Ig by Ly-1 B cells can be modulated by T cells. This modulation can take the form of enhanced isotype production or isotype-switch induction and to some extent appears to be dependent on the activation state of the T cells. Furthermore we have shown that Ly-1 B cells can mount an idiotypically restricted T cell-dependent immune response to the antigen PC-KLH. This result suggests that the previous failure to observe T cell-dependent responses by Ly-1 B cells has been due to these B cells being "blind" to the antigens used and is not due to some inherent property of these B cells. When one considers the previous reports of the substantial contribution of Ly-1 B cells to the natural serum immunoglobulin levels and the ability of T cells to affect Ig production by Ly-1 B cells documented in this report, it is clear that the interaction of T cells with the Ly-1 B-cell population is important in determining the "natural" serum Ig repertoire of the mouse.

  4. 2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

    PubMed Central

    Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

    2005-01-01

    Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with ≤ 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3− CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0·001) but increased to a normal level during the 24 months’ follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0·001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0·05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells. PMID:16045743

  5. 2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

    PubMed

    Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

    2005-09-01

    Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

  6. Down-regulation of CD73 on B cells of patients with viremic HIV correlates with B cell activation and disease progression.

    PubMed

    Kim, Eun-Seong; Ackermann, Christin; Tóth, Ilona; Dierks, Patrick; Eberhard, Johanna M; Wroblewski, Raluca; Scherg, Felix; Geyer, Matthias; Schmidt, Reinhold E; Beisel, Claudia; Bockhorn, Maximilian; Haag, Friedrich; van Lunzen, Jan; Schulze Zur Wiesch, Julian

    2017-05-01

    Recently, alterations of the T cell expression of the ectonucleotidases, CD39 and CD73, during HIV infection have been described. Here, peripheral ( n = 70) and lymph nodal B cells ( n = 10) of patients with HIV at different stages of disease as well as uninfected individuals were analyzed via multicolor flow cytometry with regard to expression of CD39 and CD73 and differentiation, proliferation, and exhaustion status. Patients with chronic, untreated HIV showed a significantly decreased frequency of CD73-expressing B cells ( P < 0.001) compared with healthy controls. Decreased frequencies of CD39 + CD73 + B cells in patients with HIV correlated with low CD4 + counts ( P < 0.0256) as well as increased proliferation and exhaustion status as determined by Ki-67 and programmed death-1 expression. Down-regulation of CD73 was observed in naive and memory B cells as determined by CD27 and CD21. Neither HIV elite controller patients nor antiretroviral therapy-treated patients had significantly lower CD39 and CD73 expression on B cells compared with healthy controls. Of importance, low CD73 + expression on B cells was associated with modulated in vitro B cell function. Further in vivo studies are warranted to evaluate the in vivo role of phenotypic loss of CD73 in B cell dysregulation in HIV. © Society for Leukocyte Biology.

  7. Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK.

    PubMed

    Kim, Ha-Na; Kim, Do-Hee; Kim, Eun-Hee; Lee, Mee-Hyun; Kundu, Joydeb Kumar; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

    2014-08-28

    Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK-IKKβ. Copyright © 2014 Elsevier Ireland Ltd. All rights

  8. Antiproliferative activity of Alisol B in MDA-MB-231 cells is mediated by apoptosis, dysregulation of mitochondrial functions, cell cycle arrest and generation of reactive oxygen species.

    PubMed

    Zhang, Aifeng; Sheng, Yuqing; Zou, Mingchang

    2017-03-01

    Previous studies have demonstrated that Alisol B has inhibitory activity in cancer cells. However, the exact mechanism through which inhibition is achieved is still poorly understood. In the present study, the authors examined the effects of Alisol B in human breast cancer cells. Alisol B showed significant anticancer activity in MDA-MB-231 cells. The results demonstrated that the cytotoxicity induced by Alisol B was mediated by induction of apoptosis, decrease in mitochondrial membrane potential, cell cycle arrest, activation of caspases and accumulation of ROS (reactive oxygen species) level. Interestingly, pretreatment of cells with the general caspase inhibitor z-VAD-FMK significantly prevented Alisol B-induced apoptosis. Furthermore, western blot analysis revealed the upregulation of p-p38 and downregulation of p-AKT, p-p65 and p-mTOR. Taken together, the above results suggest that Alisol B suppresses the growth of MDA-MB-231 cells mainly through induction of apoptosis; this outcome may represent the major mechanism of Alisol B-mediated apoptosis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Read, David J.; Langford, Lynda; Barbour, Helen R.

    2007-03-15

    Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [{sup 14}C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [{sup 14}C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTEmore » inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [{sup 14}C]phosphatidylcholine to [{sup 14}C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with > 80% inhibition of [{sup 14}C]GroPCho production, increased [{sup 14}C]phosphatidylcholine was not detected. At concentrations of 1-10 {mu}M, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.« less

  10. The Deadly Dance of B Cells with Trypanosomatids.

    PubMed

    Silva-Barrios, Sasha; Charpentier, Tania; Stäger, Simona

    2018-02-01

    B cells are notorious actors for the host's protection against several infectious diseases. So much so that early vaccinology seated its principles upon their long-term protective antibody secretion capabilities. Indeed, there are many examples of acute infectious diseases that are combated by functional humoral responses. However, some chronic infectious diseases actively induce immune deregulations that often lead to defective, if not deleterious, humoral immune responses. In this review we summarize how Leishmania and Trypanosoma spp. directly manipulate B cell responses to induce polyclonal B cell activation, hypergammaglobulinemia, low-specificity antibodies, limited B cell survival, and regulatory B cells, contributing therefore to immunopathology and the establishment of persistent infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, I-Min; Chu, Ken; Ratner, Anna

    2014-10-28

    In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorwaymore » to a new era in the discovery of novel molecules.« less

  12. PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zheng, Peihao; Guo, Honggang; Li, Guangchao

    2015-03-06

    Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expressionmore » of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.« less

  13. Tangeretin Inhibits IL-12 Expression and NF-κB Activation in Dendritic Cells and Attenuates Colitis in Mice.

    PubMed

    Eun, Su-Hyeon; Woo, Je-Te; Kim, Dong-Hyun

    2017-04-01

    In the preliminary study, tangeretin (5,6,7,8,4'-pentamethoxy flavone), a major constituent of the pericarp of Citrus sp., inhibited TNF- α , IL-12, and IL-23 expression and nuclear factor kappa-B activation in lipopolysaccharide-stimulated dendritic cells; however, it did not affect IL-10 expression. Furthermore, tangeretin (5, 10, and 20 µM) suppressed the activation and translocation of nuclear factor kappa-B (p65) into the nuclei in vitro by inhibiting the binding of lipopolysaccharide on dendritic cells. Oral administration of tangeretin (10 and 20 mg/kg) suppressed the inflammatory responses, such as nuclear factor kappa-B and mitogen-activated protein kinase activation and myeloperoxidase activity, in the colon of mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed expression of tight junction proteins occludin, claudin-1, and ZO-1. Tangeretin also inhibited 2,4,6-trinitrobenzene sulfonic acid-induced differentiation of Th1 and Th17 cells as well as the expression of T-bet, ROR γ t, interferon- γ , IL-12, IL-17, and TNF- α . However, tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed differentiation of regulatory T cells as well as the expression of Foxp3 and IL-10. These results suggest that oral administration of tangeretin may attenuate colitis by suppressing IL-12 and TNF- α expression and nuclear factor kappa-B activation through the inhibition of lipopolysaccharide binding on immune cells such as dendritic cells. Georg Thieme Verlag KG Stuttgart · New York.

  14. Murine Visceral Leishmaniasis: IgM and Polyclonal B-Cell Activation Lead to Disease Exacerbation

    PubMed Central

    Deak, Eszter; Jayakumar, Asha; Wing Cho, Ka; Goldsmith-Pestana, Karen; Dondji, Blaise; Lambris, John D.; McMahon-Pratt, Diane

    2010-01-01

    In visceral leishmaniasis, the draining lymph node (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with L. infantum. Although not unexpected, at early times post-infection there is a marked B cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JHD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JHD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JHD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to wild type BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5aR (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-γ). Overall these studies indicate that polyclonal B cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention. PMID:20213734

  15. Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation.

    PubMed

    Cerqueira, Sofia A; Tan, Min; Li, Shijun; Juillard, Franceline; McVey, Colin E; Kaye, Kenneth M; Simas, J Pedro

    2016-09-01

    Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5S(mLANA)) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on

  16. The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells

    PubMed Central

    Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F.; Schett, Georg

    2014-01-01

    The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression. PMID:25288397

  17. MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells

    PubMed Central

    Botta, C; Cucè, M; Pitari, M R; Caracciolo, D; Gullà, A; Morelli, E; Riillo, C; Biamonte, L; Gallo Cantafio, M E; Prabhala, R; Mignogna, C; Di Vito, A; Altomare, E; Amodio, N; Di Martino, M T; Correale, P; Rossi, M; Giordano, A; Munshi, N C; Tagliaferri, P; Tassone, P

    2018-01-01

    Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients’ adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM. PMID:29158557

  18. Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors

    PubMed Central

    Scharfstein, Julio; Schmitz, Veronica; Morandi, Veronica; Capella, Marcia M. A.; Lima, Ana Paula C. A.; Morrot, Alexandre; Juliano, Luiz; Müller-Esterl, Werner

    2000-01-01

    The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease. PMID:11067878

  19. IkappaB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-kappaB activity by glycine chloramine through methionine oxidation.

    PubMed

    Midwinter, Robyn G; Cheah, Fook-Choe; Moskovitz, Jackob; Vissers, Margret C; Winterbourn, Christine C

    2006-05-15

    Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.

  20. The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

    PubMed Central

    Rauert-Wunderlich, Hilka; Siegmund, Daniela; Maier, Eduard; Giner, Tina; Bargou, Ralf C.; Wajant, Harald; Stühmer, Thorsten

    2013-01-01

    Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. PMID:23527154

  1. ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet–Drug Interactions

    PubMed Central

    Telang, Urvi; Ji, Yan; Morris, Marilyn E.

    2013-01-01

    Isothiocyanates, a class of anti-cancer agents, are derived from cruciferous vegetables such as broccoli, cabbage and watercress, and have demonstrated chemopreventive activity in a number of cancer models and epidemiologic studies. Due to public interest in cancer prevention and alternative therapies in cancer, the consumption of herbal supplements and vegetables containing these compounds is widespread and increasing. Isothiocyanates interact with ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, MRP1, MRP2 and BCRP, and may influence the pharmacokinetics of substrates of these transporters. This review discusses the pharmacokinetic properties of isothiocyanates, their interactions with ABC transporters, and presents some data describing the potential for isothiocyanate-mediated diet–drug interactions. PMID:19623673

  2. Anti-inflammatory activities of fenoterol through β-arrestin-2 and inhibition of AMPK and NF-κB activation in AICAR-induced THP-1 cells.

    PubMed

    Wang, Wei; Chen, Jing; Li, Xiao Guang; Xu, Jie

    2016-12-01

    The AMP-activated protein kinase (AMPK) pathway has been shown to be able to regulate inflammation in several cell lines. We reported that fenoterol, a β 2 -adrenergic receptor (β 2 -AR) agonist, inhibited lipopolysaccharide (LPS)-induced AMPK activation and inflammatory cytokine production in THP-1 cells, a monocytic cell line in previous studies. 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an agonist of AMPK. Whether AICAR induced AMPK activation and inflammatory cytokine production in THP-1 cells can be inhibited by fenoterol is unknown. In this study, we explored the mechanism of β 2 -AR stimulation with fenoterol in AICAR-induced inflammatory cytokine secretion in THP-1 cells. We studied AMPK activation using p-AMPK and AMPK antibodies, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion in THP-1 cells stimulated by β 2 -AR in the presence or absence of AICAR and small interfering RNA (siRNA)-mediated knockdown of β-arrestin-2 or AMPKα1 subunit. AICAR-induced AMPK activation, NF-κB activation and tumor necrosis factor (TNF)-α release were reduced by fenoterol. In addition, siRNA-mediated knockdown of β-arrestin-2 abolished fenoterol's inhibition of AICAR-induced AMPK activation and TNF-α release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol in AICAR-treated THP-1 cells. Furthermore, siRNA-mediated knockdown of AMPKα1 significantly attenuated AICAR-induced NF-κB activation and TNF-α release, so AMPKα1 was a key signaling molecule involved in AICAR-induced inflammatory cytokine production. These data suggested that fenoterol inhibited AICAR-induced AMPK activation and TNF-α release through β-arrestin-2 in THP-1 cells. Management especially inhibition of AMPK signaling may provide new approaches and strategies for the treatments of immune diseases including inflammatory diseases and other critical illness. Published by Elsevier Masson SAS.

  3. Artesunate protects pancreatic beta cells against cytokine-induced damage via SIRT1 inhibiting NF-κB activation.

    PubMed

    Yu, L; Chen, J F; Shuai, X; Xu, Y; Ding, Y; Zhang, J; Yang, W; Liang, X; Su, D; Yan, C

    2016-01-01

    Artesunate (ART) has been known as the most effective and safe reagents to treat malaria for many years. In this study, we explored whether ART could protect pancreatic beta-cell against cytokine-induced damage. The production of nitrite (NO) was detected with the Griess Assay Kit. SIRT1 and inducible nitric oxide synthase (iNOS) expression were determined with Western blot. The transcriptional activity of NF-κB was evaluated by luciferase reporter assay. The expression of Sirt1 was silenced by RNA interference. Glucose-stimulated insulin secretion (GSIS) and potassium-stimulated insulin secretion (KSIS) assays were performed to measure the effect of ART on pancreatic beta-cells' function. The effect of ART on beta-cells apoptosis was evaluated by using Hochest/PI staining and TUNEL assay. ART enhanced GSIS (KSIS) and reduced apoptosis of pancreatic beta-cells induced by IL-1β. Further study showed that ART inhibited IL-1β-induced increase of NF-κB activity, iNOS expression, and NO production. Moreover, ART up-regulated SIRT1 expression in INS-1 cells and islets exposed to IL-1β. Inhibition of SIRT1 expression could partially abolished the inhibitory effect of ART on NF-κB activity in IL-1β-treated beta-cells. More importantly, the protective effect of ART on cytokine-induced damage was reversed by silencing SIRT1 expression. ART can elicit a protective effect on beta-cells exposed to IL-1β by stimulating SIRT1 expression, which resulted in the decrease of NF-κB activity, iNOS expression, and NO production. Hence, ART might be an effective drug for diabetes.

  4. NF-{kappa}B signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sakuma, Yuji, E-mail: ysakuma@gancen.asahi.yokohama.jp; Yamazaki, Yukiko; Nakamura, Yoshiyasu

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer EGFR-mutant cells in 3D culture resist EGFR inhibition compared with suspended cells. Black-Right-Pointing-Pointer Degradation of I{kappa}B and activation of NF-{kappa}B are observed in 3D-cultured cells. Black-Right-Pointing-Pointer Inhibiting NF-{kappa}B enhances the efficacy of the EGFR inhibitor in 3D-cultured cells. -- Abstract: Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cellsmore » cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of I{kappa}B{alpha}, the inhibitor of nuclear factor (NF)-{kappa}B, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-{kappa}B. Moreover, the inhibition of NF-{kappa}B with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-{kappa}B signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.« less

  5. T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

    PubMed

    Bell, Catherine C; Faulkner, Lee; Martinsson, Klara; Farrell, John; Alfirevic, Ana; Tugwood, Jonathan; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

    2013-05-20

    Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

  6. NF-κB activation primes cells to a pro-inflammatory polarized response to a TLR7 agonist

    PubMed Central

    Lee, Jongdae; Hayashi, Masaaki; Lo, Jeng-Fan; Fearns, Colleen; Chu, Wen-Ming; Luo, Yunping; Xiang, Rong; Chuang, Tsung-Hsien

    2009-01-01

    Toll-like receptor 7 (TLR7) mediates anti-viral immunity by recognizing ssRNA viruses. Small molecular weight TLR7 agonists have been approved, or are being evaluated, for treatment of cancers or infectious diseases. Although TLR7 is predominantly expressed in a restricted set of immune cell types including plasmacytoid dendritic cells (pDCs), it is also expressed in non-native expressing cells (e.g., hepatocytes) under certain circumstances. To elucidate the molecular basis of TLR7 induction by pro-inflammatory stimulation and the subsequent cellular responses in these non-native TLR7-expressing cell types, we firstly cloned and characterized the 5′-promoter region of TLR7. The proximal region of this promoter drives the transcription of the TLR7 gene. Pro-inflammatory stimuli activated TLR7 transcription via a NF-κB binding motif in this region, and this activation could be blocked by mutation of the NF-κB binding site or addition of NF-κB inhibitors. Further studies showed that pretreatment of the Hep3B hepatocytes with TNF-α or IL-1 rendered them responsive to TLR7 activation by a TLR7 agonist. However, distinct from TLR7 activation in pDCs, which respond to stimulation with Th1 polarized cytokine production, TLR7 induction by pro-inflammatory signals in hepatocytes reconstitutes the NF-κB-dependent cascade but not the IRF7-dependent cascade, resulting in a pro-inflammatory polarized response rather than a Th1 polarized response. These results indicate that inflammatory stimulation is capable of priming cells to respond to TLR7 agonist with an immune response that differs from that in native TLR7-expressing cells. PMID:19426145

  7. CD40-Mediated NF-κB Activation in B Cells Is Increased in Multiple Sclerosis and Modulated by Therapeutics.

    PubMed

    Chen, Ding; Ireland, Sara J; Remington, Gina; Alvarez, Enrique; Racke, Michael K; Greenberg, Benjamin; Frohman, Elliot M; Monson, Nancy L

    2016-12-01

    CD40 interacts with CD40L and plays an essential role in immune regulation and homeostasis. Recent research findings, however, support a pathogenic role of CD40 in a number of autoimmune diseases. We previously showed that memory B cells from relapsing-remitting multiple sclerosis (RRMS) patients exhibited enhanced proliferation with CD40 stimulation compared with healthy donors. In this study, we used a multiparameter phosflow approach to analyze the phosphorylation status of NF-κB and three major MAPKs (P38, ERK, and JNK), the essential components of signaling pathways downstream of CD40 engagement in B cells from MS patients. We found that memory and naive B cells from RRMS and secondary progressive MS patients exhibited a significantly elevated level of phosphorylated NF-κB (p-P65) following CD40 stimulation compared with healthy donor controls. Combination therapy with IFN-β-1a (Avonex) and mycophenolate mofetil (Cellcept) modulated the hyperphosphorylation of P65 in B cells of RRMS patients at levels similar to healthy donor controls. Lower disease activity after the combination therapy correlated with the reduced phosphorylation of P65 following CD40 stimulation in treated patients. Additionally, glatiramer acetate treatment also significantly reduced CD40-mediated P65 phosphorylation in RRMS patients, suggesting that reducing CD40-mediated p-P65 induction may be a general mechanism by which some current therapies modulate MS disease. Copyright © 2016 by The American Association of Immunologists, Inc.

  8. In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex.

    PubMed

    Dong, Ming; Gonzalez, Tara D; Klems, Meghan M; Steinberg, Lisa M; Chen, Wilfred; Papoutsakis, Eleftherios T; Bahnson, Brian J

    2017-09-01

    Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243-1249, 2017. © 2017 American Institute of Chemical Engineers.

  9. Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis

    PubMed Central

    Chousterman, Benjamin G.; Hilgendorf, Ingo; Robbins, Clinton S.; Theurl, Igor; Gerhardt, Louisa M.S.; Iwamoto, Yoshiko; Quach, Tam D.; Ali, Muhammad; Chen, John W.; Rothstein, Thomas L.; Nahrendorf, Matthias; Weissleder, Ralph

    2014-01-01

    Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity. PMID:24821911

  10. The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

    PubMed

    Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

    2011-06-17

    The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

  11. Control of B-cell responses by Toll-like receptors

    NASA Astrophysics Data System (ADS)

    Pasare, Chandrashekhar; Medzhitov, Ruslan

    2005-11-01

    Toll-like receptors (TLRs) detect microbial infection and have an essential role in the induction of immune responses. TLRs can directly induce innate host defence responses, but the mechanisms of TLR-mediated control of adaptive immunity are not fully understood. Although TLR-induced dendritic cell maturation is required for activation of T-helper (TH) cells, the role of TLRs in B-cell activation and antibody production in vivo is not yet known. Here we show that activation and differentiation of TH cells is not sufficient for the induction of T-dependent B-cell responses. We find that, in addition to CD4+ T-cell help, generation of T-dependent antigen-specific antibody responses requires activation of TLRs in B cells.

  12. Decursinol angelate blocks transmigration and inflammatory activation of cancer cells through inhibition of PI3K, ERK and NF-kappaB activation.

    PubMed

    Kim, Won-Jung; Lee, Min-Young; Kim, Jung-Hee; Suk, Kyoungho; Lee, Won-Ha

    2010-10-01

    Inflammation is known to be closely associated with the development of cancer. Decursinol angelate (DA), a coumarin compound isolated from Angelica gigas and related compounds have been shown to possess potent anti-inflammatory activities. However, little is known about their effects on the inflammatory processes associated with cancer. In this study, the anti-inflammatory effect of DA was evaluated in cancer cell lines with respect to cellular invasion through the extracellular matrix (ECM) and the expression of pro-inflammatory mediators such as cytokine, cell adhesion molecules and matrix metalloproteinase (MMP)-9. DA inhibited the invasion of fibrosarcoma cell line, HT1080 and breast cancer cell line, MDA-MB-231 in the Matrigel invasion assay. DA-mediated suppression of cancer cell invasion was accomplished by suppression of PI3K activity known to be associated with cytoskeletal rearrangement related to cellular migration. DA also suppressed the adhesion of cancer cells to ECM mediated by down-regulation of beta(1)-integrin expression levels. Furthermore, DA inhibited the expression of pro-inflammatory cytokines and MMP-9 through suppression of PI3K, ERK and NF-kappaB activation. These results demonstrate that DA suppresses invasion and inflammatory activation of cancer cells through modulation of PI3K/AKT, ERK and NF-kappaB. These anti-inflammatory activities of DA may contribute to its anti-cancer activity. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  13. Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

    PubMed

    Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

    2012-01-01

    Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

  14. Anticancer Effects of the Nitric Oxide-Modified Saquinavir Derivative Saquinavir-NO against Multidrug-Resistant Cancer Cells12

    PubMed Central

    Rothweiler, Florian; Michaelis, Martin; Brauer, Peter; Otte, Jürgen; Weber, Kristoffer; Fehse, Boris; Doerr, Hans Wilhelm; Wiese, Michael; Kreuter, Jörg; Al-Abed, Yousef; Nicoletti, Ferdinando; Cinatl, Jindrich

    2010-01-01

    The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors. PMID:21170266

  15. Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

    PubMed

    Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

    2008-07-01

    Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

  16. B-Cell and T-Cell Immune Responses to Experimental Helicobacter pylori Infection in Humans

    PubMed Central

    Nurgalieva, Zhannat Z.; Conner, Margaret E.; Opekun, Antone R.; Zheng, Carl Q.; Elliott, Susan N.; Ernst, Peter B.; Osato, Michael; Estes, Mary K.; Graham, David Y.

    2005-01-01

    The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development. PMID:15845507

  17. Interleukin-5 regulates genes involved in B-cell terminal maturation.

    PubMed

    Horikawa, Keisuke; Takatsu, Kiyoshi

    2006-08-01

    Interleukin (IL)-5 induces CD38-activated splenic B cells to differentiate into immunoglobulin M-secreting cells and undergo micro to gamma 1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL-4, a well-known IgG1-inducing factor does not induce immunoglobulin production or micro to gamma 1 CSR in CD38-activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL-5-induced gene expression in CD38-stimulated B cells to immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR. IL-5 and IL-4 stimulation of CD38-activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL-5-inducible 78 genes were redundantly regulated by IL-4. IL-5 and IL-4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL-5 include immunoglobulin-related genes such as J chain and immunoglobulinkappa, and genes involved in B-cell maturation such as BCL6, activation-induced cytidine deaminase (Aid) and B lymphocyte-induced maturation protein-1 (Blimp-1) and tend to be induced slowly after IL-5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp-1 and Aid in CD38-activated B cells could induce IL-4-dependent maturation to Syndecan-1+ antibody-secreting cells and micro to gamma 1 CSR, respectively, in CD38-activated B cells. Taken together, preferential Aid and Blimp-1 expression plays a critical role in IL-5-induced immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR in CD38-activated B cells.

  18. Selective blockade of B7-H3 enhances antitumour immune activity by reducing immature myeloid cells in head and neck squamous cell carcinoma.

    PubMed

    Mao, Liang; Fan, Teng-Fei; Wu, Lei; Yu, Guang-Tao; Deng, Wei-Wei; Chen, Lei; Bu, Lin-Lin; Ma, Si-Rui; Liu, Bing; Bian, Yansong; Kulkarni, Ashok B; Zhang, Wen-Feng; Sun, Zhi-Jun

    2017-09-01

    Immature myeloid cells including myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7-H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7-H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7-H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7-H3 blockade as a future therapeutic strategy to treat patients with HNSCC. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  19. A Critical Role for IL-17RB Signaling in HTLV-1 Tax-Induced NF-κB Activation and T-Cell Transformation

    PubMed Central

    Lavorgna, Alfonso; Matsuoka, Masao; Harhaj, Edward William

    2014-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis. PMID:25340344

  20. Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

    PubMed

    Yang, Po-Min; Wu, Zhi-Zhen; Zhang, Yu-Qi; Wung, Being-Sun

    2016-06-15

    Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. SHIP-1 Deficiency in AID+ B Cells Leads to the Impaired Function of B10 Cells with Spontaneous Autoimmunity.

    PubMed

    Chen, Yingjia; Hu, Fanlei; Dong, Xuejiao; Zhao, Meng; Wang, Jing; Sun, Xiaolin; Kim, Tae Jin; Li, Zhanguo; Liu, Wanli

    2017-11-01

    Unlike conventional B cells, regulatory B cells exhibit immunosuppressive functions to downregulate inflammation via IL-10 production. However, the molecular mechanism regulating the production of IL-10 is not fully understood. In this study, we report the finding that activation-induced cytidine deaminase (AID) is highly upregulated in the IL-10-competent B cell (B10) cell from Innp5d fl/fl Aicda Cre/+ mice, whereas the 5' inositol phosphatase SHIP-1 is downregulated. Notably, SHIP-1 deficiency in AID + B cells leads to a reduction in cell count and impaired IL-10 production by B10 cells. Furthermore, the Innp5d fl/fl Aicda Cre/+ mouse model shows B cell-dependent autoimmune lupus-like phenotypes, such as elevated IgG serum Abs, formation of spontaneous germinal centers, production of anti-dsDNA and anti-nuclear Abs, and the obvious deposition of IgG immune complexes in the kidney with age. We observe that these lupus-like phenotypes can be reversed by the adoptive transfer of B10 cells from control Innp5d fl/fl mice, but not from the Innp5d fl/fl Aicda Cre/+ mice. This finding highlights the importance of defective B10 cells in Innp5d fl/fl Aicda Cre/+ mice. Whereas p-Akt is significantly upregulated, MAPK and AP-1 activation is impaired in B10 cells from Innp5d fl/fl Aicda Cre/+ mice, resulting in the reduced production of IL-10. These results show that SHIP-1 is required for the maintenance of B10 cells and production of IL-10, and collectively suggests that SHIP-1 could be a new potential therapeutic target for the treatment of autoimmune diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

  2. p38 mitogen-activated protein kinase-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury.

    PubMed

    Jiang, Shao-Yun; Zou, Yuan-Yuan; Wang, Jian-Tao

    2012-01-01

    In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. These findings provide evidence of crosstalk between p38 MAPK and NF-κB p65 and

  3. p38 mitogen-activated protein kinase–induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury

    PubMed Central

    Jiang, Shao-Yun; Zou, Yuan-Yuan

    2012-01-01

    Purpose In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Methods Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. Results The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. Conclusions These findings provide evidence of crosstalk

  4. TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells

    PubMed Central

    Barr, Tom A; Brown, Sheila; Ryan, Gemma; Zhao, Jiexin; Gray, David

    2007-01-01

    In addition to their role in humoral immunity, B lymphocytes are important antigen-presenting cells (APC). In the same way as other APC, B cells make cytokines upon activation and have the potential to modulate T cell responses. In this study, we investigated which mouse B cell subsets are the most potent cytokine producers, and examined the role of Toll-like receptors (TLR) in the control of secretion of IL-6, IL-10, IL-12 and IFN-γ by B cells. Production of some cytokines was restricted to particular subsets. Marginal zone and B1 cells were the predominant source of B cell IL-10 in the spleen. Conversely, follicular B cells were found to express IFN-γ mRNA directly ex vivo. The nature of the activating stimulus dramatically influenced the cytokine made by B cells. Thus, in response to combined TLR stimulation, or via phorbol esters, IFN-γ was secreted. IL-10 was elicited by T-dependent activation or stimulation through TLR2, 4 or 9. This pattern of cytokine expression contrasts with that elicited from dendritic cells. QRT-PCR array data indicate that this may be due to differential expression of TLR signalling molecules, effectors and adaptors. Our data highlight the potentially unique nature of immune modulation when B cells act as APC. PMID:17918201

  5. Starting with ABC and Finishing with XYZ: What Financial Reporting Model Best Fits a Faculty and Why?

    ERIC Educational Resources Information Center

    Berry, Prudence Jane

    2014-01-01

    This article looks at the range of financial reporting models available for use in the Australian higher education sector, the possible application of activity-based costing (ABC) in faculties and the eventual rejection of ABC in favour of a more qualitative model designed specifically for use in one institution, in a particular Faculty. The…

  6. 7,8-Dihydroxyflavone ameliorates high-glucose induced diabetic apoptosis in human retinal pigment epithelial cells by activating TrkB.

    PubMed

    Yu, Xiaoyi; Liu, Qiuhong; Wang, Xiaochuan; Liu, Hong; Wang, Yan

    2018-01-01

    In diabetic retinopathy, prolonged high-level blood glucose induced significant impairments among various retinal tissues, including retinal pigment epithelial (RPE) cells. In an in vitro model of human RPE cells, we evaluated whether 7,8-Dihydroxyflavone (DHF) may effectively prevent high glucose-induced diabetic apoptosis among human RPE cells. ARPE-19 cells, a Human RPE cell line, were treated with d-glucose (50 mM) to induce apoptosis in vitro. Prior to glucose, ARPE-19 cells were pre-incubated with various concentrations of DHF. The effect of DHF on d-glucose-induced apoptosis was examined by TUNEL assay, in a concentration-dependent manner. The biological effects of DHF on Caspase-9 (Casp-9) and TrkB signaling pathways in d-glucose-injured ARPE-19 cells were evaluated by qRT-PCR and western blot (WB) assays. A TrkB antagonist, K252a, was also applied in DHF and d-glucose treated ARPE-19 cells. Possible effect of K252a blocking TrkB signaling pathway, thus reversing DHF-modulated apoptosis prevention was also examined by TUNEL and WB assays. DHF ameliorated d-glucose-induced diabetic apoptosis in ARPE-19 cells. Apoptotic factor Casp-9, at both mRNA and protein levels, were drastically inhibited by DHF in d-glucose-injured ARPE-19 cells. Also, DHF activated TrkB signaling pathway through phosphorylation. K252a dramatically reversed the preventive effect of DHF on d-glucose-induced apoptosis in ARPE-19 cells. Further investigation showed that K252a functioned through de-activating or de-phosphorylating TrkB signaling pathway. This work demonstrates that DHF, through activation of TrkB signaling pathway, has a preventive function in d-glucose-induced apoptosis in PRE cells in diabetic retinopathy. Copyright © 2017. Published by Elsevier Inc.

  7. Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

    PubMed

    Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

    1992-01-01

    Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

  8. SerpinB1 Promotes Pancreatic β Cell Proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    El Ouaamari, Abdelfattah; Dirice, Ercument; Gedeon, Nicholas

    2016-01-01

    Compensatory β-cell growth in response to insulin resistance is a common feature in diabetes. We recently reported that liver-derived factors participate in this compensatory response in the liver insulin receptor knockout (LIRKO) mouse, a model of significant islet hyperplasia. Here we show that serpinB1 is a liver-derived secretory protein that controls β-cell proliferation. SerpinB1 is abundant in the hepatocyte secretome and sera derived from LIRKO mice. SerpinB1 and small molecule compounds that partially mimic serpinB1 activity enhanced proliferation of zebrafish, mouse and human β-cells. We report that serpinB1-induced β-cell replication requires protease inhibition activity and mice lacking serpinB1 exhibit attenuatedmore » β-cell replication in response to insulin resistance. Finally, SerpinB1-treatment of islets modulated signaling proteins in growth and survival pathways such as MAPK, PKA and GSK3. Together, these data implicate SerpinB1 as a protein that can potentially be harnessed to enhance functional β-cell mass in patients with diabetes.« less

  9. Expression of aberrant HLA-B27 molecules is dependent on B27 dosage and peptide supply.

    PubMed

    McHugh, Kirsty; Rysnik, Oliwia; Kollnberger, Simon; Shaw, Jacqueline; Utriainen, Lotta; Al-Mossawi, Mohammad Hussein; Payeli, Sravan; Belaunzaran, Osiris Marroquin; Milling, Simon; Renner, Christoph; Bowness, Paul

    2014-04-01

    Cellular expression of non-classical forms of human leukocyte antigen (HLA)-B27 (NC-B27) may be involved in spondyloarthritis (SpA) pathogenesis. We used a novel B27-specific monoclonal antibody, HD6, to ask if B27 transgenic (TG) rat splenocytes express these NC-B27 molecules. We also investigated whether B27-binding peptides could affect the expression and functional immune recognition of HD6-reactive B27 molecules. Splenocytes from B27-TG, B7-TG and non-transgenic rats, and HLA-B27+ cell lines were stained with monoclonal antibodies recognising classical (ME-1, HLA-ABC-m1) and non-classical (HD6, HC10) B27. Cells were further cultured in the presence of HLA-B27-binding peptides, or subjected to brief low pH treatment prior to mAb staining and/or immunoprecipitation or co-culture with KIR3DL2-CD3ε-expressing Jurkat reporter cells. HD6-reactive molecules were detected in the majority of adult B27-TG rat splenocyte cell subsets, increasing with age and concomitant increased B27 expression. HD6 staining was inhibited by incubation with B27-binding peptides and induced by low pH treatment. HD6 staining correlated with KIR3DL2-CD3ε-expressing Jurkat reporter cell activity. Thus, IL-2 production was decreased when B27-expressing antigen-presenting cells were preincubated with B27-binding peptides, but increased following pretreatment with low pH buffer. Surface expression of HD6-reactive B27 molecules on B27-TG rat splenocytes is consistent with a pathogenic role for NC-B27 in SpA. Interaction of NC-B27 with innate immune receptors could be critical in SpA pathogenesis, and we show that this may be influenced by the availability and composition of the B27-binding peptide pool.

  10. B cell-intrinsic TLR7 signaling is required for optimal B cell responses during chronic viral infection

    PubMed Central

    Clingan, Jonathan M.; Matloubian, Mehrdad

    2013-01-01

    The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. Toll-like receptors (TLRs) have been demonstrated to be critical for antibody responses to a variety of immunizations. In particular, recent evidence suggests that B cell-intrinsic TLR signaling is required for optimal responses to virus-like antigens, but mechanisms by which TLR signaling impacts antibody responses during infection in vivo is unclear. In the present study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact antibody responses in mice during chronic viral infection. This effect was independent of T follicular helper cells, and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone GC B cells. These results suggest that B cell-intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of antigen-specific B cells in the germinal center. PMID:23761632

  11. B-Cell Activation and Tolerance Mediated by B-Cell Receptor, Toll-Like Receptor, and Survival Signal Crosstalk in SLE Pathogenesis

    DTIC Science & Technology

    2017-09-01

    Dec, 2016 "Integrating innate , adaptive, & survival signals to control B cell selection, homeostasis and tolerance" Pasteur Institute of Shanghai...secondary lymphoid tissues. Aging Dis. 2: 361–373. 8. Goenka, R., J. L. Scholz, M. S. Naradikian, and M. P. Cancro. 2014. Memory B cells form in aged...Scholz, and M. P. Cancro. 2011. A B- cell subset uniquely responsive to innate stimuli accumulates in aged mice. Blood 118: 1294–1304. 10. Rubtsov, A

  12. Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-κB pathways in TNF-α-activated endothelial cells

    PubMed Central

    Chang, Ying-ling; Chen, Chien-lin; Kuo, Chao-Lin; Chen, Bor-chyuan; You, Jyh-sheng

    2010-01-01

    Aim: To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC). Methods: ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [3H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay. Results: GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α. Conclusion: GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis. PMID:20418897

  13. As Easy as ABC: Re-engineering the Cost Accounting System.

    ERIC Educational Resources Information Center

    Trussel, John M.; Bitner, Larry N.

    1996-01-01

    To be useful for management decision making, the college or university's cost accounting system must capture and measure improvements. Activity-based costing (ABC), which determines more accurately the full costs of services and products, tracks improvements and should proceed alongside reengineering of institutional accounting. Guidelines are…

  14. Imatinib Enhances Docetaxel-Induced Apoptosis Through Inhibition of Nuclear Factor-κB Activation in Anaplastic Thyroid Carcinoma Cells

    PubMed Central

    Kim, EunSook; Matsuse, Michiko; Saenko, Vladimir; Suzuki, Keiji; Ohtsuru, Akira; Yamashita, Shunichi

    2012-01-01

    Background We previously reported the partial effectiveness of imatinib (also known as STI571, Glivec, or Gleevec) on anaplastic thyroid cancer (ATC) cells. Imatinib is a selective tyrosine kinase inhibitor that has been used for various types of cancer treatments. Recently, several reports have demonstrated that imatinib enhanced the sensitivity of cancer cells to other anticancer drugs. In this study, therefore, we investigated whether imatinib enhances the antitumor activity of docetaxel in ATC cells. Methods Two ATC cell lines, FRO and KTC-2, were treated with imatinib and/or docetaxel. Cell survival assay and flow cytometry for annexin V were used to assess the induction of apoptosis. Changes of pro- and antiapoptotic factors were determined by Western blot. Nuclear factor-κB (NF-κB) activity was measured by DNA-binding assay. Tumor growth was also investigated in vivo. Results The combined treatment significantly enhanced apoptosis compared with single treatment. ATC cells themselves expressed high levels of antiapoptotic factors, X-linked inhibitor of apoptosis (XIAP), and survivin. The treatment with docetaxel alone further increased their expressions; however, the combined treatment blocked the inductions. Although imatinib alone had no effect on NF-κB background levels, combined treatment significantly suppressed the docetaxel-induced NF-κB activation. Further, the combined administration of the drugs also showed significantly greater inhibitory effect on tumor growth in mice xenograft model. Conclusions Imatinib enhanced antitumor activity of docetaxel in ATC cells. Docetaxel seemed to induce both pro- and antiapoptotic signaling pathways in ATC cells, and imatinib blocked the antiapoptotic signal. Thus, docetaxel combined with imatinib emerges as an attractive strategy for the treatment of ATC. PMID:22650230

  15. B Cell Receptor Signaling-Based Index as a Biomarker for the Loss of Peripheral Immune Tolerance in Autoreactive B Cells in Rheumatoid Arthritis

    PubMed Central

    Lyubchenko, Taras; Zerbe, Gary O.

    2014-01-01

    This study examines the loss of peripherally induced B cell immune tolerance in Rheumatoid arthritis (RA) and establishes a novel signaling-based measure of activation in a subset of autoreactive B cells - the Induced tolerance status index (ITSI). Naturally occurring naïve autoreactive B cells can escape the “classical” tolerogenic mechanisms of clonal deletion and receptor editing, but remain peripherally tolerized through B cell receptor (BCR) signaling inhibition (postdevelopmental “receptor tuning” or anergy). ITSI is a statistical index that numerically determines the level of homology between activation patterns of BCR signaling intermediaries in B cells that are either tolerized or activated by auto antigen exposure, and thus quantifies the level of peripheral immune tolerance. The index is based on the logistic regression analysis of phosphorylation levels in a panel of BCR signaling proteins. Our results demonstrate a new approach to identifying autoreactive B cells based on their BCR signaling features. PMID:25057856

  16. Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jackson, Shawn S.; Medical Scientist Training Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705

    2015-02-01

    The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced bymore » TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO–IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO{sup Y308S} mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo. - Highlights: • Withaferin A, a NF-κB inhibitor, disrupts signaling induced NEMO localization, a novel point of inhibition. • NEMO can be localized to distinct signaling foci after treatment with TNF. • ABC-type DLCBL cells can be sensitized to apoptosis after treatment with Withaferin A.« less

  17. Sustainable urban stormwater management in the tropics: An evaluation of Singapore's ABC Waters Program

    NASA Astrophysics Data System (ADS)

    Lim, H. S.; Lu, X. X.

    2016-07-01

    The Active Beautiful Clean (ABC) Waters Program was implemented in 2006 as part of Singapore's stormwater management strategy and reflects the country's move towards Water Sensitive Urbanism through the adoption of Low-Impact Development (LID) ideology and practices. It is the first holistic and comprehensive LID program in the tropics and holds promise for extension to other tropical cities. This paper presents a comprehensive summary of the goals, LID practices (ABC design features) and design considerations as well as results of several monitored sites, including a constructed wetland, two rain gardens, green roofs and three canal restoration projects. We evaluate the ABC Waters Program based on these initial results and consider the challenges, issues and the research needs for it to meet its hydrological and water quality remediation goals. So far, the ABC design features evaluated perform well in removing particulates. Performance in nutrient removal is poor. With over 60 projects completed within 10 years, post-project monitoring and evaluation is necessary and complements on-going laboratory and modelling research projects conducted by local academic institutions.

  18. ABCdb: an online resource for ABC transporter repertories from sequenced archaeal and bacterial genomes.

    PubMed

    Fichant, Gwennaele; Basse, Marie-Jeanne; Quentin, Yves

    2006-03-01

    The ATP-binding cassette (ABC) transporters are one of the major classes of active transporters. They are widespread in archaea, bacteria, and eukaryota, indicating that they have arisen early in evolution. They are involved in many essential physiological processes, but the majority import or export a wide variety of compounds across cellular membranes. These systems share a common architecture composed of four (exporters) or five (importers) domains. To identify and reconstruct functional ABC transporters encoded by archaeal and bacterial genomes, we have developed a bioinformatic strategy. Cross-reference to the transport classification system is used to predict the type of compound transported. A high quality of annotation is achieved by manual verification of the predictions. However, in order to face the rapid increase in the number of published genomes, we also include analyses of genomes issuing directly from the automated strategy. Querying the database (http://www-abcdb.biotoul.fr) allows to easily retrieve ABC transporter repertories and related data. Additional query tools have been developed for the analysis of the ABC family from both functional and evolutionary perspectives.

  19. Acetylshikonin Inhibits Human Pancreatic PANC-1 Cancer Cell Proliferation by Suppressing the NF-κB Activity.

    PubMed

    Cho, Seok-Cheol; Choi, Bu Young

    2015-09-01

    Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-κB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-α (TNF-α)-induced NF-κB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-κB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer.

  20. Effect of gemfibrozil on apolipoprotein B secretion and diacylglycerol acyltransferase activity in human hepatoblastoma (HepG2) cells.

    PubMed

    Zhu, Daming; Ganji, Shobha H; Kamanna, Vaijinath S; Kashyap, Moti L

    2002-10-01

    The mechanism of action of a widely used drug gemfibrozil to reduce triglycerides (TG) and apolipoprotein B (apo B) is incompletely understood. Using human hepatoblastoma (HepG2) cells, we examined the effect of gemfibrozil on apo B secretion and TG synthesis catalyzed by diacylglycerol acyltransferase (DGAT), primary processes associated with the secretion of LDL. Gemfibrozil significantly decreased apo B secretion by HepG2 cells. It decreased oleate-induced stimulation of apo B secretion, suggesting that gemfibrozil-mediated inhibition of apo B secretion may be dependent on the synthesis of TG catalyzed by DGAT. Pre-incubation of HepG2 cells with gemfibrozil (200-400 micromol/l for 48 h) significantly inhibited microsomal DGAT activity. When added directly to the DGAT assay system containing control microsomes, gemfibrozil significantly inhibited the activity of DGAT by 14-25%. Gemfibrozil (200-400 micromol/l) inhibited TG synthesis by 47-50% as measured by the incorporation of 3H-oleic acid into TG. The data indicate that gemfibrozil inhibits DGAT activity resulting in decreased synthesis of TG and its availability for apo B lipidation rendering it susceptible to intracellular apo B degradation leading to the decreased secretion. These in-vitro data suggest a novel additional mechanism by which gemfibrozil lowers plasma TG and atherogenic apo B lipoproteins in dyslipidemic patients.

  1. Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

    PubMed

    Kiss, Izabella; Unger, Christine; Huu, Chi Nguyen; Atanasov, Atanas Georgiev; Kramer, Nina; Chatruphonprasert, Waranya; Brenner, Stefan; McKinnon, Ruxandra; Peschel, Andrea; Vasas, Andrea; Lajter, Ildiko; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

    2015-01-28

    An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this

  2. Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

    PubMed

    Lee, Edmund C; Fitzgerald, Michael; Bannerman, Bret; Donelan, Jill; Bano, Kristen; Terkelsen, Jennifer; Bradley, Daniel P; Subakan, Ozlem; Silva, Matthew D; Liu, Ray; Pickard, Michael; Li, Zhi; Tayber, Olga; Li, Ping; Hales, Paul; Carsillo, Mary; Neppalli, Vishala T; Berger, Allison J; Kupperman, Erik; Manfredi, Mark; Bolen, Joseph B; Van Ness, Brian; Janz, Siegfried

    2011-12-01

    The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Both cell line-derived OCI-Ly10 and primary human lymphoma-derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) was used to determine antitumor activity and effects on osteolytic bone disease. MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMyc(Cα)/Bcl-X(L) GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. ©2011 AACR.

  3. Antitumor Activity of the Investigational Proteasome Inhibitor MLN9708 in Mouse Models of B-cell and Plasma Cell Malignancies

    PubMed Central

    Lee, Edmund C.; Fitzgerald, Michael; Bannerman, Bret; Donelan, Jill; Bano, Kristen; Terkelsen, Jennifer; Bradley, Daniel P.; Subakan, Ozlem; Silva, Matthew D.; Liu, Ray; Pickard, Michael; Li, Zhi; Tayber, Olga; Li, Ping; Hales, Paul; Carsillo, Mary; Neppalli, Vishala T.; Berger, Allison J.; Kupperman, Erik; Manfredi, Mark; Bolen, Joseph B.; Van Ness, Brian; Janz, Siegfried

    2012-01-01

    Purpose The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/ Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. PMID:21903769

  4. Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux

    PubMed Central

    Gangavarapu, Kalyan J.; Huss, Wendy J.

    2011-01-01

    This unit describes methods for the digestion of human prostate clinical specimens, dye cycle violet (DCV) staining procedure for the identification, isolation, and quantitation of radiolabeled dihydrotestosterone (DHT) retention of side population cells. The principle of the side population assay is based on differential efflux of DCV, a cell membrane permeable fluorescent dye, by cells with high ATP binding cassette (ABC) transporter activity. Cells with high ABC transporter activity efflux DCV and fall in the lower left quadrant of a flow cytograph are designated as “side population” cells. This unit emphasizes tissue digestion, DCV staining, flow settings for sorting side population cells and quantitation of radiolabeled DHT retention. PMID:21400686

  5. Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Ge; Wan, Rong; Hu, Yanling

    2014-01-31

    Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration ofmore » sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.« less

  6. Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

    PubMed

    Kochenderfer, James N; Rosenberg, Steven A

    2013-05-01

    Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

  7. 6-Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase-9 expression via blockade of nuclear factor-κB activation

    PubMed Central

    Ling, H; Yang, H; Tan, S-H; Chui, W-K; Chew, E-H

    2010-01-01

    BACKGROUND AND PURPOSE Shogaols are reported to possess anti-inflammatory and anticancer activities. However, the antimetastatic potential of shogaols remains unexplored. This study was performed to assess the effects of shogaols against breast cancer cell invasion and to investigate the underlying mechanisms. EXPERIMENTAL APPROACH The anti-invasive effect of a series of shogaols was initially evaluated on MDA-MB-231 breast cancer cells using the matrigel invasion assay. The suppressive effects of 6-shogaol on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) gelatinolytic activity and nuclear factor-κB (NF-κB) activation were further determined. KEY RESULTS Shogaols (6-, 8- and 10-shogaol) inhibited PMA-stimulated MDA-MB-231 cell invasion with an accompanying decrease in MMP-9 secretion. 6-Shogaol was identified to display the greatest anti-invasive effect in association with a dose-dependent reduction in MMP-9 gene activation, protein expression and secretion. The NF-κB transcriptional activity was decreased by 6-shogaol; an effect mediated by inhibition of IκB phosphorylation and degradation that subsequently led to suppression of NF-κB p65 phosphorylation and nuclear translocation. In addition, 6-shogaol was found to inhibit JNK activation with no resulting reduction in activator protein-1 transcriptional activity. By using specific inhibitors, it was demonstrated that ERK and NF-κB signalling, but not JNK and p38 signalling, were involved in PMA-stimulated MMP-9 activation. CONCLUSIONS AND IMPLICATIONS 6-Shogaol is a potent inhibitor of MDA-MB-231 cell invasion, and the molecular mechanism involves at least in part the down-regulation of MMP-9 transcription by targeting the NF-κB activation cascade. This class of naturally occurring small molecules thus have potential for clinical use as antimetastatic treatments. PMID:20718733

  8. 6-Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase-9 expression via blockade of nuclear factor-κB activation.

    PubMed

    Ling, H; Yang, H; Tan, S-H; Chui, W-K; Chew, E-H

    2010-12-01

    Shogaols are reported to possess anti-inflammatory and anticancer activities. However, the antimetastatic potential of shogaols remains unexplored. This study was performed to assess the effects of shogaols against breast cancer cell invasion and to investigate the underlying mechanisms. The anti-invasive effect of a series of shogaols was initially evaluated on MDA-MB-231 breast cancer cells using the matrigel invasion assay. The suppressive effects of 6-shogaol on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) gelatinolytic activity and nuclear factor-κB (NF-κB) activation were further determined. Shogaols (6-, 8- and 10-shogaol) inhibited PMA-stimulated MDA-MB-231 cell invasion with an accompanying decrease in MMP-9 secretion. 6-Shogaol was identified to display the greatest anti-invasive effect in association with a dose-dependent reduction in MMP-9 gene activation, protein expression and secretion. The NF-κB transcriptional activity was decreased by 6-shogaol; an effect mediated by inhibition of IκB phosphorylation and degradation that subsequently led to suppression of NF-κB p65 phosphorylation and nuclear translocation. In addition, 6-shogaol was found to inhibit JNK activation with no resulting reduction in activator protein-1 transcriptional activity. By using specific inhibitors, it was demonstrated that ERK and NF-κB signalling, but not JNK and p38 signalling, were involved in PMA-stimulated MMP-9 activation. 6-Shogaol is a potent inhibitor of MDA-MB-231 cell invasion, and the molecular mechanism involves at least in part the down-regulation of MMP-9 transcription by targeting the NF-κB activation cascade. This class of naturally occurring small molecules thus have potential for clinical use as antimetastatic treatments. © 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.

  9. EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

    PubMed Central

    Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Cai, Qiliang; Banerjee, Shuvomoy; Prasad, Mahadesh A. J.

    2013-01-01

    Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis. PMID:23986604

  10. Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes.

    PubMed

    Smal, Caroline; Van Den Neste, Eric; Maerevoet, Marie; Poiré, Xavier; Théate, Ivan; Bontemps, Françoise

    2007-08-08

    Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.

  11. Mechanical stretch endows mesenchymal stem cells stronger angiogenic and anti-apoptotic capacities via NFκB activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Zhuoli; Gan, Xueqi; Fan, Hongyi

    Mesenchymal stem cells (MSCs) have been broadly used for tissue regeneration and repair due to their broad differentiation potential and potent paracrine properties such as angiogenic capacity. Strategies to increase their survival rate after transplantation and the angiogenic ability are of priority for the utility of MSCs. In this study, we found that mechanical stretch (10% extension, 30 cycles/min cyclic stretch) preconditioning increase the angiogenic capacity via VEGFA induction. In addition, mechanical stretch also increases the survival rate of mesenchymal stem cells under nutrients deprivation. Consistent with the increase VEGFA expression and resistance to apoptosis, nuclear localization of NFκB activity p65more » increased upon mechanical stretch. Inhibition of NFκB activity by BAY 11-708 blocks the pro-angiogenesis and anti-apoptosis function of mechanical stretch. Taken together, our findings here raise the possibility that mechanical stretch preconditioning might enhance the therapeutic efficacy of mesenchymal stem cells. - Highlights: • Mechanical stretch increases the angiogenic capacity via VEGFA induction in MSCs. • Mechanical stretch increases the survival rate of MSCs under nutrients deprivation. • Mechanical stretch manipulates MSCs via the activation of NFκB.« less

  12. EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

    PubMed

    Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

    2013-01-01

    Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

  13. In vitro anticancer property of a novel thalidomide analogue through inhibition of NF-kappaB activation in HL-60 cells.

    PubMed

    Li, Min; Sun, Wan; Yang, Ya-ping; Xu, Bo; Yi, Wen-yuan; Ma, Yan-xia; Li, Zhong-jun; Cui, Jing-rong

    2009-01-01

    To investigate the anticancer property and possible mechanism of action of a novel sugar-substituted thalidomide derivative (STA-35) on HL-60 cells in vitro. TNF-alpha-induced NF-kappaB activation was determined using a reporter gene assay. The MTT assay was used to measure cytotoxicity of the compound. The appearance of apoptotic Sub-G1 cells was detected by flow cytometry analysis. PARP cleavage and protein expression of NF-kappaB p65 and its inhibitor IkappaB were viewed by Western blotting. TA-35 (1-20 micromol/L) suppressed TNF-alpha-induced NF-kappaB activation in transfected cells (HEK293/pNiFty-SEAP) in a dose- (1-20 micromol/L) and time-dependent (0-48 h) manner. It was also shown that STA-35 exerted a dose-dependent inhibitory effect on HL-60 cell proliferation with an IC(50) value of 9.05 micromol/L. In addition, STA-35 induced apoptosis in HL-60 cells, as indicated by the appearance of a Sub-G1 peak in the cell cycle distribution, as well as poly ADP-ribose polymerase (PARP) cleavage. Subsequently, both NF-kappaB p65 and its inhibitor IkappaB gradually accumulated in cytoplasmic extracts in a dose- and time-dependent manner, indicating the blockage of NF-kappaB translocation induced by TNF-alpha from the cytoplasm to the nucleus. A novel sugar-substituted thalidomide derivative, STA-35, is potent toward HL-60 cells in vitro and induces apoptosis by the suppression of NF-kappaB activation.

  14. CREATING AN IPHONE APPLICATION FOR COLLECTING CONTINUOUS ABC DATA

    PubMed Central

    Whiting, Seth W; Dixon, Mark R

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data-collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to an e-mail account after observations have concluded. Further suggestions are provided to customize the ABC data- collection system for individual preferences and clinical needs. PMID:23060682

  15. ABCs of Being Smart: S Is for Supporting

    ERIC Educational Resources Information Center

    Foster, Joanne

    2014-01-01

    Joanne Foster's article "R We There Yet?" was first published in "Parenting for High Potential" ("PHP") in 2006, which became the springboard for the "ABCs of Being Smart" series of columns. At that time, Foster invited "PHP" readers to think about their own versions of the "ABCs of Being…

  16. UNG protects B cells from AID-induced telomere loss

    PubMed Central

    Cortizas, Elena M.; Zahn, Astrid; Safavi, Shiva

    2016-01-01

    Activation-induced deaminase (AID) initiates antibody gene diversification by creating G:U mismatches in the immunoglobulin loci. However, AID also deaminates nonimmunoglobulin genes, and failure to faithfully repair these off-target lesions can cause B cell lymphoma. In this study, we identify a mechanism by which processing of G:U produced by AID at the telomeres can eliminate B cells at risk of genomic instability. We show that telomeres are off-target substrates of AID and that B cell proliferation depends on protective repair by uracil-DNA glycosylase (UNG). In contrast, in the absence of UNG activity, deleterious processing by mismatch repair leads to telomere loss and defective cell proliferation. Indeed, we show that UNG deficiency reduces B cell clonal expansion in the germinal center in mice and blocks the proliferation of tumor B cells expressing AID. We propose that AID-induced damage at telomeres acts as a fail-safe mechanism to limit the tumor promoting activity of AID when it overwhelms uracil excision repair. PMID:27697833

  17. Reciprocal inhibition of p53 and nuclear factor-kappaB transcriptional activities determines cell survival or death in neurons.

    PubMed

    Culmsee, Carsten; Siewe, Jan; Junker, Vera; Retiounskaia, Marina; Schwarz, Stephanie; Camandola, Simonetta; El-Metainy, Shahira; Behnke, Hagen; Mattson, Mark P; Krieglstein, Josef

    2003-09-17

    The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 precedes apoptosis in many cell types. Controversial reports exist on the role of the transcription factor nuclear factor-kappaB (NF-kappaB) in p53-mediated apoptosis, depending on the cell type and experimental conditions. Therefore, we sought to elucidate the role of NF-kappaB in p53-mediated neuron death. In cultured neurons DNA damaging compounds induced activation of p53, whereas NF-kappaB activity declined significantly. The p53 inhibitor pifithrin-alpha (PFT) preserved NF-kappaB activity and protected neurons against apoptosis. Immunoprecipitation experiments revealed enhanced p53 binding to the transcriptional cofactor p300 after induction of DNA damage, whereas binding of p300 to NF-kappaB was reduced. In contrast, PFT blocked the interaction of p53 with the cofactor, whereas NF-kappaB binding to p300 was enhanced. Most interestingly, similar results were observed after oxygen glucose deprivation in cultured neurons and in ischemic brain tissue. Ischemia-induced repression of NF-kappaB activity was prevented and brain damage was reduced by the p53 inhibitor PFT in a dose-dependent manner. It is concluded that a balanced competitive interaction of p53 and NF-kappaB with the transcriptional cofactor p300 exists in neurons. Exposure of neurons to lethal stress activates p53 and disrupts NF-kappaB binding to p300, thereby blocking NF-kappaB-mediated survival signaling. Inhibitors of p53 provide pronounced neuroprotective effects because they block p53-mediated induction of cell death and concomitantly enhance NF-kappaB-induced survival signaling.

  18. Autocrine activity of BDNF induced by the STAT3 signaling pathway causes prolonged TrkB activation and promotes human non-small-cell lung cancer proliferation

    PubMed Central

    Chen, Bo; Liang, Yan; He, Zheng; An, Yunhe; Zhao, Weihong; Wu, Jianqing

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily, which has been implicated in the pathophysiology of the nervous system. Recently, several studies have suggested that BDNF and/or its receptor, tropomyosin related kinase B (TrkB), are involved in tumor growth and metastasis in several cancers, including prostate cancer, neuroblastoma, pancreatic ductal carcinoma, hepatocellular carcinoma, and lung cancer. Despite the increasing emphasis on BDNF/TrkB signaling in human tumors, how it participates in primary tumors has not yet been determined. Additionally, little is known about the molecular mechanisms that elicit signaling downstream of TrkB in the progression of non-small-cell lung cancer (NSCLC). In this study, we report the significant expression of BDNF in NSCLC samples and show that BDNF stimulation increases the synthesis of BDNF itself through activation of STAT3 in lung cancer cells. The release of BDNF can in turn activate TrkB signaling. The activation of both TrkB and STAT3 contribute to downstream signaling and promote human non-small-cell lung cancer proliferation. PMID:27456333

  19. Circulating Th1 cell-type Tfh cells that exhibit impaired B cell help are preferentially activated during acute malaria in children

    PubMed Central

    Obeng-Adjei, Nyamekye; Portugal, Silvia; Tran, Tuan M.; Yazew, Takele B.; Skinner, Jeff; Li, Shanping; Jain, Aarti; Felgner, Philip L.; Doumbo, Ogobara K.; Kayentao, Kassoum; Ongoiba, Aissata; Traore, Boubacar; Crompton, Peter D.

    2015-01-01

    SUMMARY Malaria-specific antibody responses are short-lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1+CXCR5+CD4+ Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population PD-1+CXCR5+CXCR3− Tfh cells are superior to Th1-polarized PD-1+CXCR5+CXCR3+ Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children, and suggest that vaccine strategies that promote CXCR3− Tfh cell responses may improve malaria vaccine efficacy. PMID:26440897

  20. Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

    PubMed

    Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

    2017-11-01

    CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. γδ T Cells Shape Preimmune Peripheral B Cell Populations.

    PubMed

    Huang, Yafei; Getahun, Andrew; Heiser, Ryan A; Detanico, Thiago O; Aviszus, Katja; Kirchenbaum, Greg A; Casper, Tamara L; Huang, Chunjian; Aydintug, M Kemal; Carding, Simon R; Ikuta, Koichi; Huang, Hua; Wysocki, Lawrence J; Cambier, John C; O'Brien, Rebecca L; Born, Willi K

    2016-01-01

    We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations. Copyright © 2015 by The American Association of Immunologists, Inc.

  2. CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells

    PubMed Central

    Mariño, Eliana; Villanueva, Jeanette; Walters, Stacey; Liuwantara, David; Mackay, Fabienne; Grey, Shane T.

    2009-01-01

    OBJECTIVE Tumor necrosis factor ligand family members B-cell–activating factor (BAFF) and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and autoimmunity. We examined the effect of blocking BAFF and APRIL on the development of autoimmune diabetes. RESEARCH DESIGN AND METHODS Female NOD mice were administered B-cell maturation antigen (BCMA)-Fc from 9 to 15 weeks of age. Diabetes incidence, islet pathology, and T- and B-cell populations were examined. RESULTS BCMA-Fc treatment reduced the severity of insulitis and prevented diabetes development in NOD mice. BCMA-Fc–treated mice showed reduced follicular, marginal-zone, and T2MZ B-cells. B-cell reduction was accompanied by decreased frequencies of pathogenic CD4+CD40+ T-cells and reduced Th1 cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was blunted with reduced B-cells. However, BCMA-Fc–treated mice still harbored detectable diabetogenic T-cells, suggesting that regulatory mechanisms contributed to diabetes prevention. Indeed, BCMA-Fc–treated mice accumulated increased CD4+CD25+ regulatory T-cells (Tregs) with age. CD4+CD25+ cells were essential for maintaining euglycemia because their depletion abrogated BCMA-Fc–mediated protection. BCMA-Fc did not directly affect Treg homeostasis given that CD4+CD25+Foxp3+ T-cells did not express TACI or BR3 receptors and that CD4+CD25+Foxp3+ T-cell frequencies were equivalent in wild-type, BAFF−/−, TACI−/−, BCMA−/−, and BR3−/− mice. Rather, B-cell depletion resulted in CD4+CD25+ T-cell–mediated protection from diabetes because anti-CD25 monoclonal antibody treatment precipitated diabetes in both diabetes-resistant NOD.μMT−/− and BCMA-Fc–treated mice. CONCLUSIONS BAFF/APRIL blockade prevents diabetes. BCMA-Fc reduces B-cells, subsequently blunting autoimmune activity and allowing endogenous regulatory mechanisms to preserve a

  3. Curcumin Modulates the Radiosensitivity of Colorectal Cancer Cells by Suppressing Constitutive and Inducible NF-{kappa}B Activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sandur, Santosh K.; Deorukhkar, Amit; Pandey, Manoj K.

    2009-10-01

    Purpose: Radiation therapy is an integral part of the preoperative treatment of rectal cancers. However, only a minority of patients achieve a complete pathologic response to therapy because of resistance of these tumors to radiation therapy. This resistance may be mediated by constitutively active pro-survival signaling pathways or by inducible/acquired mechanisms in response to radiation therapy. Simultaneous inhibition of these pathways can sensitize these tumors to radiation therapy. Methods and Materials: Human colorectal cancer cells were exposed to clinically relevant doses of gamma rays, and the mechanism of their radioresistance was investigated. We characterized the transcription factor nuclear factor-{kappa}B (NF-{kappa}B)more » activation as a mechanism of inducible radioresistance in colorectal cancer and used curcumin, the active ingredient in the yellow spice turmeric, to overcome this resistance. Results: Curcumin inhibited the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation stimulated NF-{kappa}B activity in a dose- and time-dependent manner, whereas curcumin suppressed this radiation-induced NF-{kappa}B activation via inhibition of radiation-induced phosphorylation and degradation of inhibitor of {kappa}B alpha, inhibition of inhibitor of {kappa}B kinase activity, and inhibition of Akt phosphorylation. Curcumin also suppressed NF-{kappa}B-regulated gene products (Bcl-2, Bcl-x{sub L}, inhibitor of apoptosis protein-2, cyclooxygenase-2, and cyclin D1). Conclusions: Our results suggest that transient inducible NF-{kappa}B activation provides a prosurvival response to radiation that may account for development of radioresistance. Curcumin blocks this signaling pathway and potentiates the antitumor effects of radiation therapy.« less

  4. Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper, Th17- and Activated B-Cells and Correlates with Progression

    PubMed Central

    Christensen, Jeppe Romme; Börnsen, Lars; Ratzer, Rikke; Piehl, Fredrik; Khademi, Mohsen; Olsson, Tomas; Sørensen, Per Soelberg; Sellebjerg, Finn

    2013-01-01

    Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (TFH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4+ and CD8+T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS+TFH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 TFH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 TFH-cells. The Th17-subset, interleukin-23-receptor+CD4+T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN+ and CD83+B-cells in SPMS. ICOS+TFH-cells and DC-SIGN+B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4+T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of TFH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated TFH-cells in MS. The increased frequencies of Th17-cells, activated TFH- and B-cells parallel findings from pathology studies

  5. Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

    PubMed Central

    Grigoryev, Yevgeniy A.; Kurian, Sunil M.; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L.; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M.; Kantor, Aaron B.; Marsh, Christopher; Salomon, Daniel R.

    2010-01-01

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant. PMID:20976225

  6. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    PubMed

    Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

    2010-10-14

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  7. BCR mediated signal transduction in immature and mature B cells.

    PubMed

    Koncz, Gábor; Bodor, Csaba; Kövesdi, Dorottya; Gáti, Róbert; Sármay, Gabriella

    2002-06-03

    Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death

  8. pH-responsive supramolecular self-assembly of well-defined zwitterionic ABC miktoarm star terpolymers.

    PubMed

    Liu, Hao; Li, Changhua; Liu, Hewen; Liu, Shiyong

    2009-04-21

    We report the first example of the synthesis and pH-responsive supramolecular self-assembly of double hydrophilic ABC miktoarm star terpolymers. Well-defined ABC miktoarm star terpolymers consisting of poly(ethylene glycol), poly(tert-butyl methacrylate), and poly(2-(diethylamino)ethyl methacrylate) arms [PEG(-b-PtBMA)-b-PDEA] were synthesized via the combination of consecutive click reactions and atom transfer radical polymerization (ATRP), starting from a trifunctional core molecule, 1-azido-3-chloro-2-propanol (ACP). The click reaction of monoalkynyl-terminated PEG with an excess of ACP afforded difunctional PEG bearing a chlorine and a secondary hydroxyl moiety at the chain end, PEG113(-Cl)-OH (1). After azidation with NaN3, PEG-based macroinitiator PEG113(-N3)-Br (3) was prepared by the esterification of PEG113(-N3)-OH (2) with 2-bromoisobutyryl bromide and then employed in the ATRP of tert-butyl methacrylate (tBMA). The obtained PEG(-N3)-b-PtBMA copolymers (4) possessed an azido moiety at the diblock junction point. The preparation of PEG(-b-PtBMA)-b-PDEA miktoarm star terpolymers was then achieved via the click reaction of 4 with an excess of monoalkynyl-terminated PDEA. The obtained miktoarm star terpolymers were successfully converted into PEG(-b-PMAA)-b-PDEA, where PMAA is poly(methacrylic acid). In aqueous solution, PEG(-b-PMAA)-b-PDEA zwitterionic ABC miktoarm star terpolymers can self-assemble into three types of micellar aggregates by simply adjusting solution pH at room temperature. Above pH 8, PDEA-core micelles stabilized by PEG/ionized PMAA hybrid coronas were formed due to the insolubility of PDEA block. In the range of pH 5-7, micelles possessing polyion complex cores formed as a result of charge compensation between partially ionized PMAA and partially protonated PDEA sequences. At pH<4, hydrogen bonding interactions between fully protonated PMAA and PEG led to the formation of another type of micellar aggregates possessing hydrogen

  9. Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

    PubMed

    Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

    2018-05-01

    Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

  10. The PI3K Isoforms p110α and p110δ are Essential for Pre-B Cell Receptor Signaling and B Cell Development

    PubMed Central

    Ramadani, Faruk; Bolland, Daniel J.; Garcon, Fabien; Emery, Juliet L.; Vanhaesebroeck, Bart; Corcoran, Anne E.; Okkenhaug, Klaus

    2013-01-01

    B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes are assembled in frame to produce a functional B cell receptor (BCR) and antibodies. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igα and Igβ chains. Whereas the activation of Src and Syk tyrosine kinases is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110δ isoform of phosphoinositide 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on tonic BCR signaling, are not substantially affected by a deficiency in p110δ. Here, we show that in the absence of p110δ, p110α, but not p110β, can compensate to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110α and p110δ activities, pre-BCR signaling fails to suppress the production of recombination-activating gene (Rag) protein and to promote developmental progression of B cell progenitors. By contrast, p110α does not contribute to agonist-induced BCR signaling. These studies indicate that either p110α or p110δ can mediate tonic signaling from the BCR, but that only p110δ can contribute to antigen-dependent activation of B cells. PMID:20699475

  11. The P20R mutation of αB-crystallin diminishes its anti-apoptotic activity in human lens epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Peiran; Li, Weiwei; Ni, Mengxia

    αB-crystallin acts as an anti-apoptosis protein in human lens epithelial (HLE) cells. We recently identified a missense mutation in αB-crystallin that changes proline 20 to an arginine (P20R) in a Chinese family with autosomal dominant congenital posterior polar cataract. The impact of the P20R mutation on the anti-apoptosis function remains unclear. To explore the anti-apoptotic activity of αB-crystallin wild type (αB-wt) and its P20R mutant under oxidative stress, HLE cells were transfected with αB-wt and αB-P20R constructs and expression was measured by western blotting. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL) staining were performed tomore » investigate apoptosis. We found that αB-wt performed a dominant role in inhibiting stress-induced apoptosis, but this function was impeded in cells expressing αB-P20R. The P20R mutant of αB-crystallin exhibits diminished anti-apoptotic activity compared with the native protein. - Highlights: • We identified a novel mutation (P20R) in αB-crystallin. • The mutation decreased anti-apoptotic activity in αB-crystallin, which compared with the native protein. • We speculate that the mutation may influence phosphorylation, especially Ser19.« less

  12. Porcine circovirus type 2 induces the activation of nuclear factor kappa B by I{kappa}B{alpha} degradation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei Li; Kwang, Jimmy; Wang Jin

    The transcription factor NF-{kappa}B is commonly activated upon virus infection and a key player in the induction and regulation of the host immune response. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), which is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome, can activate NF-{kappa}B in PCV2-infected PK15 cells. In PCV2-infected cells, NF-{kappa}B was activated concomitantly with viral replication, which was characterized by increased DNA binding activity, translocation of NF-{kappa}B p65 from the cytoplasm to the nucleus, as well as degradation and phosphorylation of I{kappa}B{alpha} protein. We further demonstratedmore » PCV2-induced activation of NF-{kappa}B and colocalization of p65 nuclear translocation with virus replication in cultured cells. Treatment of cells with CAPE, a selective inhibitor of NF-{kappa}B activation, reduced virus protein expression and progeny production followed by decreasing PCV2-induced apoptotic caspase activity, indicating the involvement of this transcription factor in induction of cell death. Taken together, these data suggest that NF-{kappa}B activation is important for PCV2 replication and contributes to virus-mediated changes in host cells. The results presented here provide a basis for understanding molecular mechanism of PCV2 infection.« less

  13. Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goldman, M.; Rose, L.M.; Hochmann, A.

    1982-05-01

    We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us tomore » detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.« less

  14. B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

    PubMed

    Marasco, Emiliano; Farroni, Chiara; Cascioli, Simona; Marcellini, Valentina; Scarsella, Marco; Giorda, Ezio; Piano Mortari, Eva; Leonardi, Lucia; Scarselli, Alessia; Valentini, Diletta; Cancrini, Caterina; Duse, Marzia; Grimsholm, Ola; Carsetti, Rita

    2017-01-01

    Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system. © 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death

    PubMed Central

    Morgan, Jessica A.; Lynch, John; Panetta, John C.; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T.; Janke, Laura; Green, Daniel M.; Chemaitilly, Wassim; Schuetz, John D.

    2015-01-01

    Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer. PMID:26576726

  16. Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death.

    PubMed

    Morgan, Jessica A; Lynch, John; Panetta, John C; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T; Janke, Laura; Green, Daniel M; Chemaitilly, Wassim; Schuetz, John D

    2015-11-18

    Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer.

  17. Bacterially activated B-cells drive T cell differentiation towards Tr1 through PD-1/PD-L1 expression.

    PubMed

    Said, Sawsan Sudqi; Barut, Guliz Tuba; Mansur, Nesteren; Korkmaz, Asli; Sayi-Yazgan, Ayca

    2018-04-01

    Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter -stimulated B cells. Our results demonstrate that Helicobacter felis -stimulated IL-10- producing B cells (Hf stim - IL-10 + B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter felis -stimulated IL-10- nonproducing B (Hf stim - IL-10 - B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-β, IL-6, and TNF-α, and IgM and IgG2b. Furthermore, we show that both Hf stim - IL-10 + B cells and Hf stim - IL-10 - B cells induce CD49b + LAG-3 + Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hf stim - IL-10 - B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-β. Taken together, the current study demonstrates that Hf stim - B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-β. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent mu to gamma1 class switch recombination in CD38-activated B cells.

    PubMed

    Tsukamoto, Yumiko; Uehara, Shoji; Mizoguchi, Chieko; Sato, Atsushi; Horikawa, Keisuke; Takatsu, Kiyoshi

    2005-10-21

    Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to mu to gamma1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce mu to gamma1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces mu to gamma1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce mu to gamma1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce mu to gamma1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR.

  19. Epstein-Barr virus EBNA2 directs doxorubicin resistance of B cell lymphoma through CCL3 and CCL4-mediated activation of NF-κB and Btk.

    PubMed

    Kim, Joo Hyun; Kim, Won Seog; Hong, Jung Yong; Ryu, Kung Ju; Kim, Seok Jin; Park, Chaehwa

    2017-01-17

    Epstein-Barr virus (EBV)-encoded nuclear antigen, EBNA2, expressed in EBV-infected B lymphocytes is critical for lymphoblastoid cell growth. Microarray profiling and cytokine array screening revealed that EBNA2 is associated with upregulation of the chemokines CCL3 and CCL4 in lymphoma cells. Depletion or inactivation of CCL3 or CCL4 sensitized DLBCL cells to doxorubicin. Our results indicate that EBV influences cell survival via an autocrine mechanism whereby EBNA2 increases CCL3 and CCL4, which in turn activate the Btk and NF-κB pathways, contributing to doxorubicin resistance of B lymphoma cells. Western blot data further confirmed that CCL3 and CCL4 direct activation of Btk and NF-κB. Based on these findings, we propose that a pathway involving EBNA2/Btk/NF-κB/CCL3/CCL4 plays a key role in doxorubicin resistance, and therefore, inhibition of specific components of this pathway may sensitize lymphoma cells to doxorubicin. Evaluation of the relationship between CCL3 expression and EBV infection revealed high CCL3 levels in EBV-positive patients. Our data collectively suggest that doxorubicin treatment for EBNA2-positive DLBCL cells may be effectively complemented with a NF-κB or Btk inhibitor. Moreover, evaluation of the CCL3 and CCL4 levels may be helpful for selecting DLBCL patients likely to benefit from doxorubicin treatment in combination with the velcade or ibrutinib.

  20. CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent.

    PubMed

    D'Souza, Lucas; Gupta, Sneh Lata; Bal, Vineeta; Rath, Satyajit; George, Anna

    2017-12-01

    B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM + cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73 + IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory. © 2017 John Wiley & Sons Ltd.