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Sample records for potency regulate subsets

  1. Glycolysis determines dichotomous regulation of T cell subsets in hypoxia.

    PubMed

    Xu, Yang; Chaudhury, Arindam; Zhang, Ming; Savoldo, Barbara; Metelitsa, Leonid S; Rodgers, John; Yustein, Jason T; Neilson, Joel R; Dotti, Gianpietro

    2016-07-01

    Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (TN and TCM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (TEM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in TN and TCM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate-rich elements in the 3'-UTR region of HIF1A mRNA in glycolytically inactive TN and TCM. Conversely, active glycolysis with decreased GAPDH availability in TEM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance TEM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue. PMID:27294526

  2. Regulation of T Helper Cell Subsets by Cyclooxygenases and Their Metabolites

    PubMed Central

    Li, Hong; Edin, Matthew L.; Gruzdev, Artiom; Cheng, Jennifer; Bradbury, J. Alyce; Graves, Joan P.; DeGraff, Laura M.; Zeldin, Darryl C.

    2013-01-01

    Cyclooxygenases and their metabolites are important regulators of inflammatory responses and play critical roles in regulating the differentiation of T helper cell subsets in inflammatory diseases. In this review, we highlight new information on regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostanoids influence cytokine production on both antigen presenting cells and T cells to regulate the differentiation of naïve CD4+ T cells to Th1, Th2 and Th17 cell phenotypes. Cyclooxygenases and PGE2 generally exacerbate Th2 and Th17 phenotypes, while suppressing Th1 differentiation. Thus, cycloxygenases may play a critical role in diseases that involve immune cell dysfunction. Targeting of cyclooxygenases and their eicosanoid products may represent a new approach for treatment of inflammatory diseases, tumors and autoimmune disorders. PMID:23201570

  3. NF-κB: Roles and Regulation In Different CD4+ T cell subsets

    PubMed Central

    Oh, Hyunju; Ghosh, Sankar

    2012-01-01

    Summary The nuclear factor-κB (NF-κB) family of transcription factors plays important roles in various biological processes including apoptosis, stress response, immunity, and inflammation. NF-κB signaling is involved in both immune cell development and function, and it is critical in modulation of the immune response through the transcriptional regulation of cytokine and chemokine expression. An area of great interest in T-cell-mediated adaptive immunity is the ability of naive CD4+ T cells generated in the thymus to differentiate into various subsets including T-helper 1 (Th1), Th2, Th17, Th9, follicular helper T (Tfh), Th22 and regulatory T (Treg) cells, upon encountering different pathogens and microenvironments. In this review, we discuss the role of NF-κB pathway in the development and functional divergence of the different helper T-cell subsets as well as in regulatory T cells. PMID:23405894

  4. Interferon Regulatory Factor (IRF)-4 regulates the expression of a subset of Th2 cytokines 1

    PubMed Central

    Ahyi, Ayele-Nati N.; Chang, Hua-Chen; Dent, Alexander L.; Nutt, Stephen L.; Kaplan, Mark H.

    2009-01-01

    Th2 cells can be subdivided into subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN-regulatory factor (IRF)-4. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. Infection of total Th2 cells, and IL-10 non-secreting cells, with retrovirus expressing IRF4, resulted in increased IL-4 and IL-10 expression, no change in IL-5 or IL-13 production and decreased Il9 transcription. Transfection of an IRF4-specific siRNA into Th2 cells decreases IL-10 production. IRF4 directly binds the Il10 gene as evidenced by ChIP assay, and regulates Il10 control elements in a reporter assay. IRF4 interacts with PU.1, and in PU.1-deficient T cells there was an increase in IRF4 binding to the Il10 gene, and in the ability of IRF4 to induce IL-10 production compared to wild type cells and Il10 promoter activity in a reporter assay. Further heterogeneity of IRF4 expression was observed in Th2 cells analyzed for expression of multiple Th2 cytokines. Thus, IRF4 promotes the expression of a subset of Th2 cytokines and contributes to Th2 heterogeneity. PMID:19592658

  5. T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

    PubMed

    Grassi, Francesco; Cattini, Luca; Gambari, Laura; Manferdini, Cristina; Piacentini, Anna; Gabusi, Elena; Facchini, Andrea; Lisignoli, Gina

    2016-04-01

    T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23653421

  6. Functional Specialization of Skin Dendritic Cell Subsets in Regulating T Cell Responses

    PubMed Central

    Clausen, Björn E.; Stoitzner, Patrizia

    2015-01-01

    Dendritic cells (DC) are a heterogeneous family of professional antigen-presenting cells classically recognized as most potent inducers of adaptive immune responses. In this respect, Langerhans cells have long been considered to be prototypic immunogenic DC in the skin. More recently this view has considerably changed. The generation of in vivo cell ablation and lineage tracing models revealed the complexity of the skin DC network and, in particular, established the existence of a number of phenotypically distinct Langerin+ and negative DC populations in the dermis. Moreover, by now we appreciate that DC also exert important regulatory functions and are required for the maintenance of tolerance toward harmless foreign and self-antigens. This review summarizes our current understanding of the skin-resident DC system in the mouse and discusses emerging concepts on the functional specialization of the different skin DC subsets in regulating T cell responses. Special consideration is given to antigen cross-presentation as well as immune reactions toward contact sensitizers, cutaneous pathogens, and tumors. These studies form the basis for the manipulation of the human counterparts of the murine DC subsets to promote immunity or tolerance for the treatment of human disease. PMID:26557117

  7. Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression.

    PubMed

    Miniard, Angela C; Middleton, Lisa M; Budiman, Michael E; Gerber, Carri A; Driscoll, Donna M

    2010-08-01

    Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level. PMID:20385601

  8. Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes.

    PubMed

    Arbouzova, Natalia I; Bach, Erika A; Zeidler, Martin P

    2006-01-10

    A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. Here we describe a new negative regulator of JAK/STAT signaling and identify a potential mechanism by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, we have identified Ken & Barbie (Ken) , which is an ortholog of the mammalian proto-oncogene BCL6 , as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, we show that endogenous Ken specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo. PMID:16401426

  9. Ubiquitin binds to and regulates a subset of SH3 domains

    PubMed Central

    Stamenova, Svetoslava D.; French, Michael E.; He, Yuan; Francis, Smitha A.; Kramer, Zachary B.; Hicke, Linda

    2009-01-01

    Summary SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that binds to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain, and that ubiquitin-binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands. PMID:17244534

  10. Retinoic acid differentially regulates the migration of innate lymphoid cell subsets to the gut

    PubMed Central

    Kim, Myung H.; Taparowsky, Elizabeth J.; Kim, Chang H.

    2015-01-01

    Summary Distinct groups of innate lymphoid cells (ILCs) such as ILC1, ILC2 and ILC3 populate the intestine, but how these ILCs develop tissue tropism for this organ is unclear. We report that prior to migration to the intestine ILCs first undergo a `switch' in their expression of homing receptors from lymphoid to gut homing receptors. This process is regulated by mucosal dendritic cells and the gut-specific tissue factor retinoic acid (RA). This change in homing receptors is required for long-term population and effector function of ILCs in the intestine. Only ILC1 and ILC3, but not ILC2, undergo the RA-dependent homing receptor switch in gut-associated lymphoid tissues. In contrast, ILC2 acquire gut homing receptors in a largely RA-independent manner during their development in the bone marrow and can migrate directly to the intestine. Thus, distinct programs regulate the migration of ILC subsets to the intestine for regulation of innate immunity. PMID:26141583

  11. Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi.

    PubMed

    Cox, John V; Kansal, Rita; Whitt, Michael A

    2016-06-01

    To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE)). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and G(AE), we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi. We show that the expression of GFP-Rab43 arrested the anterograde transport of G(AE) in a Rab43-positive medial Golgi compartment. GFP-Rab43 expression also inhibited the acquisition of endoH-resistant sugars and the surface delivery of G(AE), as well as the surface delivery of the AE1-4 anion exchanger. In contrast, GFP-Rab43 expression did not affect the glycosylation or surface delivery of G. Unexpectedly, down-regulation of endogenous Rab43 using small interfering RNA resulted in an increase in the accumulation of G(AE) on the cell surface while having minimal effect on the surface levels of G. Our data demonstrate that Rab43 regulates the sorting of a subset of membrane-spanning cargo as they progress through the medial Golgi. PMID:27053659

  12. The Transcriptional Repressor EUO Regulates Both Subsets of Chlamydia Late Genes

    PubMed Central

    Rosario, Christopher J.; Hanson, Brett R.; Tan, Ming

    2014-01-01

    SUMMARY The pathogenic bacterium Chlamydia replicates in a eukaryotic host cell via a developmental cycle marked by temporal waves of gene expression. We have previously shown that late genes transcribed by the major chlamydial RNA polymerase, σ66 RNA polymerase, are regulated by a transcriptional repressor EUO. We now report that EUO also represses promoters for a second subset of late genes that are transcribed by an alternative polymerase called σ28 RNA polymerase. EUO bound in the vicinity of six σ28-dependent promoters and inhibited transcription of each promoter. We used a mutational analysis to demonstrate that the EUO binding site functions as an operator that is necessary and sufficient for EUO-mediated repression of σ28-dependent transcription. We also verified specific binding of EUO to σ66-dependent and σ28-dependent promoters with a DNA immunoprecipitation assay. These findings support a model in which EUO represses expression of both σ66-dependent and σ28-dependent late genes. We thus propose that EUO is the master regulator of late gene expression in the chlamydial developmental cycle. PMID:25250726

  13. TASK-1 Regulates Apoptosis and Proliferation in a Subset of Non-Small Cell Lung Cancers.

    PubMed

    Leithner, Katharina; Hirschmugl, Birgit; Li, Yingji; Tang, Bi; Papp, Rita; Nagaraj, Chandran; Stacher, Elvira; Stiegler, Philipp; Lindenmann, Jörg; Olschewski, Andrea; Olschewski, Horst; Hrzenjak, Andelko

    2016-01-01

    Lung cancer is the leading cause of cancer deaths worldwide; survival times are poor despite therapy. The role of the two-pore domain K+ (K2P) channel TASK-1 (KCNK3) in lung cancer is at present unknown. We found that TASK-1 is expressed in non-small cell lung cancer (NSCLC) cell lines at variable levels. In a highly TASK-1 expressing NSCLC cell line, A549, a characteristic pH- and hypoxia-sensitive non-inactivating K+ current was measured, indicating the presence of functional TASK-1 channels. Inhibition of TASK-1 led to significant depolarization in these cells. Knockdown of TASK-1 by siRNA significantly enhanced apoptosis and reduced proliferation in A549 cells, but not in weakly TASK-1 expressing NCI-H358 cells. Na+-coupled nutrient transport across the cell membrane is functionally coupled to the efflux of K+ via K+ channels, thus TASK-1 may potentially influence Na+-coupled nutrient transport. In contrast to TASK-1, which was not differentially expressed in lung cancer vs. normal lung tissue, we found the Na+-coupled nutrient transporters, SLC5A3, SLC5A6, and SLC38A1, transporters for myo-inositol, biotin and glutamine, respectively, to be significantly overexpressed in lung adenocarcinomas. In summary, we show for the first time that the TASK-1 channel regulates apoptosis and proliferation in a subset of NSCLC. PMID:27294516

  14. TASK-1 Regulates Apoptosis and Proliferation in a Subset of Non-Small Cell Lung Cancers

    PubMed Central

    Leithner, Katharina; Hirschmugl, Birgit; Li, Yingji; Tang, Bi; Papp, Rita; Nagaraj, Chandran; Stacher, Elvira; Stiegler, Philipp; Lindenmann, Jörg; Olschewski, Andrea; Olschewski, Horst; Hrzenjak, Andelko

    2016-01-01

    Lung cancer is the leading cause of cancer deaths worldwide; survival times are poor despite therapy. The role of the two-pore domain K+ (K2P) channel TASK-1 (KCNK3) in lung cancer is at present unknown. We found that TASK-1 is expressed in non-small cell lung cancer (NSCLC) cell lines at variable levels. In a highly TASK-1 expressing NSCLC cell line, A549, a characteristic pH- and hypoxia-sensitive non-inactivating K+ current was measured, indicating the presence of functional TASK-1 channels. Inhibition of TASK-1 led to significant depolarization in these cells. Knockdown of TASK-1 by siRNA significantly enhanced apoptosis and reduced proliferation in A549 cells, but not in weakly TASK-1 expressing NCI-H358 cells. Na+-coupled nutrient transport across the cell membrane is functionally coupled to the efflux of K+ via K+ channels, thus TASK-1 may potentially influence Na+-coupled nutrient transport. In contrast to TASK-1, which was not differentially expressed in lung cancer vs. normal lung tissue, we found the Na+-coupled nutrient transporters, SLC5A3, SLC5A6, and SLC38A1, transporters for myo-inositol, biotin and glutamine, respectively, to be significantly overexpressed in lung adenocarcinomas. In summary, we show for the first time that the TASK-1 channel regulates apoptosis and proliferation in a subset of NSCLC. PMID:27294516

  15. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    PubMed

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective. PMID:25055961

  16. Protection of human myeloid dendritic cell subsets against influenza A virus infection is differentially regulated upon TLR stimulation.

    PubMed

    Baharom, Faezzah; Thomas, Saskia; Bieder, Andrea; Hellmér, Maria; Volz, Julia; Sandgren, Kerrie J; McInerney, Gerald M; Karlsson Hedestam, Gunilla B; Mellman, Ira; Smed-Sörensen, Anna

    2015-05-01

    The proinflammatory microenvironment in the respiratory airway induces maturation of both resident and infiltrating dendritic cells (DCs) upon influenza A virus (IAV) infection. This results in upregulation of antiviral pathways as well as modulation of endocytic processes, which affect the susceptibility of DCs to IAV infection. Therefore, it is highly relevant to understand how IAV interacts with and infects mature DCs. To investigate how different subsets of human myeloid DCs (MDCs) involved in tissue inflammation are affected by inflammatory stimulation during IAV infection, we stimulated primary blood MDCs and inflammatory monocyte-derived DCs (MDDCs) with TLR ligands, resulting in maturation. Interestingly, MDDCs but not MDCs were protected against IAV infection after LPS (TLR4) stimulation. In contrast, stimulation with TLR7/8 ligand protected MDCs but not MDDCs from IAV infection. The reduced susceptibility to IAV infection correlated with induction of type I IFNs. We found that differential expression of TLR4, TRIF, and MyD88 in the two MDC subsets regulated the ability of the cells to enter an antiviral state upon maturation. This difference was functionally confirmed using small interfering RNA and inhibitors. Our data show that different human MDC subsets may play distinct roles during IAV infection, as their capacity to induce type I IFNs is dependent on TLR-specific maturation, resulting in differential susceptibility to IAV infection. PMID:25801434

  17. IFN-γ differentially regulates subsets of Gr-1(+)CD11b(+) myeloid cells in chronic inflammation.

    PubMed

    Zhan, Xiaoxia; Fang, Yimin; Hu, Shengfeng; Wu, Yongjian; Yang, Kun; Liao, Chunxin; Zhang, Yuanqing; Huang, Xi; Wu, Minhao

    2015-08-01

    During chronic inflammation, prolonged over-reactive immune response may lead to tissue destruction, while immune suppression hinders tissue repair and pathogen elimination. Therefore, precise regulation of the immune response is needed to avoid immuno-pathology. Interferon-gamma (IFN-γ) is widely used in clinical treatment of inflammatory diseases. However, the underlying mechanism remains unclear. Here, we evaluated the role of IFN-γ on CD11b(+)Gr-1(+) myeloid cell differentiation and function, using a heat-killed Mycobacterium bovis BCG-induced chronic inflammation model. After challenge with heat-killed BCG, two subpopulations of CD11b(+)Gr-1(+) myeloid cells were generated in the mouse spleen. Phenotypical, morphological and functional analysis indicated that the CD11b(+)Gr-1(high) Ly6G(high) Ly6C(low) subset was neutrophil-like myeloid-derived inducer cells (N-MDICs), which promoted T cell activation, while the other subset was CD11b(+)Gr-1(low) Ly6G(neg) Ly6C(high) monocyte-like myeloid-derived suppressor cells (M-MDSCs) that displayed extensive suppressor function. IFN-γ treatment dampened N-MDICs-mediated T cell activation through up-regulating T cell suppressive mediators, reactive oxygen species (ROS) and arginase I. While for M-MDSCs, IFN-γ reduced their suppressing activity by decreasing the arginase activity. Our study provides evidence that IFN-γ balances the over-reactive vs compromised immune response through different regulation of distinct myeloid subsets, and therefore displays significant therapeutic potential for effective immuno-therapy of chronic inflammatory diseases. PMID:26021804

  18. B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets.

    PubMed

    Luo, Liqun; Zhu, Gefeng; Xu, Haiying; Yao, Sheng; Zhou, Gang; Zhu, Yuwen; Tamada, Koji; Huang, Lanqing; Flies, Andrew D; Broadwater, Megan; Ruff, William; van Deursen, Jan M A; Melero, Ignacio; Zhu, Zhou; Chen, Lieping

    2015-01-01

    B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3's role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease. PMID:26065426

  19. Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators.

    PubMed

    Dodt, J; Hubbard, A R; Wicks, S J; Gray, E; Neugebauer, B; Charton, E; Silvester, G

    2015-07-01

    A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed. PMID:25623631

  20. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  1. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.

    PubMed

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco As; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. PMID:26824392

  2. Regulation of the migration and survival of monocyte subsets by chemokine receptors and its relevance to atherosclerosis

    PubMed Central

    Gautier, Emmanuel L.; Jakubzick, Claudia; Randolph, Gwendalyn J.

    2009-01-01

    Monocytes are central mediators in the advance of atherosclerotic plaque, making them a natural therapeutic target for reducing disease burden. Here, we highlight recent advances in our current understanding of monocyte heterogeneity and its relevance to regulation of monocyte accumulation and function within atherosclerotic plaques. Differences that distinguish monocyte subsets include differential expression of chemokine receptors, especially CCR2 and CX3CR1. Ablation of expression of these two receptors (or their ligands) in mice has an additive inhibition on monocyte recruitment to atherosclerotic plaques. Moreover, simultaneously interfering with three key pathways—CCR2, CX3CR1, and CCR5--essentially abolishes atherosclerosis in mice. Here, we discuss how these chemokine receptors act at multiple points on at least one monocyte subset, regulating their mobilization from bone marrow, survival, and/or recruitment to plaques. Finally, we discuss how this knowledge may be useful clinically, emphasizing that CX3CR1 may in particular be a viable target for therapeutic manipulation of monocyte-derived cell fate in cardiovascular disease. PMID:19759373

  3. Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets

    PubMed Central

    Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E.; Whiteside, Theresa L.

    2016-01-01

    Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4+ Tconv, CD8+ T or CD4+ CD39+ Treg were isolated from normal donors’ peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24–27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4+ Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes. PMID:26842680

  4. Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling*

    PubMed Central

    Cheng, Yu-Hong; Ho, Mei-Shang; Huang, Wei-Ting; Chou, Ying-Ting; King, Klim

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) analogs are approved for treatment of type 2 diabetes and are in clinical trials for disorders including neurodegenerative diseases. GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Given a normally basal circulating level of GLP-1, knowledge of mechanisms regulating GLP-1R signaling, which has diverse functions in extrapancreatic tissues, remains elusive. In this study, we found that the potency of GLP-1, not exendin 4, is specifically enhanced by the endocannabinoid-like lipids oleoylethanolamide (OEA) and 2-oleoylglycerol but not by stearoylethanolamide (SEA) or palmitoylethanolamide. 9.2 μm OEA enhances the potency of GLP-1 in stimulating cAMP production by 10-fold but does not affect its receptor binding affinity. OEA and 2-oleoylglycerol, but not SEA, bind to GLP-1 in a dose-dependent and saturable manner. OEA but not SEA promoted GLP-1(7–36) amide to trypsin inactivation in a dose-dependent and saturable manner. Susceptibility of GLP-1(7–36) amide to trypsin inactivation is increased 40-fold upon binding to OEA but not to SEA. Our findings indicate that OEA binds to GLP-1(7–36) amide and enhances the potency that may result from a conformational change of the peptide. In conclusion, modulating potency of GLP-1 by physiologically regulated endocannabinoid-like lipids allows GLP-1R signaling to be regulated spatiotemporally at a constant basal GLP-1 level. PMID:25903129

  5. Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling.

    PubMed

    Cheng, Yu-Hong; Ho, Mei-Shang; Huang, Wei-Ting; Chou, Ying-Ting; King, Klim

    2015-06-01

    Glucagon-like peptide-1 (GLP-1) analogs are approved for treatment of type 2 diabetes and are in clinical trials for disorders including neurodegenerative diseases. GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Given a normally basal circulating level of GLP-1, knowledge of mechanisms regulating GLP-1R signaling, which has diverse functions in extrapancreatic tissues, remains elusive. In this study, we found that the potency of GLP-1, not exendin 4, is specifically enhanced by the endocannabinoid-like lipids oleoylethanolamide (OEA) and 2-oleoylglycerol but not by stearoylethanolamide (SEA) or palmitoylethanolamide. 9.2 μM OEA enhances the potency of GLP-1 in stimulating cAMP production by 10-fold but does not affect its receptor binding affinity. OEA and 2-oleoylglycerol, but not SEA, bind to GLP-1 in a dose-dependent and saturable manner. OEA but not SEA promoted GLP-1(7-36) amide to trypsin inactivation in a dose-dependent and saturable manner. Susceptibility of GLP-1(7-36) amide to trypsin inactivation is increased 40-fold upon binding to OEA but not to SEA. Our findings indicate that OEA binds to GLP-1(7-36) amide and enhances the potency that may result from a conformational change of the peptide. In conclusion, modulating potency of GLP-1 by physiologically regulated endocannabinoid-like lipids allows GLP-1R signaling to be regulated spatiotemporally at a constant basal GLP-1 level. PMID:25903129

  6. Regulation of Autocrine Signaling in Subsets of Sympathetic Neurons Has Regional Effects on Tissue Innervation

    PubMed Central

    McWilliams, Thomas G.; Howard, Laura; Wyatt, Sean; Davies, Alun M.

    2015-01-01

    Summary The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cornerstone of neurotrophic theory. Whereas autocrine signaling in neurons affecting survival and axon growth has been described, it is difficult to reconcile autocrine signaling with the idea that targets control their innervation. Here, we report that an autocrine signaling loop in developing mouse sympathetic neurons involving CD40L (TNFSF5) and CD40 (TNFRSF5) selectively enhances NGF-promoted axon growth and branching, but not survival, via CD40L reverse signaling. Because NGF negatively regulates CD40L and CD40 expression, this signaling loop operates only in neurons exposed to low levels of NGF. Consequently, the sympathetic innervation density of tissues expressing low NGF is significantly reduced in CD40-deficient mice, whereas the innervation density of tissues expressing high levels of NGF is unaffected. Our findings reveal how differential regulation of autocrine signaling in neurons has region-specific effects on axon growth and tissue innervation. PMID:25753410

  7. Folate Receptor β Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen.

    PubMed

    Machacek, Christian; Supper, Verena; Leksa, Vladimir; Mitulovic, Goran; Spittler, Andreas; Drbal, Karel; Suchanek, Miloslav; Ohradanova-Repic, Anna; Stockinger, Hannes

    2016-09-15

    Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ(+) macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRβ(-) counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen. PMID:27534550

  8. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    PubMed

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-04-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis. PMID:26984507

  9. Yupingfeng Pulvis Regulates the Balance of T Cell Subsets in Asthma Mice

    PubMed Central

    Wang, Zhigang; Cai, Xueting; Pang, Zhonghua; Wang, Dawei; Ye, Juan; Su, Kelei; Sun, Xiaoyan; Li, Jing; Cao, Peng; Hu, Chunping

    2016-01-01

    Background. Yupingfeng Pulvis (HFBP) had played an active role in many diseases, especially respiratory tract infections. Exploring the possible prevention mechanism of HFBP may provide new ideas in clinical applications for this well-known herbal formula. Purpose. To study the possible mechanisms of therapy effect of HFBP on asthma mice via regulating the balance of Tregs and Th17 cells. Method. The female BALB/c mice were divided into five groups: control group, model group, prednisone (5.5 mg/kg) group, and 22 g/kg HFBP and 44 g/kg HFBP groups. Ovalbumin was used to make the asthma model of mice; the drug was ig administered daily after atomization for consecutive 15 d. The mice were killed after the last administration. The paraffin-embedded tissue sections of the lungs were stained by H&E. Tregs and Th17 cells in bronchoalveolar lavage fluid were detected by flow cytometry. IL-4, TGF-β, and TNF-α in the serum were detected by ELISA assay. Results. HFBP could alleviate the inflammation in the lung tissue of mice, decrease the proportion of Th17 cells, and increase the proportion of Treg cells in bronchoalveolar lavage fluid. HFBP could decrease IL-4 and TNF-α level and increase TGF-β level in blood. Conclusion. HFBP could treat the asthma through impacting the balance of Th17 cells and Treg cells as well as the levels of related inflammatory cytokines in asthma mice. PMID:27143988

  10. Yupingfeng Pulvis Regulates the Balance of T Cell Subsets in Asthma Mice.

    PubMed

    Wang, Zhigang; Cai, Xueting; Pang, Zhonghua; Wang, Dawei; Ye, Juan; Su, Kelei; Sun, Xiaoyan; Li, Jing; Cao, Peng; Hu, Chunping

    2016-01-01

    Background. Yupingfeng Pulvis (HFBP) had played an active role in many diseases, especially respiratory tract infections. Exploring the possible prevention mechanism of HFBP may provide new ideas in clinical applications for this well-known herbal formula. Purpose. To study the possible mechanisms of therapy effect of HFBP on asthma mice via regulating the balance of Tregs and Th17 cells. Method. The female BALB/c mice were divided into five groups: control group, model group, prednisone (5.5 mg/kg) group, and 22 g/kg HFBP and 44 g/kg HFBP groups. Ovalbumin was used to make the asthma model of mice; the drug was ig administered daily after atomization for consecutive 15 d. The mice were killed after the last administration. The paraffin-embedded tissue sections of the lungs were stained by H&E. Tregs and Th17 cells in bronchoalveolar lavage fluid were detected by flow cytometry. IL-4, TGF-β, and TNF-α in the serum were detected by ELISA assay. Results. HFBP could alleviate the inflammation in the lung tissue of mice, decrease the proportion of Th17 cells, and increase the proportion of Treg cells in bronchoalveolar lavage fluid. HFBP could decrease IL-4 and TNF-α level and increase TGF-β level in blood. Conclusion. HFBP could treat the asthma through impacting the balance of Th17 cells and Treg cells as well as the levels of related inflammatory cytokines in asthma mice. PMID:27143988

  11. Auxin Is Rapidly Induced by Herbivore Attack and Regulates a Subset of Systemic, Jasmonate-Dependent Defenses.

    PubMed

    Machado, Ricardo A R; Robert, Christelle A M; Arce, Carla C M; Ferrieri, Abigail P; Xu, Shuqing; Jimenez-Aleman, Guillermo H; Baldwin, Ian T; Erb, Matthias

    2016-09-01

    Plant responses to herbivore attack are regulated by phytohormonal networks. To date, the role of the auxin indole-3-acetic acid (IAA) in this context is not well understood. We quantified and manipulated the spatiotemporal patterns of IAA accumulation in herbivore-attacked Nicotiana attenuata plants to unravel its role in the regulation of plant secondary metabolism. We found that IAA is strongly, rapidly, and specifically induced by herbivore attack. IAA is elicited by herbivore oral secretions and fatty acid conjugate elicitors and is accompanied by a rapid transcriptional increase of auxin biosynthetic YUCCA-like genes. IAA accumulation starts 30 to 60 s after local induction and peaks within 5 min after induction, thereby preceding the jasmonate (JA) burst. IAA accumulation does not require JA signaling and spreads rapidly from the wound site to systemic tissues. Complementation and transport inhibition experiments reveal that IAA is required for the herbivore-specific, JA-dependent accumulation of anthocyanins and phenolamides in the stems. In contrast, IAA does not affect the accumulation of nicotine or 7-hydroxygeranyllinalool diterpene glycosides in the same tissue. Taken together, our results uncover IAA as a rapid and specific signal that regulates a subset of systemic, JA-dependent secondary metabolites in herbivore-attacked plants. PMID:27485882

  12. Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

    PubMed

    Ma, Cindy S; Wong, Natalie; Rao, Geetha; Nguyen, Akira; Avery, Danielle T; Payne, Kathryn; Torpy, James; O'Young, Patrick; Deenick, Elissa; Bustamante, Jacinta; Puel, Anne; Okada, Satoshi; Kobayashi, Masao; Martinez-Barricarte, Ruben; Elliott, Michael; Sebnem Kilic, Sara; El Baghdadi, Jamila; Minegishi, Yoshiyuki; Bousfiha, Aziz; Robertson, Nic; Hambleton, Sophie; Arkwright, Peter D; French, Martyn; Blincoe, Annaliesse K; Hsu, Peter; Campbell, Dianne E; Stormon, Michael O; Wong, Melanie; Adelstein, Stephen; Fulcher, David A; Cook, Matthew C; Stepensky, Polina; Boztug, Kaan; Beier, Rita; Ikincioğullari, Aydan; Ziegler, John B; Gray, Paul; Picard, Capucine; Boisson-Dupuis, Stéphanie; Phan, Tri Giang; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M; Uzel, Gulbu; Casanova, Jean-Laurent; Tangye, Stuart G

    2016-07-25

    Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation. PMID:27401342

  13. Cross talk between cytokine and hyperthermia-induced pathways: identification of different subsets of NF-κB-dependent genes regulated by TNFα and heat shock.

    PubMed

    Janus, Patryk; Stokowy, Tomasz; Jaksik, Roman; Szoltysek, Katarzyna; Handschuh, Luiza; Podkowinski, Jan; Widlak, Wieslawa; Kimmel, Marek; Widlak, Piotr

    2015-10-01

    Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions. PMID:25944781

  14. Regulation of Mouse Retroelement MuERV-L/MERVL Expression by REX1 and Epigenetic Control of Stem Cell Potency

    PubMed Central

    Schoorlemmer, Jon; Pérez-Palacios, Raquel; Climent, María; Guallar, Diana; Muniesa, Pedro

    2014-01-01

    About half of the mammalian genome is occupied by DNA sequences that originate from transposable elements. Retrotransposons can modulate gene expression in different ways and, particularly retrotransposon-derived long terminal repeats, profoundly shape expression of both surrounding and distant genomic loci. This is especially important in pre-implantation development, during which extensive reprograming of the genome takes place and cells pass through totipotent and pluripotent states. At this stage, the main mechanism responsible for retrotransposon silencing, i.e., DNA methylation, is inoperative. A particular retrotransposon called muERV-L/MERVL is expressed during pre-implantation stages and contributes to the plasticity of mouse embryonic stem cells. This review will focus on the role of MERVL-derived sequences as controlling elements of gene expression specific for pre-implantation development, two-cell stage-specific gene expression, and stem cell pluripotency, the epigenetic mechanisms that control their expression, and the contributions of the pluripotency marker REX1 and the related Yin Yang 1 family of transcription factors to this regulation process. PMID:24567914

  15. MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome

    PubMed Central

    Wang, Qian-fei; Wu, George; Mi, Shuangli; He, Fuhong; Wu, Jun; Dong, Jingfang; Luo, Roger T.; Mattison, Ryan; Kaberlein, Joseph J.; Prabhakar, Shyam; Ji, Hongkai

    2011-01-01

    MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL–bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes. PMID:21518926

  16. A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

    PubMed

    Hanley, Christopher J; Noble, Fergus; Ward, Matthew; Bullock, Marc; Drifka, Cole; Mellone, Massimiliano; Manousopoulou, Antigoni; Johnston, Harvey E; Hayden, Annette; Thirdborough, Steve; Liu, Yuming; Smith, David M; Mellows, Toby; Kao, W John; Garbis, Spiros D; Mirnezami, Alex; Underwood, Tim J; Eliceiri, Kevin W; Thomas, Gareth J

    2016-02-01

    Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure. PMID:26716418

  17. A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers

    PubMed Central

    Hanley, Christopher J.; Noble, Fergus; Ward, Matthew; Bullock, Marc; Drifka, Cole; Mellone, Massimiliano; Manousopoulou, Antigoni; Johnston, Harvey E.; Hayden, Annette; Thirdborough, Steve; Liu, Yuming; Smith, David M.; Mellows, Toby; Kao, W. John; Garbis, Spiros D.; Mirnezami, Alex; Underwood, Tim J.

    2016-01-01

    Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure. PMID:26716418

  18. Relative glucocorticoid potency revisited.

    PubMed

    Tanaka, H; Hirano, F; Nomura, Y; Miura, T; Makino, Y; Fukawa, E; Makino, I

    1994-01-01

    To determine the relative potency of synthetic glucocorticoids, glucocorticoid receptor expressing cells were transfected with a hormone-inducible reporter gene, and were cultured in the presence of various glucocorticoid ligands. Hormonal inducibility was determined by means of a chloramphenicol acetyltransferase assay. Dexamethasone and prednisolone, as well as cortisol, induced the expression of the reporter gene in a dose-dependent fashion. The relative potency of each ligand was in this order when inducibility was quantitatively assessed. In conclusion, the transcription assay described here may be a convenient and alternative method to evaluate the relative potency of given glucocorticoids. PMID:7939139

  19. A Subset of Patients with Acute Myeloid Leukemia Has Leukemia Cells Characterized by Chemokine Responsiveness and Altered Expression of Transcriptional as well as Angiogenic Regulators

    PubMed Central

    Brenner, Annette K.; Reikvam, Håkon; Bruserud, Øystein

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized in vitro suspension cultures were used to investigate AML cell proliferation, and global gene expression profiles were compared for chemokine responders and non-responders identified through the proliferation assays. CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to

  20. Stress-mediated Sin3B activation leads to negative regulation of subset of p53 target genes

    PubMed Central

    Kadamb, Rama; Mittal, Shilpi; Bansal, Nidhi; Saluja, Daman

    2015-01-01

    The multiprotein SWI-independent 3 (Sin3)–HDAC (histone deacetylase) corepressor complex mediates gene repression through its interaction with DNA-binding factors and recruitment of chromatin-modifying proteins on to the promoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor protein, p53 has been established upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulated under variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expression is significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, a radiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associated with enhanced interaction of Sin3B with Ser15 phosphorylated p53. Binding of Sin3–HDAC repressor complex on to the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 and H3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated gene repression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation is p53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has an implication in understanding the transrepression mechanism of p53 under DNA damaging conditions. PMID:26181367

  1. Identification of KCa3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines.

    PubMed

    Kovalenko, Ilya; Glasauer, Andrea; Schöckel, Laura; Sauter, Daniel R P; Ehrmann, Alexander; Sohler, Florian; Hägebarth, Andrea; Novak, Ivana; Christian, Sven

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC. PMID:27494181

  2. Identification of KCa3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines

    PubMed Central

    Kovalenko, Ilya; Glasauer, Andrea; Schöckel, Laura; Sauter, Daniel R. P.; Ehrmann, Alexander; Sohler, Florian; Hägebarth, Andrea; Novak, Ivana; Christian, Sven

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC. PMID:27494181

  3. Dynamic regulation of effector IFN-γ-producing and IL-17-producing T cell subsets in the development of acute graft-versus-host disease.

    PubMed

    Zhao, Kai; Ruan, Suhong; Yin, Lingling; Zhao, Dongmei; Chen, Chong; Pan, Bin; Zeng, Lingyu; Li, Zhenyu; Xu, Kailin

    2016-02-01

    Graft-versus-host disease (GVHD) as the predominant complication of allogeneic hematopoietic stem cell transplantation remains to be fully understood. It is known that the cytokines produced by allogeneic reactive effector CD4+ and CD8+ T cells are involved in GVHD. However, the regulation and coordination of IFN-γ-producing and IL-17-producing effector T cells remain unclear. The present study aimed to investigate the dynamic changes of alloantigen-specific effector CD4+ T and CD8+ T cell subsets by flow cytometry, which produce inflammatory cytokines involved in the multistep GVHD pathogenesis progress. The results demonstrated that IL-17-producing CD8+ T (Tc17) cells and IFN-γ+CD8+ T (Tc1) cells were detected in the early stage of GVHD. The differentiation of CD4+ T cells into Th1 cell (IFN-γ+CD4+ T) and Th17 (IL-17+CD4+ T) cells was later than that of the Tc1 and Tc17 cells. The effector CD4+ T and CD8+ T cell subsets either became exhausted or became memory cells, exhibiting a CD62L-CD44+ phenotype following marked expansion during GVHD. Furthermore, T cell-associated type I (IL-2 and IFN-γ) and type II (IL-4 and IL-10) classical cytokines exhibited coordinated dynamic regulation. It was concluded that the differentiation of cytokine-producing Tc1 and Tc17 cells may be the key step in the initiation of GVHD, whereas CD4+ effector Th1 and Th17 cells are considered to be pathophysiological factors leading to the continuous aggravation of GVHD. PMID:26647759

  4. Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways

    PubMed Central

    Garcia-Mata, Carlos; Gay, Robert; Sokolovski, Sergei; Hills, Adrian; Lamattina, Lorenzo; Blatt, Michael R.

    2003-01-01

    Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements. PMID:12949257

  5. Nitrated Alpha Synuclein Induced Alterations in Microglial Immunity is Regulated by CD4+ T Cell Subsets1

    PubMed Central

    Reynolds, Ashley D.; Stone, David K.; Mosley, R. Lee; Gendelman, Howard E.

    2009-01-01

    Microglial inflammatory neuroregulatory activities affect the tempo of nigrostriatal degeneration during Parkinson's disease (PD). Such activities are induced, in part, by misfolded, nitrated alpha-synuclein (N-α-syn) within Lewy bodies released from dying or dead dopaminergic neurons. Such pathobiologic events initiate innate and adaptive immune responses affecting neurodegeneration. We posit that the neurobiological activities of activated microglia are affected by cell-protein and cell-cell contacts, in that microglial interactions with N-α-syn and CD4+ T cells substantively alter the microglial proteome. This leads to alterations in cell homeostatic functions and disease. CD4+CD25+ regulatory T cells (Treg) suppress N-α-syn microglial induced reactive oxygen species and nuclear factor kappa B activation by modulating redox-active enzymes, cell migration, phagocytosis, and bioenergetic protein expression and cell function. In contrast, CD4+CD25− effector T cells exacerbate microglial inflammation and induce “putative” neurotoxic responses. These data support the importance of adaptive immunity in the regulation of PD-associated microglial inflammation. PMID:19299711

  6. Recurrent MALAT1-GLI1 oncogenic fusion and GLI1 up-regulation define a subset of plexiform fibromyxoma.

    PubMed

    Spans, Lien; Fletcher, Christopher Dm; Antonescu, Cristina R; Rouquette, Alexandre; Coindre, Jean-Michel; Sciot, Raf; Debiec-Rychter, Maria

    2016-07-01

    Plexiform fibromyxomas are rare neoplasms, being officially recognized as a distinct entity among benign mesenchymal gastric tumours in the 2010 WHO Classification of Tumours of the Digestive System. Characteristically, these tumours have a multinodular/plexiform growth pattern, and histologically contain variably cellular areas of bland myofibroblastic-type spindle cells embedded in an abundant myxoid matrix, rich in capillary-type vessels. As yet, the molecular and/or genetic features of these tumours are unknown. Here we describe a recurrent translocation, t(11;12)(q11;q13), involving the long non-coding gene metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the gene glioma-associated oncogene homologue 1 (GLI1) in a subgroup of these tumours. The presence of the fusion transcript in our index case was confirmed using polymerase chain reaction (PCR) on genomic DNA, followed by Sanger sequencing. We showed that the truncated GLI1 protein is overexpressed and retains its capacity to transcriptionally activate its target genes. A specific FISH assay was developed to detect the novel MALAT1-GLI1 translocation in formalin-fixed, paraffin-embedded (FFPE) material. This resulted in the identification of two additional cases with this fusion and two cases with polysomy of the GLI1 gene. Finally, immunohistochemistry revealed that the GLI1 protein is exclusively overexpressed in those cases that harbour GLI1/12q13 genomic alterations. In conclusion, overexpression of GLI1 through a recurrent MALAT1-GLI1 translocation or GLI1 up-regulation delineates a pathogenically distinct subgroup of plexiform fibromyxomas with activation of the Sonic Hedgehog signalling pathway. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27101025

  7. Oncoprotein E7 from Beta Human Papillomavirus 38 Induces Formation of an Inhibitory Complex for a Subset of p53-Regulated Promoters

    PubMed Central

    Saidj, Djamel; Cros, Marie-Pierre; Hernandez-Vargas, Hector; Guarino, Francesca; Sylla, Bakary S.; Tommasino, Massimo

    2013-01-01

    Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of IκB kinase beta (IKKβ) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ΔNp73α, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKKβ nuclear form. We show that IKKβ nuclear translocation and ΔNp73α accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ΔNp73α and IKKβ are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ΔNp73α strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ΔNp73α/IKKβ/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes. PMID:24006445

  8. Potency in psychotherapy.

    PubMed

    Gilchrist, A A

    1976-06-01

    This paper defines therapeutic potency in terms of constructive as opposed to destructive intervention. In judging the degree of success in psychotherapy we may use either a medical or a growth model. Whichever criterion is adopted the conclusion stands that potency will be at a maximum whenever the therapist selects the most efficient technique for working with his client regardless of the school which gave birth to it. An eclectic approach views the differences between schools as exaggerated by the use of divergent psychological models and terminology, and sees value in various explanatory concepts such as 'reinforcement' 'conditioning', 'insight' and 'cognitive restructuring'. PMID:1067838

  9. Oligosaccharide-specific induction of interleukin 10 production by B220+ cells from schistosome-infected mice: a mechanism for regulation of CD4+ T-cell subsets.

    PubMed Central

    Velupillai, P; Harn, D A

    1994-01-01

    Defining the factors and/or mechanisms that lead to the predominance of a particular CD4+ T-cell subset (Th-1 vs. Th-2) is an area of intense investigation. In murine schistosomiasis, Th-2-type T cells become predominant after deposition of eggs. The most immunoreactive egg components are glycoproteins. Previously we identified two interesting oligosaccharides found on schistosome eggs and schistosomula. One, lacto-N-fucopentaose III (LNFP-III) contains the interesting trisaccharide Lewisx, which is a weak ligand for P-selectin and is a sugar also found on the alpha and beta chains of the integrin lymphocyte function-associated molecule 1, a ligand for intercellular adhesion molecule 1. Because of the correlation between schistosome egg glycoproteins and Th-2 dominance, the present study examined whether LNFP-III and structurally related oligosaccharides were lymphostimulatory and/or able to induce factors known to down-regulate Th-1 cells. We found that LNFP-III and related sugars did induce proliferation of splenic non-T cells, B220+,CD4-,CD8- cells (B cells) of schistosome-infected and naive mice. In contrast to proliferation, LNFP-III was the only oligosaccharide that induced spleen cells to produce large amounts of interleukin 10 and prostaglandin E2, two molecules known to down-regulate Th-1 cells. Further, only spleen cells from infected mice produced cytokines after oligosaccharide stimulation. Interestingly, LNFP-III stimulation did not induce production of interleukin 4. Thus, a specific carbohydrate ligand has been identified that stimulates B cells to proliferate and produce factors that down-regulate Th-1 T cells. Further, we suggest that identical or structurally related ligands may contribute to the known Th-1 down-regulation in other parasitic diseases and in chronic blood-vascular diseases such as human immunodeficiency virus infection and a number of metastatic carcinomas and that this effect may, therefore, be a general phenomenon. PMID:7904066

  10. NeuroD1 mediates nicotine-induced migration and invasion via regulation of the nicotinic acetylcholine receptor subunits in a subset of neural and neuroendocrine carcinomas

    PubMed Central

    Osborne, Jihan K.; Guerra, Marcy L.; Gonzales, Joshua X.; McMillan, Elizabeth A.; Minna, John D.; Cobb, Melanie H.

    2014-01-01

    Cigarette smoking is a major risk factor for acquisition of small cell lung cancer (SCLC). A role has been demonstrated for the basic helix-loop-helix transcription factor NeuroD1 in the pathogenesis of neural and neuroendocrine lung cancer, including SCLC. In the present study we investigate the possible function of NeuroD1 in established tumors, as well as actions early on in pathogenesis, in response to nicotine. We demonstrate that nicotine up-regulates NeuroD1 in immortalized normal bronchial epithelial cells and a subset of undifferentiated carcinomas. Increased expression of NeuroD1 subsequently leads to regulation of expression and function of the nicotinic acetylcholine receptor subunit cluster of α3, α5, and β4. In addition, we find that coordinated expression of these subunits by NeuroD1 leads to enhanced nicotine-induced migration and invasion, likely through changes in intracellular calcium. These findings suggest that aspects of the pathogenesis of neural and neuroendocrine lung cancers may be affected by a nicotine- and NeuroD1-induced positive feedback loop. PMID:24719457

  11. TFII-I down-regulates a subset of estrogen-responsive genes through its interaction with an initiator element and estrogen receptor alpha.

    PubMed

    Ogura, Yuji; Azuma, Motoki; Tsuboi, Yasunori; Kabe, Yasuaki; Yamaguchi, Yuki; Wada, Tadashi; Watanabe, Hajime; Handa, Hiroshi

    2006-04-01

    TFII-I was initially identified as the general transcription factor that binds to initiator (Inr) elements in vitro. Subsequent studies have shown that TFII-I activates transcription of various genes either through Inr elements or through other upstream elements in vivo. Since, however, most studies so far on TFII-I have been limited to over-expression and reporter gene assays, we reevaluated the role of TFII-I in vivo by using stable knockdown with siRNA and by examining the expression of endogenous genes. Contrary to the widely accepted view, here we show that TFII-I is not important for cell viability in general but rather inhibits the growth of MCF-7 human breast cancer cells. MCF-7 cells are known to proliferate in an estrogen-dependent manner. Through analysis of TFII-I's cell-type specific growth inhibitory effect, we show evidence that TFII-I down-regulates a subset of estrogen-responsive genes, only those containing Inr elements, by recruiting estrogen receptor (ER) alpha and corepressors to these promoters. Thus, this study has revealed an unexpected new role of TFII-I as a negative regulator of transcription and cell proliferation. PMID:16611241

  12. Exploration of Dimensions of Estrogen Potency

    PubMed Central

    Jeyakumar, M.; Carlson, Kathryn E.; Gunther, Jillian R.; Katzenellenbogen, John A.

    2011-01-01

    The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity. PMID:21321128

  13. CPDB: Carcinogenic Potency Database.

    PubMed

    Fitzpatrick, Roberta Bronson

    2008-01-01

    The Carcinogenic Potency Database reports analyses of animal cancer tests on 1,547 chemicals. These tests are used in support of cancer risk assessments for humans. Results are searchable and are made available via the National Library of Medicine's (NLM) TOXNET system. This column will provide background information on the database, as well as present search basics. PMID:19042710

  14. Studies on human blood lymphocytes with iC3b (type 3) complement receptors. II. Characterization of subsets which regulate pokeweed mitogen-induced lymphocyte proliferation and immunoglobulin synthesis.

    PubMed Central

    Abo, W; Gray, J D; Bakke, A C; Horwitz, D A

    1987-01-01

    Human blood lymphocytes that express Type 3 complement receptors (CR3) can be divided into a major subset with high density Fc receptors for IgG (FcR) identified with the monoclonal antibody Leu 11 and two minor subsets which display either CD8 (Leu 2) or CD4 (Leu 3) markers. We isolated CR3+ lymphocyte subsets and examined them for regulatory effects on pokeweed mitogen (PWM) stimulated cells. The FCR CR3+ cell suppressed PWM-induced proliferation and Ig production. Pretreatment of these lymphocytes with immune complexes was required to suppress proliferation, but not IgG production. The CR3+ Leu 2+ FCR- subset also had suppressive activity, but this effect was not observed unless the CR3+ Leu 3+ enriched subset was removed. In fact, the CR3+ Leu 3+ enriched subset enhanced IgG synthesis. Brief exposure of CR3+ lymphocytes to recombinant interleukin 2, recombinant alpha-interferon, but not gamma-interferon, markedly enhanced the inhibitory effect. Time course studies and a comparison of inhibition of Ig synthesis with natural killer cell activity suggested that CR3+ lymphocytes act shortly after lymphocytes are exposed to PWM and that Ig production was regulated by suppression rather than cytotoxicity. These CR3+ lymphocyte subsets may have broad antigen non-specific effects on immunoglobulin synthesis. PMID:2955973

  15. Evidence that the familial adenomatous polyposis gene is involved in a subset of colon cancers with a complementable defect in c-myc regulation

    SciTech Connect

    Erisman, M.D.; Scott, J.K.; Astrin, S.M. )

    1989-06-01

    Human colorectal carcinomas frequently express elevated levels of c-myc mRNA in the absence of a gross genetic change at the c-myc locus. To test the hypothesis that these tumors are defective in a gene function necessary for the regulation of c-myc expression, the authors fused an osteosarcoma cell line that exhibits normal c-myc regulation with two colon carcinoma cell lines that express deregulated levels of c-myc mRNA. Since rates of c-myc mRNA turnover in the colon carcinoma cells were found to be comparable to those in normal cells, increased message stability cannot account for the increased steady-state levels of transcripts. These finding suggest that loss of function of a trans-acting regulator is responsible for the deregulation of c-myc expression in a major fraction of colorectal carcinomas. Analysis of restriction fragment length polymorphisms in tumor/normal tissue pairs from patients with primary colorectal lesions indicated that deregulation of c-myc expression in the tumors is correlated with frequent loss of alleles of syntenic markers on chromosome 5q. Chromosome 5q is the region known to contain the gene for familial adenomatous polyposis, an inherited predisposition to colon cancer. These findings, together with the arlier finding that the colonic distribution of tumors exhibiting deregulated c-myc expression is similar to that reported for familial polyposis, provide evidence that loss of function of the familial adenomatous polyposis gene is involved in a subset of colorectal cancers in which c-myc expression is deregulated.

  16. P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

    PubMed

    Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

    2015-02-01

    Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

  17. P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    PubMed Central

    Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

    2015-01-01

    Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

  18. Potency disorder among Pathans.

    PubMed

    Khan, M F; Ahmed, S H

    1990-01-01

    In a working class industrial area of Karachi hundred consecutive Pathans presenting to a family physician with potency disorder were examined. After exclusion of those with structural or drug related conditions, a structured proforma was introduced. Their presentation, associated symptoms and background pointed to masked depression and lack of sex education. Symptoms of anxiety were noticed in 49% and depressive features in 43%. The guilt feelings were reinforced by Hakims and lay literature which stress more on masturbation (79%) and spermatorrhoea (60%) and not extra-marital intercourse (52%) or bestiality (39%). PMID:2109124

  19. Probabilistic Subset Conjunction

    ERIC Educational Resources Information Center

    Kohli, Rajeev; Jedidi, Kamel

    2005-01-01

    The authors introduce subset conjunction as a classification rule by which an acceptable alternative must satisfy some minimum number of criteria. The rule subsumes conjunctive and disjunctive decision strategies as special cases. Subset conjunction can be represented in a binary-response model, for example, in a logistic regression, using only…

  20. Ratcheting up cancer potency estimates

    PubMed Central

    Crouch, Edmund A.C.; Omenn, Gilbert S

    2014-01-01

    Summary The current paradigm for cancer risk assessment in the United States (U.S.) typically requires selection of representative rodent bioassay dose-response data for extrapolation to a single cancer potency estimate for humans. In the absence of extensive further information, the chosen bioassay result generally is taken to be that which gives the highest extrapolated result from the “most sensitive” species or strain. The estimated human cancer potency is thus derived from an upper-bound value on animal cancer potency that is technically similar to an extreme value statistic. Thus additional information from further bioassays can only lead to equal or larger cancer potency estimates. We here calculate the size of this effect using the collected results of a large number of bioassays. Since many standards are predicated on the value of the cancer potency, this effect is undesirable in producing a strong counter-incentive to performing further bioassays. PMID:22296526

  1. Opioid analgesics: does potency matter?

    PubMed

    Passik, Steven D; Webster, Lynn

    2014-01-01

    Prescription opioid analgesics with a wide range of potencies are currently used for the treatment of chronic pain. Yet understanding the clinical relevance and therapeutic consequences of opioid potency remains ill defined. Both patients and clinicians alike have misperceptions about opioid potency, expecting that less-potent opioids will be less effective or fearing that more-potent opioids are more dangerous or more likely to be abused. In this review, common myths about the potency of opioid analgesics will be discussed. Clinicians should understand that pharmacologic potency per se does not necessarily imply more effective analgesia or higher abuse liability. Published dose conversion tables may not accurately calculate the dose for effective and safe rotation from one opioid to another in patients receiving long-term opioid therapy because they are based on limited data that may not apply to chronic pain. Differences in pharmacologic potency are largely accounted for by the actual doses prescribed, according to individualized patient need. Factors for achieving effective analgesia and reducing the risks involved with opioid use include careful medication selection based on patient characteristics, appropriate dosing titration and opioid rotation practices, knowledge of product formulation characteristics (eg, extended release, immediate release, and tamper-resistant features), and an awareness of differences in opioid pharmacokinetics and metabolism. Clinicians should remain vigilant in monitoring patients on any opioid medication, regardless of classification along the opioid potency continuum. PMID:25162606

  2. Shen-Qi-Jie-Yu-Fang has antidepressant effects in a rodent model of postpartum depression by regulating the immune organs and subsets of T lymphocytes

    PubMed Central

    Qu, Miao; Tang, Qisheng; Li, Xiaoli; Zhao, Ruizhen; Li, Jingya; Xu, Hong; Gao, Yushan; Mao, Yingqiu

    2015-01-01

    Background Shen-Qi-Jie-Yu-Fang (SJ Fang) is a herbal preparation used in traditional Chinese medicine, and is a potentially important new therapeutic agent in postpartum depression (PPD). Previously, we have elucidated the effects of SJ Fang on hormone receptors and monoamine neurotransmitters involved in the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes in PPD rats. However, the immune-modulating effects of SJ Fang in PPD are still unknown. In this study, we explored the effects of SJ Fang on the immune organs and subsets of T lymphocytes in PPD rats. Methods PPD was created in Sprague-Dawley rats by inducing hormone-simulated pregnancy followed by hormone withdrawal. After hormone withdrawal, the PPD rats were then treated with fluoxetine at 1, 2, and 4 weeks, and the SJ Fang rats were also treated at 1, 2, and 4 weeks. Depressive behavior in the rats was evaluated by the forced swim test, sucrose consumption test, and open field test. The thymus index and spleen index were calculated. Hematoxylin-eosin staining was used to identify pathological features in the thymus and spleen. CD3, CD4, and CD8 lymphocyte subsets were analyzed by flow cytometry. Results Both fluoxetine and SJ Fang decreased immobility time, increased sucrose consumption, an horizontal and vertical movement. After 4 weeks of treatment with fluoxetine or SJ Fang, the thymus index and spleen index were significantly higher than at baseline, and the morphology of the thymus and spleen were returning to normal. Two weeks after hormone withdrawal, subsets of T lymphocytes indicated a shift from immune activation to immune suppression, which was reversed by 4 weeks of treatment with fluoxetine or SJ Fang. Conclusion It is suggested that T-cell mediate immune responses which may play a role in the etiopathology of postpartum depression. SJ Fang had an antidepressant effect on the immune system in rats with PPD. PMID:26203247

  3. CERES Search and Subset Tool

    Atmospheric Science Data Center

    2016-02-18

    Description:  Search and subset CERES Level 2 SSF and FLASHFlux data Customization Options Search and subset by date, time, and geolocation Subset by parameter and ... HDF and NetCDF Details:  CERES Search and Subset Tool Screenshot:  ...

  4. Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

    PubMed

    Abd El-Hamid, M I; Abd El-Aziz, N K; Ali, H A

    2016-01-01

    Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems. PMID:27609474

  5. CTLA4Ig inhibits airway eosinophilia and hyperresponsiveness by regulating the development of Th1/Th2 subsets in a murine model of asthma.

    PubMed

    Padrid, P A; Mathur, M; Li, X; Herrmann, K; Qin, Y; Cattamanchi, A; Weinstock, J; Elliott, D; Sperling, A I; Bluestone, J A

    1998-04-01

    Complete T-cell activation requires two distinct signals, one delivered via the T-cell receptor, and the second "co-stimulatory" signal through CD28/B7 ligation. Previous studies showed that the blockade of CD28/B7 ligation alters differentiation of Th1/Th2 lymphocyte subsets in vitro and in vivo. The present study was designed to determine the effect of a CD28/B7 antagonist (CTLA4Ig) on Th1/Th2 development in Schistosoma mansoni-sensitized and airway-challenged mice. Treatment of mice with CTLA4Ig beginning 1 wk after sensitization abolished airway responsiveness to intravenous methacholine determined 96 h following antigen challenge. We also found a significant reduction in bronchoalveolar lavage (BAL) eosinophilia, and reduced peribronchial eosinophilic infiltration and mucoid-cell hyperplasia. Furthermore, CTLA4Ig treatment significantly decreased interleukin (IL)-4 and IL-5 content in BAL fluid in vivo, and the production of IL-5 by lung lymphocytes stimulated with soluble egg antigen (SEA) in vitro. In contrast, the content of interferon-gamma in BAL fluid and supernatant from SEA-stimulated lung lymphocytes from CTLA4Ig-treated mice was increased significantly compared with untreated animals. Thus, CTLA4Ig inhibits eosinophilic airway inflammation and airway hyperresponsiveness in S. mansoni-sensitized and airway-challenged mice, most likely due to attenuated secretion of Th2-type cytokines and increased secretion of Th1-type cytokines. PMID:9533932

  6. The Subset Sum game☆

    PubMed Central

    Darmann, Andreas; Nicosia, Gaia; Pferschy, Ulrich; Schauer, Joachim

    2014-01-01

    In this work we address a game theoretic variant of the Subset Sum problem, in which two decision makers (agents/players) compete for the usage of a common resource represented by a knapsack capacity. Each agent owns a set of integer weighted items and wants to maximize the total weight of its own items included in the knapsack. The solution is built as follows: Each agent, in turn, selects one of its items (not previously selected) and includes it in the knapsack if there is enough capacity. The process ends when the remaining capacity is too small for including any item left. We look at the problem from a single agent point of view and show that finding an optimal sequence of items to select is an NP-hard problem. Therefore we propose two natural heuristic strategies and analyze their worst-case performance when (1) the opponent is able to play optimally and (2) the opponent adopts a greedy strategy. From a centralized perspective we observe that some known results on the approximation of the classical Subset Sum can be effectively adapted to the multi-agent version of the problem. PMID:25844012

  7. Redefining Myeloid Cell Subsets in Murine Spleen

    PubMed Central

    Hey, Ying-Ying; Tan, Jonathan K. H.; O’Neill, Helen C.

    2016-01-01

    Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types. PMID:26793192

  8. CERES Search and Subset Tool

    Atmospheric Science Data Center

    2016-06-24

    ... data granules using a high resolution spatial metadata database and directly accessing the archived data granules. Subset results are ... data granules using a high resolution spatial metadata database and directly accessing the archived data granules. Subset results are ...

  9. MOPITT Search and Subset Tool

    Atmospheric Science Data Center

    2016-07-06

    Description:  Search and subset MOPITT Version 5 & 6 Level 2 data Customization Options Search and subset MOPITT Version 5 and Version 6 Level 2 data Update the ... NetCDF3 and NetCDF4 Details:  MOPITT Search and Subset Tool Acronyms/Abbreviations:  MOPITT ...

  10. RELATIVE POTENCY RANKING FOR CHLOROPHENOLS

    EPA Science Inventory

    Recently the National Center for Environmental Assessment-Cincinnati completed a feasibility study for developing a toxicity related relative potency ranking scheme for chlorophenols. In this study it was concluded that a large data base exists pertaining to the relative toxicity...

  11. The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters.

    PubMed

    Haakonsen, Diane L; Yuan, Andy H; Laub, Michael T

    2015-11-01

    Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively. PMID:26545812

  12. The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters

    PubMed Central

    Haakonsen, Diane L.; Yuan, Andy H.; Laub, Michael T.

    2015-01-01

    Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ70-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the −10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively. PMID:26545812

  13. NaJAZh Regulates a Subset of Defense Responses against Herbivores and Spontaneous Leaf Necrosis in Nicotiana attenuata Plants[C][W][OA

    PubMed Central

    Oh, Youngjoo; Baldwin, Ian T.; Gális, Ivan

    2012-01-01

    The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development. PMID:22496510

  14. 21 CFR 610.10 - Potency.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Potency. 610.10 Section 610.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.10 Potency. Tests for potency shall consist of...

  15. 21 CFR 610.10 - Potency.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Potency. 610.10 Section 610.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.10 Potency. Tests for potency shall consist of...

  16. 21 CFR 610.10 - Potency.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Potency. 610.10 Section 610.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.10 Potency. Tests for potency shall consist of...

  17. 21 CFR 610.10 - Potency.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Potency. 610.10 Section 610.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.10 Potency. Tests for potency shall consist of...

  18. 21 CFR 610.10 - Potency.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Potency. 610.10 Section 610.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.10 Potency. Tests for potency shall consist of...

  19. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes

    PubMed Central

    Li, Xuan; Chatterjee, Nirmalya; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to “stress-defence mode” when challenged by external stress. PMID:26911346

  20. A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line.

    PubMed

    Benabbou, Nadia; Mirshahi, Pezhman; Bordu, Camille; Faussat, Anne-Marie; Tang, Ruoping; Therwath, Amu; Soria, Jeannette; Marie, Jean-Pierre; Mirshahi, Massoud

    2014-10-01

    The importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called 'Hospicells' (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells. PMID:25095896

  1. Programmed death 1 regulates memory phenotype CD4 T cell accumulation, inhibits expansion of the effector memory phenotype subset and modulates production of effector cytokines.

    PubMed

    Charlton, Joanna J; Tsoukatou, Debbie; Mamalaki, Clio; Chatzidakis, Ioannis

    2015-01-01

    Memory phenotype CD4 T cells are found in normal mice and arise through response to environmental antigens or homeostatic mechanisms. The factors that regulate the homeostasis of memory phenotype CD4 cells are not clear. In the present study we demonstrate that there is a marked accumulation of memory phenotype CD4 cells, specifically of the effector memory (T(EM)) phenotype, in lymphoid organs and tissues of mice deficient for the negative co-stimulatory receptor programmed death 1 (PD-1). This can be correlated with decreased apoptosis but not with enhanced homeostatic turnover potential of these cells. PD-1 ablation increased the frequency of memory phenotype CD4 IFN-γ producers but decreased the respective frequency of IL-17A-producing cells. In particular, IFN-γ producers were more abundant but IL-17A producing cells were more scarce among PD-1 KO T(EM)-phenotype cells relative to WT. Transfer of peripheral naïve CD4 T cells suggested that accumulated PD-1 KO T(EM)-phenotype cells are of peripheral and not of thymic origin. This accumulation effect was mediated by CD4 cell-intrinsic mechanisms as shown by mixed bone marrow chimera experiments. Naïve PD-1 KO CD4 T cells gave rise to higher numbers of TEM-phenotype lymphopenia-induced proliferation memory cells. In conclusion, we provide evidence that PD-1 has an important role in determining the composition and functional aspects of memory phenotype CD4 T cell pool. PMID:25803808

  2. MOPITT Search and Subset Tool

    Atmospheric Science Data Center

    2016-06-24

    ... 6 data is now available in both HDF5 and netCDF4 formats Login is no longer required to search and subset, only when placing an order ... 6 data is now available in both HDF5 and netCDF4 formats Login is no longer required to search and subset, only when placing an order ...

  3. [Immune stimulative potency of milk proteins].

    PubMed

    Ambroziak, Adam; Cichosz, Grazyna

    2014-02-01

    Milk proteins are characterized by the highest immune stimulative potency from among all the proteins present in human diet. Whey proteins and numerous growth factors that regulate insulin secretion, differentiation of intestine epithelium cells, and also tissue restoration, are priceless in stimulation the immune system. Lactoferrin shows the most comprehensive pro-health properties: antioxidative, anticancer, immune stimulative and even chemopreventive. Also peptides and amino acids formed from casein and whey proteins possess immune stimulative activity. The most valuable proteins, i.e. lactoferrin, immune globulins, lactoperoxidase and lisozyme, together with bioactive peptides, are resistant to pepsin and trypsin activity. This is why they maintain their exceptional biological activity within human organism. Properly high consumption of milk proteins conditions correct function of immune system, especially at children and elderly persons. PMID:24720113

  4. Newcastle disease virus vaccine potency determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potency of inactivated Newcastle disease virus (NDV) vaccines is determined using vaccination and challenge. If the minimum killed viral antigen necessary for clinical protection can be determined, vaccines meeting or exceeding this dose might be considered of adequate potency. In these studies, c...

  5. 21 CFR 660.4 - Potency test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Potency test. 660.4 Section 660.4 Food and Drugs... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface...

  6. 21 CFR 660.43 - Potency test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Potency test. 660.43 Section 660.43 Food and Drugs... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test... antibody in the appropriate sera of the reference panel by all test methods recommended by the...

  7. New TES Search and Subset Application

    Atmospheric Science Data Center

    2013-08-06

    New TES Search and Subset Application Search & Subset Application Wednesday, September 19, 2012 The Atmospheric Science Data Center (ASDC) at NASA Langley Research Center in collaboration with the ...

  8. VARIATIONS IN THE NEUROTOXIC POTENCY OF TRIMETHYLTIN

    EPA Science Inventory

    Seven samples of trimethyltin obtained from three commercial sources were evaluated for neurotoxic potency in the rat. Hippocampus weight, histology and assays of the astrocyte protein, glial fibrillary acidic protein, were used as indices of neurotoxicity. A single administratio...

  9. Potency classification of topical corticosteroids: modern perspectives.

    PubMed

    Ashworth, J

    1989-01-01

    The effects of topical corticosteroid therapy upon human epidermal Langerhans cell surface markers were studied. The reduction in numbers of human leukocyte antigen-DR+/T6+ Langerhands cells proved to be a potency-related effect and Langerhans cell enumeration may therefore have some use as an adjunct in the assessment of steroid potency. Further studies showed that corticosteroid therapy also reduced the function of Langerhans cells in terms of epidermal cell alloantigen-presenting capacity. However, experimental variation and the high antigen-presenting potency of residual flow cytomterically sorted human leukocyte antigen-DR+/T6+ Langerhans cells following steroid application (1) mean that such functional assessments of epidermal cell alloantigen-presenting capacity cannot be recommended as an easily reproducible method for measuring steroid potency. PMID:2696307

  10. Lower bounds for identifying subset members with subset queries

    SciTech Connect

    Knill, E.

    1994-04-01

    An instance of a group testing problem is a set of objects {Omicron}and an unknown subset P of {Omicron}.The task is to determine P by using queries of the type ``does P intersect ``Q``, where Q is a subset of {Omicron}. This problem occurs in areas such as fault detection, multiaccess communications, optimal search, blood testing and chromosome mapping. Consider the two stage algorithm for solving a group testing problem where in the first stage, a predetermined set of queries, are asked in parallel, and in the second stage, P is determined by testing individual objects. Let n = {vert_bar}{Omicron}{vert_bar}. Suppose that P is generated by independently adding each {chi} {element_of}{Omicron} to P with probability p/n. Let q{sub 1} (q{sub 2}) be the number of queries asked in the first (second) stage of this algorithm. We show that if q{sub 1} = o(log(n) log(n)/log log(n)), then Exp(q{sub 2}) = n{sup l{minus}0(1)}, while there exist algorithms with q{sub 1} = O(log(n)log(n)/loglog(n)) and Exp(q{sub 2}) = o(l). The proof involves a relaxation technique which can be used with arbitrary distributions. The best previously known bound is q{sub 1} + Exp(q{sub 2}) = {Omega}(p log(n)). For general group testing algorithms, our results imply that if the average number of queries over the course of n{sup {gamma}} ({gamma} > 0) independent experiments is O n{sup l{minus}{element_of}}, then with high probability {Omega}(log(n)log(n)/loglog(n)) non-singleton subsets are queried. This settles a conjecture of Bill Bruno and David Torney and has important consequences for the use of group testing in screening DNA libraries and other applications where its is more cost effective to use non-adaptive algorithms and/or expensive to prepare a subset Q for its first test.

  11. Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

    PubMed

    Jeyakumar, M; Carlson, Kathryn E; Gunther, Jillian R; Katzenellenbogen, John A

    2011-04-15

    The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity. PMID:21321128

  12. 21 CFR 660.4 - Potency test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently...

  13. 21 CFR 660.43 - Potency test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect...

  14. 21 CFR 660.4 - Potency test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently...

  15. 21 CFR 660.43 - Potency test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect...

  16. RELATIVE POTENCIES OF MINERAL AND SYNTHETIC FIBERS

    EPA Science Inventory

    This paper presents a summary of the present need for the NHEERL data set, how the data are being recovered and entered into spreadsheets for analysis and modeling, and the potential for improved risk models that include consideration of differences in relative potencies based on...

  17. 21 CFR 660.43 - Potency test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect...

  18. 21 CFR 660.4 - Potency test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently...

  19. 21 CFR 660.43 - Potency test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect...

  20. 21 CFR 660.4 - Potency test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently...

  1. Macrophage subsets and microglia in multiple sclerosis.

    PubMed

    Bogie, Jeroen F J; Stinissen, Piet; Hendriks, Jerome J A

    2014-08-01

    Along with microglia and monocyte-derived macrophages, macrophages in the perivascular space, choroid plexus, and meninges are the principal effector cells in neuroinflammatory and neurodegenerative disorders. These phagocytes are highly heterogeneous cells displaying spatial- and temporal-dependent identities in the healthy, injured, and inflamed CNS. In the last decade, researchers have debated on whether phagocytes subtypes and phenotypes are pathogenic or protective in CNS pathologies. In the context of this dichotomy, we summarize and discuss the current knowledge on the spatiotemporal physiology of macrophage subsets and microglia in the healthy and diseased CNS, and elaborate on factors regulating their behavior. In addition, the impact of macrophages present in lymphoid organs on CNS pathologies is defined. The prime focus of this review is on multiple sclerosis (MS), which is characterized by inflammation, demyelination, neurodegeneration, and CNS repair, and in which microglia and macrophages have been extensively scrutinized. On one hand, microglia and macrophages promote neuroinflammatory and neurodegenerative events in MS by releasing inflammatory mediators and stimulating leukocyte activity and infiltration into the CNS. On the other hand, microglia and macrophages assist in CNS repair through the production of neurotrophic factors and clearance of inhibitory myelin debris. Finally, we define how microglia and macrophage physiology can be harnessed for new therapeutics aimed at suppressing neuroinflammatory and cytodegenerative events, as well as promoting CNS repair. We conclude that microglia and macrophages are highly dynamic cells displaying disease stage and location-specific fates in neurological disorders. Changing the physiology of divergent phagocyte subsets at particular disease stages holds promise for future therapeutics for CNS pathologies. PMID:24952885

  2. Functional and Phenotypic Plasticity of CD4+ T Cell Subsets

    PubMed Central

    Caza, Tiffany; Landas, Steve

    2015-01-01

    The remarkable plasticity of CD4+ T cells allows individuals to respond to environmental stimuli in a context-dependent manner. A balance of CD4+ T cell subsets is critical to mount responses against pathogen challenges to prevent inappropriate activation, to maintain tolerance, and to participate in antitumor immune responses. Specification of subsets is a process beginning in intrathymic development and continuing within the circulation. It is highly flexible to adapt to differences in nutrient availability and the tissue microenvironment. CD4+ T cell subsets have significant cross talk, with the ability to “dedifferentiate” given appropriate environmental signals. This ability is dependent on the metabolic status of the cell, with mTOR acting as the rheostat. Autoimmune and antitumor immune responses are regulated by the balance between regulatory T cells and Th17 cells. When a homeostatic balance of subsets is not maintained, immunopathology can result. CD4+ T cells carry complex roles within tumor microenvironments, with context-dependent immune responses influenced by oncogenic drivers and the presence of inflammation. Here, we examine the signals involved in CD4+ T cell specification towards each subset, interconnectedness of cytokine networks, impact of mTOR signaling, and cellular metabolism in lineage specification and provide a supplement describing techniques to study these processes. PMID:26583116

  3. Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.

    PubMed

    Schwartz, Phillip A; Kuzmic, Petr; Solowiej, James; Bergqvist, Simon; Bolanos, Ben; Almaden, Chau; Nagata, Asako; Ryan, Kevin; Feng, Junli; Dalvie, Deepak; Kath, John C; Xu, Meirong; Wani, Revati; Murray, Brion William

    2014-01-01

    Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors. PMID:24347635

  4. 21 CFR 640.56 - Quality control test for potency.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Quality control test for potency. 640.56 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.56 Quality control test for potency. (a) Quality control tests for potency of antihemophilic factor shall be...

  5. 21 CFR 640.56 - Quality control test for potency.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Quality control test for potency. 640.56 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.56 Quality control test for potency. (a) Quality control tests for potency of antihemophilic factor shall be...

  6. Dermal Vγ4(+) γδ T cells possess a migratory potency to the draining lymph nodes and modulate CD8(+) T-cell activity through TNF-α production.

    PubMed

    Nakamizo, Satoshi; Egawa, Gyohei; Tomura, Michio; Sakai, Shunsuke; Tsuchiya, Soken; Kitoh, Akihiko; Honda, Tetsuya; Otsuka, Atsushi; Nakajima, Saeko; Dainichi, Teruki; Tanizaki, Hideaki; Mitsuyama, Masao; Sugimoto, Yukihiko; Kawai, Kazuhiro; Yoshikai, Yasunobu; Miyachi, Yoshiki; Kabashima, Kenji

    2015-04-01

    A large number of gamma delta T cells (γδ T cells) are located within epithelial tissues including the skin. In mice, epidermal and dermal γδ T cells consist of distinct subsets and have specific roles in cutaneous immune responses. A recent study demonstrated that γδ T cells and cutaneous dendritic cells migrate from the skin to the draining lymph nodes (LNs). However, it remains unclear whether they regulate the antigen-specific immune response within the LNs. Herein, we investigated their properties and role in the LNs using the Mycobacterium bovis bacille Calmette-Guérin (BCG) infection model. In vivo cell labeling analysis revealed that most of the migratory subset comprised dermal Vγ4(+) cells. This population transmigrated from the skin to the LNs in a Gi-coupled chemokine receptor-independent manner. By depleting Vγ4(+) cells, the intranodal expansion of CD8(+) T cell against BCG was significantly attenuated. In addition, in vitro analysis revealed that Vγ4(+) cells produced TNF-α and enhanced IL-12 production by dendritic cells. Taken together, these findings suggest that dermal Vγ4(+) cells are a unique subset that possesses a migratory potency to the skin-draining LNs and enhances the dendritic cell function therein. PMID:25493651

  7. Rationale for Further Medical and Health Research on High-Potency Sweeteners

    PubMed Central

    2012-01-01

    High-potency or artificial sweeteners have historically been considered inert compounds without physiological consequences other than taste sensations. However, recent data suggest that some of these sweeteners have biological effects that may impact human health. Furthermore, there are significant gaps in our current knowledge of the pharmacokinetics of these sweeteners, their potential for “sweetener–drug interactions” and their impact on appetite and body weight regulation. Nine research needs are described that address some of the major unknown issues associated with ingestion of high-potency sweeteners. PMID:22539626

  8. Digital Image Correlation with Dynamic Subset Selection

    NASA Astrophysics Data System (ADS)

    Hassan, Ghulam Mubashar; MacNish, Cara; Dyskin, Arcady; Shufrin, Igor

    2016-09-01

    The quality of the surface pattern and selection of subset size play a critical role in achieving high accuracy in Digital Image Correlation (DIC). The subset size in DIC is normally selected by testing different subset sizes across the entire image, which is a laborious procedure. This also leads to the problem that the worst region of the surface pattern influences the performance of DIC across the entire image. In order to avoid these limitations, a Dynamic Subset Selection (DSS) algorithm is proposed in this paper to optimize the subset size for each point in an image before optimizing the correlation parameters. The proposed DSS algorithm uses the local pattern around the point of interest to calculate a parameter called the Intensity Variation Ratio (Λ), which is used to optimize the subset size. The performance of the DSS algorithm is analyzed using numerically generated images and is compared with the results of traditional DIC. Images obtained from laboratory experiments are also used to demonstrate the utility of the DSS algorithm. Results illustrate that the DSS algorithm provides a better alternative to subset size "guessing" and finds an appropriate subset size for each point of interest according to the local pattern.

  9. Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions

    PubMed Central

    Vu Manh, Thien-Phong; Bertho, Nicolas; Hosmalin, Anne; Schwartz-Cornil, Isabelle; Dalod, Marc

    2015-01-01

    Dendritic cells (DCs) were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T-cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks, and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization, and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation, and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes, and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types, organs, and

  10. Summary of carcinogenic potency and positivity for 492 rodent carcinogens in the carcinogenic potency database.

    PubMed Central

    Gold, L S; Slone, T H; Bernstein, L

    1989-01-01

    A tabulation of carcinogenic potency (TD50) by species for 492 chemicals that induce tumors in rats or mice is presented. With the use of the Carcinogenic Potency Database, experimental results are summarized by indicating in which sex-species groups the chemical was tested and the respective evaluations of carcinogenicity. A comparison of three summary measures of TD50 for chemicals with more than one positive experiment per species shows that the most potent TD50 value is similar to measures that average values or functions of values. This tabulation can be used to investigate associations between rodent potency and other factors such as mutagenicity, teratogenicity, chemical structure, and human exposure. PMID:2707207

  11. Potency of Animal Models in KANSEI Engineering

    NASA Astrophysics Data System (ADS)

    Ozaki, Shigeru; Hisano, Setsuji; Iwamoto, Yoshiki

    Various species of animals have been used as animal models for neuroscience and provided critical information about the brain functions. Although it seems difficult to elucidate a highly advanced function of the human brain, animal models have potency to clarify the fundamental mechanisms of emotion, decision-making and social behavior. In this review, we will pick up common animal models and point to both the merits and demerits caused by the characteristics. We will also mention that wide-ranging approaches from animal models are advantageous to understand KANSEI as well as mind in humans.

  12. Antecedents of team potency and team effectiveness: an examination of goal and process clarity and servant leadership.

    PubMed

    Hu, Jia; Liden, Robert C

    2011-07-01

    Integrating theories of self-regulation with team and leadership literatures, this study investigated goal and process clarity and servant leadership as 3 antecedents of team potency and subsequent team effectiveness, operationalized as team performance and organizational citizenship behavior. Our sample of 304 employees represented 71 teams in 5 banks. Results showed that team-level goal and process clarity as well as team servant leadership served as 3 antecedents of team potency and subsequent team performance and team organizational citizenship behavior. Furthermore, we found that servant leadership moderated the relationships between both goal and process clarity and team potency, such that the positive relationships between both goal and process clarity and team potency were stronger in the presence of servant leadership. PMID:21319877

  13. The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

    PubMed Central

    Rückert, Christian; Milse, Johanna; Albersmeier, Andreas; Koch, Daniel J; Pühler, Alfred; Kalinowski, Jörn

    2008-01-01

    Background Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. Results Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. Conclusion CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules. PMID:18854009

  14. Inhaled Anesthetic Potency in Aged Alzheimer Mice

    PubMed Central

    Bianchi, Shannon L.; Caltagarone, Breanna M.; LaFerla, Frank M.; Eckenhoff, Roderic G.; Kelz, Max B.

    2016-01-01

    BACKGROUND The number of elderly patients with frank or incipient Alzheimer’s disease (AD) requiring surgery is growing as the population ages. General anesthesia may exacerbate symptoms of and the pathology underlying AD, so minimizing anesthetic exposure may be important. This requires knowledge of whether the continuing AD pathogenesis alters anesthetic potency. METHODS We determined the induction potency and emergence time for isoflurane, halothane, and sevoflurane using the minimum alveolar anesthetic concentration for loss of righting reflex as an end point in 12- to 14-mo-old triple transgenic Alzheimer (3xTgAD) mice and wild type C57BL6 controls. 3xTgAD mice model AD by harboring three distinct mutations: the APPSwe, Tau, and PS1 human transgenes, each of which has been associated with familial forms of human AD. RESULTS The 3xTgAD mice exhibited mild resistance (from 8% to 30%) to volatile anesthetics but displayed indistinguishable emergence patterns from all three inhaled anesthetics. CONCLUSIONS These results show that the genetic vulnerabilities and neuropathology associated with AD produce a small but significant decrease in sensitivity to the hypnotic actions of three inhaled anesthetics. Emergence times were not altered. PMID:19820240

  15. New MOPITT Search and Subset Application

    Atmospheric Science Data Center

    2013-09-11

    New MOPITT Search and Subset Application New Subsetter for Level 2 Version 5 data Wednesday, September 11, 2013 The Atmospheric Science Data Center (ASDC) at NASA Langley Research Center in collaboration ...

  16. Cross-species comparison of relative potencies and relative sensitivities of fishes to dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in vitro.

    PubMed

    Eisner, Bryanna K; Doering, Jon A; Beitel, Shawn C; Wiseman, Steve; Raine, Jason C; Hecker, Markus

    2016-01-01

    Dioxin-like compounds of varying toxicities are found in complex mixtures. The toxic equivalency factor (TEF) approach was developed based on the potency of a dioxin-like compound relative to the potency of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) to streamline risk assessment. One limitation of the TEF approach is uncertainty regarding differences in the relative potency of dioxin-like compounds among different species. Relative potencies among fishes are limited, relative to relative potencies among birds and mammals, and TEFs for fishes are based entirely on the model species, rainbow trout (Oncorhynchus mykiss). An in vitro liver explant assay was used to characterize species-specific responses with regard to up-regulation of CYP1A transcript after exposure to 6 dioxin-like compounds in rainbow trout, white sturgeon (Acipenser transmontanus), lake sturgeon (Acipenser fulvescens), and northern pike (Esox lucius). Differences in sensitivities were observed among species after exposure to dioxin-like compounds. The relative potencies developed from liver explants of rainbow trout were comparable to relative potencies developed from embryo toxicity assays. Differences in relative potencies between species with the least and greatest relative potencies were up to 40-fold. To compare relative potencies among species, concentrations of dioxin-like compounds in fish eggs in the Fraser River and in Lake Ontario were used to calculate toxic equivalency quotients (TEQs) determined from TEFs or TCDD equivalents determined from relative potencies. The TEQs underestimated TCDD equivalents for white sturgeon, lake sturgeon, and northern pike, indicating uncertainty in application of TEFs to diverse fishes. PMID:26202062

  17. Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing.

    PubMed

    Walker, A; Srinivas, G B

    2013-09-01

    Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the "International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward" held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination-challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination-challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified. PMID:23891496

  18. CD27 natural killer cell subsets play different roles during the pre-onset stage of experimental autoimmune encephalomyelitis.

    PubMed

    Gao, Ming; Yang, Yan; Li, Daling; Ming, Bingxia; Chen, Huoying; Sun, Yan; Xiao, Yifan; Lai, Lin; Zou, Huijuan; Xu, Yong; Xiong, Ping; Tan, Zheng; Gong, Feili; Zheng, Fang

    2016-08-01

    NK cells participate in the development of human multiple sclerosis (MS) and mouse experimental autoimmune encephalomyelitis (EAE), but the roles of different NK cell subsets in disease onset remain poorly understood. In this study, murine NK cells were divided into CD27(high) and CD27(low/-) subsets. The CD27(high) subset was decreased and the CD27(low/-) subset was increased in lymphoid organs during the pre-onset stage of EAE. Compared with the counterpart in naïve mice, the CD27(high) subset showed lower expression of Ly49D, Ly49H and NKG2D, and less production of IFN-γ, whereas the CD27(low/-) subset showed similar expression of the above mentioned surface receptors but higher cytotoxic activity in EAE mice. Compared with the CD27(high) subset, the CD27(low/-) subset exhibited increased promotion of DC maturation and no significant inhibition of T cells proliferation and Th17 cells differentiation in vitro Additionally, adoptive transfer of the CD27(low/-) subset, but not the CD27(high) subset, exacerbated the severity of EAE. Collectively, our data suggest the CD27 NK cell subsets play different roles in controlling EAE onset, which provide a new understanding for the regulation of NK cell subsets in early autoimmune disease. PMID:27368310

  19. ALTERNATIVE APPROACH TO ESTIMATING CANCER POTENCY FOR ASBESTOS

    EPA Science Inventory

    The alternative approach for estimating cancer potency from inhalation exposure to asbestos seeks to improve the methods developed by USEPA (1986). This efforts seeks to modify the the current approach for estimating cancer potency for lung cancer and mesothelioma to account fo...

  20. 21 CFR 660.25 - Potency tests without reference preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Potency tests without reference preparations. 660.25 Section 660.25 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.25 Potency tests...

  1. 21 CFR 610.21 - Limits of potency.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Limits of potency. 610.21 Section 610.21 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Standard Preparations and Limits of Potency § 610.21 Limits...

  2. 21 CFR 610.21 - Limits of potency.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Limits of potency. 610.21 Section 610.21 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Standard Preparations and Limits of Potency § 610.21 Limits...

  3. Distinct Translational Control in CD4+ T Cell Subsets

    PubMed Central

    Yurchenko, Ekaterina; Zheng, Lei; Gandin, Valentina; Topisirovic, Ivan; Li, Shui; Wagner, Carston R.; Sonenberg, Nahum; Piccirillo, Ciriaco A.

    2013-01-01

    Regulatory T cells expressing the transcription factor Foxp3 play indispensable roles for the induction and maintenance of immunological self-tolerance and immune homeostasis. Genome-wide mRNA expression studies have defined canonical signatures of T cell subsets. Changes in steady-state mRNA levels, however, often do not reflect those of corresponding proteins due to post-transcriptional mechanisms including mRNA translation. Here, we unveil a unique translational signature, contrasting CD4+Foxp3+ regulatory T (TFoxp3+) and CD4+Foxp3− non-regulatory T (TFoxp3−) cells, which imprints subset-specific protein expression. We further show that translation of eukaryotic translation initiation factor 4E (eIF4E) is induced during T cell activation and, in turn, regulates translation of cell cycle related mRNAs and proliferation in both TFoxp3− and TFoxp3+ cells. Unexpectedly, eIF4E also affects Foxp3 expression and thereby lineage identity. Thus, mRNA–specific translational control directs both common and distinct cellular processes in CD4+ T cell subsets. PMID:23658533

  4. Ordered subsets algorithms for transmission tomography.

    PubMed

    Erdogan, H; Fessler, J A

    1999-11-01

    The ordered subsets EM (OSEM) algorithm has enjoyed considerable interest for emission image reconstruction due to its acceleration of the original EM algorithm and ease of programming. The transmission EM reconstruction algorithm converges very slowly and is not used in practice. In this paper, we introduce a simultaneous update algorithm called separable paraboloidal surrogates (SPS) that converges much faster than the transmission EM algorithm. Furthermore, unlike the 'convex algorithm' for transmission tomography, the proposed algorithm is monotonic even with nonzero background counts. We demonstrate that the ordered subsets principle can also be applied to the new SPS algorithm for transmission tomography to accelerate 'convergence', albeit with similar sacrifice of global convergence properties as for OSEM. We implemented and evaluated this ordered subsets transmission (OSTR) algorithm. The results indicate that the OSTR algorithm speeds up the increase in the objective function by roughly the number of subsets in the early iterates when compared to the ordinary SPS algorithm. We compute mean square errors and segmentation errors for different methods and show that OSTR is superior to OSEM applied to the logarithm of the transmission data. However, penalized-likelihood reconstructions yield the best quality images among all other methods tested. PMID:10588288

  5. Nfil3 is required for the development of all innate lymphoid cell subsets

    PubMed Central

    Seillet, Cyril; Rankin, Lucille C.; Groom, Joanna R.; Mielke, Lisa A.; Tellier, Julie; Chopin, Michael; Huntington, Nicholas D.

    2014-01-01

    Innate lymphoid cell (ILC) populations protect against infection and are essential for lymphoid tissue formation and tissue remodeling after damage. Nfil3 is implicated in the function of adaptive immune lineages and NK cell development, but it is not yet known if Nfil3 regulates other innate lymphoid lineages. Here, we identify that Nfil3 is essential for the development of Peyer’s patches and ILC2 and ILC3 subsets. Loss of Nfil3 selectively reduced Peyer’s patch formation and was accompanied by impaired recruitment and distribution of lymphocytes within the patches. ILC subsets exhibited high Nfil3 expression and genetic deletion of Nfil3 severely compromised the development of all subsets. Subsequently, Nfil3−/− mice were highly susceptible to disease when challenged with inflammatory or infectious agents. Thus, we demonstrate that Nfil3 is a key regulator of the development of ILC subsets essential for immune protection in the lung and gut. PMID:25092873

  6. Engineering High-Potency R-spondin Adult Stem Cell Growth Factors

    PubMed Central

    Warner, Margaret L.; Bell, Tufica

    2015-01-01

    Secreted R-spondin proteins (RSPOs1–4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1–Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein–coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1–4 using purified RSPO Fu1–Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2–4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a “Superspondin” that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4–1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a “Poorspondin” that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1–4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications. PMID:25504990

  7. Expansion of the in vitro assay for Leptospira potency testing to other Serovars: Case study with Leptospira hardjo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Code for Federal Regulations (9 CFR 113:101-104) specifies how vaccine potency is evaluated in a hamster model for evaluation of leptospiral vaccines against pomona, icterohaemorrhagiae, canicola, and grippotyphosa serotypes of Leptospira interrogans. There are several issues which complicate th...

  8. Potency of Fish Collagen as a Scaffold for Regenerative Medicine

    PubMed Central

    Yamamoto, Kohei; Yanagiguchi, Kajiro

    2014-01-01

    Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine. PMID:24982861

  9. Lymphocytes subsets reference values in childhood.

    PubMed

    Tosato, F; Bucciol, G; Pantano, G; Putti, M C; Sanzari, M C; Basso, G; Plebani, M

    2015-01-01

    Immunophenotyping of blood lymphocyte subsets and activation markers is a basic tool in the diagnostic process of primary immunodeficiency diseases, its use becoming more and more widespread as the knowledge about these illnesses increases. However, the availability of reliable reference values, which need to be age-matched for the pediatric population, is a pre-requisite for the reliable interpretation of immunophenotyping data. Aim of this study is to analyze the lymphocyte subsets and activation markers distribution in children aged 0-18 years referring to the University Hospital of Padova and to create age-matched reference values expressed by percentiles, thus providing a valuable guideline for the interpretation of the immunophenotype. PMID:25132325

  10. Homeostasis of IL-15 dependent lymphocyte subsets in the liver.

    PubMed

    Cepero-Donates, Yuneivy; Rakotoarivelo, Volatiana; Mayhue, Marian; Ma, Averil; Chen, Yi-Guang; Ramanathan, Sheela

    2016-06-01

    IL-15 is a member of the gamma chain family of cytokines (γc - CD132). The IL-15 receptor (IL-15R) complex consists of 3 subunits: the ligand-binding IL-15Rα chain (CD215), the β chain (CD122; also used by IL-2), and the common γ chain. The biological activities of IL-15 are mostly mediated by the IL-15:IL-15Rα complex, produced by the same cell and 'trans-presented' to responder cells expressing the IL-15Rβγc. The peculiar and almost unique requirement for IL-15 to be trans-presented by IL-15Rα suggests that the biological effects of IL-15 signaling are tightly regulated even at the level of availability of IL-15. Tissue-specific deletion of IL-15Rα has shown macrophage-and dendritic cell-derived IL-15Rα mediate the homeostasis of different CD8(+) T cell subsets. Here we show that hepatocyte and macrophage- specific expression of IL-15Rα is required to maintain the homeostasis of NK and NKT cells in the liver. Thus, homeostasis of IL-15-dependent lymphocyte subsets is also regulated by trans-presentation of IL-15 by non-hematopoietic cells in the tissue environment. PMID:26778709

  11. Thymic stromal cell subsets for T cell development.

    PubMed

    Nitta, Takeshi; Suzuki, Harumi

    2016-03-01

    The thymus provides a specialized microenvironment in which a variety of stromal cells of both hematopoietic and non-hematopoietic origin regulate development and repertoire selection of T cells. Recent studies have been unraveling the inter- and intracellular signals and transcriptional networks for spatiotemporal regulation of development of thymic stromal cells, mainly thymic epithelial cells (TECs), and the molecular mechanisms of how different TEC subsets control T cell development and selection. TECs are classified into two functionally different subsets: cortical TECs (cTECs) and medullary TECs (mTECs). cTECs induce positive selection of diverse and functionally distinct T cells by virtue of unique antigen-processing systems, while mTECs are essential for establishing T cell tolerance via ectopic expression of peripheral tissue-restricted antigens and cooperation with dendritic cells. In addition to reviewing the role of the thymic stroma in conventional T cell development, we will discuss recently discovered novel functions of TECs in the development of unconventional T cells, such as natural killer T cells and γδT cells. PMID:26825337

  12. Fizzy. Feature subset selection for metagenomics

    SciTech Connect

    Ditzler, Gregory; Morrison, J. Calvin; Lan, Yemin; Rosen, Gail L.

    2015-11-04

    Background: Some of the current software tools for comparative metagenomics provide ecologists with the ability to investigate and explore bacterial communities using α– & β–diversity. Feature subset selection – a sub-field of machine learning – can also provide a unique insight into the differences between metagenomic or 16S phenotypes. In particular, feature subset selection methods can obtain the operational taxonomic units (OTUs), or functional features, that have a high-level of influence on the condition being studied. For example, in a previous study we have used information-theoretic feature selection to understand the differences between protein family abundances that best discriminate between age groups in the human gut microbiome. Results: We have developed a new Python command line tool, which is compatible with the widely adopted BIOM format, for microbial ecologists that implements information-theoretic subset selection methods for biological data formats. We demonstrate the software tools capabilities on publicly available datasets. Conclusions: We have made the software implementation of Fizzy available to the public under the GNU GPL license. The standalone implementation can be found at http://github.com/EESI/Fizzy.

  13. Fizzy. Feature subset selection for metagenomics

    DOE PAGESBeta

    Ditzler, Gregory; Morrison, J. Calvin; Lan, Yemin; Rosen, Gail L.

    2015-11-04

    Background: Some of the current software tools for comparative metagenomics provide ecologists with the ability to investigate and explore bacterial communities using α– & β–diversity. Feature subset selection – a sub-field of machine learning – can also provide a unique insight into the differences between metagenomic or 16S phenotypes. In particular, feature subset selection methods can obtain the operational taxonomic units (OTUs), or functional features, that have a high-level of influence on the condition being studied. For example, in a previous study we have used information-theoretic feature selection to understand the differences between protein family abundances that best discriminate betweenmore » age groups in the human gut microbiome. Results: We have developed a new Python command line tool, which is compatible with the widely adopted BIOM format, for microbial ecologists that implements information-theoretic subset selection methods for biological data formats. We demonstrate the software tools capabilities on publicly available datasets. Conclusions: We have made the software implementation of Fizzy available to the public under the GNU GPL license. The standalone implementation can be found at http://github.com/EESI/Fizzy.« less

  14. The Effects of Medical Marijuana Laws on Potency

    PubMed Central

    Pacula, Rosalie Liccardo; Heaton, Paul

    2014-01-01

    Background Marijuana potency has risen dramatically over the past two decades. In the United States, it is unclear whether state medical marijuana policies have contributed to this increase. Methods Employing a differences-in-differences model within a mediation framework, we analyzed data on n = 39,157 marijuana samples seized by law enforcement in 51 U.S. jurisdictions between 1990-2010, producing estimates of the direct and indirect effects of state medical marijuana laws on potency, as measured by Δ9-tetrahydrocannabinol content. Results We found evidence that potency increased by a half percentage point on average after legalization of medical marijuana, although this result was not significant. When we examined specific medical marijuana supply provisions, results suggest that legal allowances for retail dispensaries had the strongest influence, significantly increasing potency by about one percentage point on average. Our mediation analyses examining the mechanisms through which medical marijuana laws influence potency found no evidence of direct regulatory impact. Rather, the results suggest that the impact of these laws occurs predominantly through a compositional shift in the share of the market captured by high-potency sinsemilla. Conclusion Our findings have important implications for policymakers and those in the scientific community trying to understand the extent to which greater availability of higher potency marijuana increases the risk of negative public health outcomes, such as drugged driving and drug-induced psychoses. Future work should reconsider the impact of medical marijuana laws on health outcomes in light of dramatic and ongoing shifts in both marijuana potency and the medical marijuana policy environment. PMID:24502887

  15. Inhaled corticosteroids: potency, dose equivalence and therapeutic index

    PubMed Central

    Daley-Yates, Peter T

    2015-01-01

    Glucocorticosteroids are a group of structurally related molecules that includes natural hormones and synthetic drugs with a wide range of anti-inflammatory potencies. For synthetic corticosteroid analogues it is commonly assumed that the therapeutic index cannot be improved by increasing their glucocorticoid receptor binding affinity. The validity of this assumption, particularly for inhaled corticosteroids, has not been fully explored. Inhaled corticosteroids exert their anti-inflammatory activity locally in the airways, and hence this can be dissociated from their potential to cause systemic adverse effects. The molecular structural features that increase glucocorticoid receptor binding affinity and selectivity drive topical anti-inflammatory activity. However, in addition, these structural modifications also result in physicochemical and pharmacokinetic changes that can enhance targeting to the airways and reduce systemic exposure. As a consequence, potency and therapeutic index can be correlated. However, this consideration is not reflected in asthma treatment guidelines that classify inhaled corticosteroid formulations as low-, mid- and high dose, and imbed a simple dose equivalence approach where potency is not considered to affect the therapeutic index. This article describes the relationship between potency and therapeutic index, and concludes that higher potency can potentially improve the therapeutic index. Therefore, both efficacy and safety should be considered when classifying inhaled corticosteroid regimens in terms of dose equivalence. The historical approach to dose equivalence in asthma treatment guidelines is not appropriate for the wider range of molecules, potencies and device/formulations now available. A more robust method is needed that incorporates pharmacological principles. PMID:25808113

  16. Toward a refined definition of monocyte subsets.

    PubMed

    Ziegler-Heitbrock, Loems; Hofer, Thomas P J

    2013-01-01

    In a nomenclature proposal published in 2010 monocytes were subdivided into classical and non-classical cells and in addition an intermediate monocyte subset was proposed. Over the last couple of years many studies have analyzed these intermediate cells, their characteristics have been described, and their expansion has been documented in many clinical settings. While these cells appear to be in transition from classical to non-classical monocytes and hence may not form a distinct cell population in a strict sense, their separate analysis and enumeration is warranted in health and disease. PMID:23382732

  17. Immunity to Pathogens Taught by Specialized Human Dendritic Cell Subsets

    PubMed Central

    Geginat, Jens; Nizzoli, Giulia; Paroni, Moira; Maglie, Stefano; Larghi, Paola; Pascolo, Steve; Abrignani, Sergio

    2015-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have a key role in immune responses because they bridge the innate and adaptive arms of the immune system. They mature upon recognition of pathogens and upregulate MHC molecules and costimulatory receptors to activate antigen-specific CD4+ and CD8+ T cells. It is now well established that DCs are not a homogeneous population but are composed of different subsets with specialized functions in immune responses to specific pathogens. Upon viral infections, plasmacytoid DCs (pDCs) rapidly produce large amounts of IFN-α, which has potent antiviral functions and activates several other immune cells. However, pDCs are not particularly potent APCs and induce the tolerogenic cytokine IL-10 in CD4+ T cells. In contrast, myeloid DCs (mDCs) are very potent APCs and possess the unique capacity to prime naive T cells and consequently to initiate a primary adaptive immune response. Different subsets of mDCs with specialized functions have been identified. In mice, CD8α+ mDCs capture antigenic material from necrotic cells, secrete high levels of IL-12, and prime Th1 and cytotoxic T-cell responses to control intracellular pathogens. Conversely, CD8α− mDCs preferentially prime CD4+ T cells and promote Th2 or Th17 differentiation. BDCA-3+ mDC2 are the human homologue of CD8α+ mDCs, since they share the expression of several key molecules, the capacity to cross-present antigens to CD8+ T-cells and to produce IFN-λ. However, although several features of the DC network are conserved between humans and mice, the expression of several toll-like receptors as well as the production of cytokines that regulate T-cell differentiation are different. Intriguingly, recent data suggest specific roles for human DC subsets in immune responses against individual pathogens. The biology of human DC subsets holds the promise to be exploitable in translational medicine, in particular for the development of vaccines against

  18. Immunity to Pathogens Taught by Specialized Human Dendritic Cell Subsets.

    PubMed

    Geginat, Jens; Nizzoli, Giulia; Paroni, Moira; Maglie, Stefano; Larghi, Paola; Pascolo, Steve; Abrignani, Sergio

    2015-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have a key role in immune responses because they bridge the innate and adaptive arms of the immune system. They mature upon recognition of pathogens and upregulate MHC molecules and costimulatory receptors to activate antigen-specific CD4(+) and CD8(+) T cells. It is now well established that DCs are not a homogeneous population but are composed of different subsets with specialized functions in immune responses to specific pathogens. Upon viral infections, plasmacytoid DCs (pDCs) rapidly produce large amounts of IFN-α, which has potent antiviral functions and activates several other immune cells. However, pDCs are not particularly potent APCs and induce the tolerogenic cytokine IL-10 in CD4(+) T cells. In contrast, myeloid DCs (mDCs) are very potent APCs and possess the unique capacity to prime naive T cells and consequently to initiate a primary adaptive immune response. Different subsets of mDCs with specialized functions have been identified. In mice, CD8α(+) mDCs capture antigenic material from necrotic cells, secrete high levels of IL-12, and prime Th1 and cytotoxic T-cell responses to control intracellular pathogens. Conversely, CD8α(-) mDCs preferentially prime CD4(+) T cells and promote Th2 or Th17 differentiation. BDCA-3(+) mDC2 are the human homologue of CD8α(+) mDCs, since they share the expression of several key molecules, the capacity to cross-present antigens to CD8(+) T-cells and to produce IFN-λ. However, although several features of the DC network are conserved between humans and mice, the expression of several toll-like receptors as well as the production of cytokines that regulate T-cell differentiation are different. Intriguingly, recent data suggest specific roles for human DC subsets in immune responses against individual pathogens. The biology of human DC subsets holds the promise to be exploitable in translational medicine, in particular for the development of

  19. Solvatochromic dyes detect the presence of homeopathic potencies.

    PubMed

    Cartwright, Steven J

    2016-02-01

    A systematic approach to the design of simple, chemical systems for investigating the nature of homeopathic medicines has led to the development of an experimental protocol in which solvatochromic dyes are used as molecular probes of serially diluted and agitated solutions. Electronic spectroscopy has been used to follow changes in the absorbance of this class of dyes across the visible spectrum in the presence of homeopathic potencies. Evidence is presented using six different solvatochromic dyes in three different solvent systems. In all cases homeopathic potencies produce consistent and reproducible changes in the spectra of the dyes. Results suggest that potencies influence the supramolecular chemistry of solvatochromic dyes, enhancing either dye aggregation or disaggregation, depending upon dye structure. Comparable dyes lacking the intramolecular charge transfer feature of solvatochromic dyes are unaffected by homeopathic potencies, suggesting potencies require the oscillating dipole of solvatochromic dyes for effective interaction. The implications of the results presented, both for an eventual understanding of the nature of homeopathic medicines and their mode of action, together with future directions for research in this area, are discussed. PMID:26827998

  20. Potency Determination of Antidandruff Shampoos in Nystatin International Unit Equivalents

    PubMed Central

    Anusha Hewage, D. B. G.; Pathirana, W.; Pinnawela, Amara

    2008-01-01

    A convenient standard microbiological potency determination test for the antidandruff shampoos was developed by adopting the pharmacopoeial microbiological assay procedure of the drug nystatin. A standard curve was drawn consisting of the inhibition zone diameters vs. logarithm of nystatin concentrations in international units using the fungus Saccharomyces cerevisiae (yeast) strain National Collection of Type Culture (NCTC) 1071606 as the test organism. From the standard curve the yeast inhibitory potencies of the shampoos in nystatin international unit equivalents were determined from the respective inhibition zones of the test samples of the shampoos. Under test conditions four shampoo samples showed remarkable fungal inhibitory potencies of 10227, 10731, 12396 and 18211 nystatin international unit equivalents/ml while two shampoo samples had extremely feeble inhibitory potencies 4.07 and 4.37 nystatin international unit equivalents/ml although the latter two products claimed antifungal activity. The potency determination method could be applied to any antidandruff shampoo with any one or a combination of active ingredients. PMID:21394271

  1. Plasticity of T helper cell subsets: Implications in periodontal disease.

    PubMed

    Talwar, Avaneendra; Arun, K V; Kumar, T S S; Clements, Jasmine

    2013-05-01

    T helper (Th) cells have an important role in host defence as well in the pathogenesis of periodontal disease. Th cells differentiate from naive cells into various subsets, each of which is associated with a set of inducing and effector cytokines. Previously, it was thought that this differentiation was an irreversible event. Recent evidence suggest that even differentiated Th cells, retain the flexibility to transform from one lineage to another, a phenomenon referred to as plasticity. This plasticity is thought to be brought about by epigenetic modifications that are regulated by external and internal signals in the micro-environment of these cells. The factors and mechanisms which affect the plasticity of these cells and their potential role in the etio-pathogenesis of periodontal disease has been described in this article. PMID:24049327

  2. Role of distinct CD4(+) T helper subset in pathogenesis of oral lichen planus.

    PubMed

    Wang, Hui; Zhang, Dunfang; Han, Qi; Zhao, Xin; Zeng, Xin; Xu, Yi; Sun, Zheng; Chen, Qianming

    2016-07-01

    Oral lichen planus (OLP) is one of the most common chronic inflammatory oral mucosal diseases with T-cell-mediated immune pathogenesis. In subepithelial and lamina propria of OLP local lesions, the presence of CD4(+) T helper (CD4(+) Th) cells appeared as the major lymphocytes. These CD4(+) T lymphocytes can differentiate into distinct Th cell types such as Th1, Th2, Treg, Th17, Th22, Th9, and Tfh within the context of certain cytokines environment. Growing evidence indicated that Th1/Th2 imbalance may greatly participate into the cytokine network of OLP immunopathology. In addition, Th1/Th2 imbalance can be regulated by the Treg subset and also greatly influenced by the emerging novel CD4(+) Th subset Th17. Furthermore, the presence of novel subsets Th22, Th9 and Tfh in OLP patients is yet to be clarified. All these Th subsets and their specific cytokines may play a critical role in determining the character, extent and duration of immune responses in OLP pathogenesis. Therefore, we review the roles of distinct CD4(+) Th subsets and their signature cytokines in determining disease severity and susceptibility of OLP and also reveal the novel therapeutic strategies based on T lymphocytes subsets in OLP treatment. PMID:26693958

  3. Standard Preparations, Limits of Potency, and Dating Period Limitations for Biological Products. Direct final rule.

    PubMed

    2016-05-01

    The Food and Drug Administration (FDA or Agency or we) is amending the general biological products standards relating to dating periods and also removing certain standards relating to standard preparations and limits of potency. FDA is taking this action to update outdated requirements, and accommodate new and evolving technology and testing capabilities, without diminishing public health protections. This action is part of FDA's retrospective review of its regulations in response to an Executive order. FDA is issuing these amendments directly as a final rule because the Agency believes they are noncontroversial and FDA anticipates no significant adverse comments. PMID:27192727

  4. Protective and pathogenic functions of macrophage subsets.

    PubMed

    Murray, Peter J; Wynn, Thomas A

    2011-11-01

    Macrophages are strategically located throughout the body tissues, where they ingest and process foreign materials, dead cells and debris and recruit additional macrophages in response to inflammatory signals. They are highly heterogeneous cells that can rapidly change their function in response to local microenvironmental signals. In this Review, we discuss the four stages of orderly inflammation mediated by macrophages: recruitment to tissues; differentiation and activation in situ; conversion to suppressive cells; and restoration of tissue homeostasis. We also discuss the protective and pathogenic functions of the various macrophage subsets in antimicrobial defence, antitumour immune responses, metabolism and obesity, allergy and asthma, tumorigenesis, autoimmunity, atherosclerosis, fibrosis and wound healing. Finally, we briefly discuss the characterization of macrophage heterogeneity in humans. PMID:21997792

  5. [Changes of Tim-3 and PD-1 on peripheral blood monocyte subsets in patients with chronic hepatitis C].

    PubMed

    Liang, Yan; Zhang, Peixin; Yi, Wenjing; Zhou, Yun; Jia, Zhansheng; Zhang, Ying

    2016-05-01

    Objective To investigate the distribution of peripheral blood monocyte subsets of chronic hepatitis C (CHC) patients and observe the expression of negative regulators T cell immunoglobulin and mucin domain-3 (Tim-3) and programmed cell death-1 (PD-1) on the monocyte subsets. Methods Flow cytometry was employed to determine the distribution of three monocyte subsets as well as Tim-3 and PD-1 expression on the three monocyte subsets. Their correlations with the clinical parameters were analyzed by Spearman test. Results Compared with healthy controls, an increased distribution of CD14(+)CD16(+) monocytes, especially CD14(++)CD16(+) monocyte subset, was observed in CHC patients. Tim-3 expression was significantly elevated on CD14(++)CD16(-) and CD14(+)CD16(++) subsets in CHC patients. Obviously increased PD-1 expression was found mainly on CD14(++)CD16(-) and CD14(++)CD16(+) subsets. There were no significant correlations between monocyte subsets, PD-1, Tim-3 and the clinical parameters. Conclusion The levels Tim-3 and PD-1 are different in three monocyte subsets. PMID:27126947

  6. Roles of participation and feedback in group potency.

    PubMed

    Gamero, Nuria; Peiró, José M; Zornoza, Ana; Picazo, Carmen

    2009-08-01

    The roles of group participation and group performance feedback were examined as antecedents of group potency, i.e., beliefs shared among a work group's members about the general effectiveness of the work group. Also examined were how group participation and the congruence of the feedback received from different sources about performance predicted convergence in members' beliefs about group effectiveness. The sample comprised 61 work groups of professionals involved in Master in Business Administration (MBA) programs (284 participants). Mean group size was 4.6 members (SD = .58). 65% of participants were male, and 51% were between 30 and 40 years of age. Data were gathered at two measurement times. Increases in group participation were positively related to increases in group potency and the convergence in beliefs about group effectiveness among group members over time. Results supported the premise that group performance feedback is an antecedent of changes in group potency over time. PMID:19810455

  7. Characterization of Dendritic Cell Subsets Through Gene Expression Analysis.

    PubMed

    Vu Manh, Thien-Phong; Dalod, Marc

    2016-01-01

    Dendritic cells (DCs) are immune sentinels of the body and play a key role in the orchestration of the communication between the innate and the adaptive immune systems. DCs can polarize innate and adaptive immunity toward a variety of functions, sometimes with opposite roles in the overall control of immune responses (e.g., tolerance or immunosuppression versus immunity) or in the balance between various defense mechanisms promoting the control of different types of pathogens (e.g., antiviral versus antibacterial versus anti-worm immunity). These multiple DC functions result both from the plasticity of individual DC to exert different activities and from the existence of various DC subsets specialized in distinct functions. Functional genomics represents a powerful, unbiased, approach to better characterize these two levels of DC plasticity and to decipher its molecular regulation. Indeed, more and more experimental immunologists are generating high-throughput data in order to better characterize different states of DC based, for example, on their belonging to a specific subpopulation and/or on their exposure to specific stimuli and/or on their ability to exert a specific function. However, the interpretation of this wealth of data is severely hampered by the bottleneck of their bioinformatics analysis. Indeed, most experimental immunologists lack advanced computational or bioinformatics expertise and do not know how to translate raw gene expression data into potential biological meaning. Moreover, subcontracting such analyses is generally disappointing or financially not sustainable, since companies generally propose canonical analysis pipelines that are often unadapted for the structure of the data to analyze or for the precise type of questions asked. Hence, there is an important need of democratization of the bioinformatics analyses of gene expression profiling studies, in order to accelerate interpretation of the results by the researchers at the origin of the

  8. MicroRNA expression profiling of human blood monocyte subsets highlights functional differences

    PubMed Central

    Dang, Truong-Minh; Wong, Wing-Cheong; Ong, Siew-Min; Li, Peng; Lum, Josephine; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Wong, Siew-Cheng

    2015-01-01

    Within human blood there are two subsets of monocytes that can be identified by differential expression of CD16. Although numerous phenotypic and functional differences between the subsets have been described, little is known of the mechanisms underlying the distinctive properties of the two subsets. MicroRNAs (miRNAs) are small non-coding RNAs that can regulate gene expression through promoting mRNA degradation or repressing translation, leading to alterations in cellular processes. Their potential influence on the functions of monocyte subsets has not been investigated. In this study, we employed microarray analysis to define the miRNA expression profile of human monocyte subsets. We identified 66 miRNAs that were differentially expressed (DE) between CD16+ and CD16− monocytes. Gene ontology analysis revealed that the predicted targets of the DE miRNAs were predominantly associated with cell death and cellular movement. We validated the functional impacts of selected DE miRNAs in CD16− monocytes, over-expression of miR-432 significantly increases apoptosis, and inhibiting miR-19a significantly reduces cell motility. Furthermore, we found that miR-345, another DE miRNA directly targets the transcription factor RelA in monocytes, which resulted in the differential expression of RelA in monocyte subsets. This implicates miR-345 indirect regulation of many genes downstream of RelA, including important inflammatory mediators. Together, our data show that DE miRNAs could contribute substantially to regulating the functions of human blood monocytes. PMID:25707426

  9. Mutagenic Potency of Food-Derived Heterocyclic Amines

    SciTech Connect

    Felton, J S; Knize, M G; Wu, R W; Colvin, M E; Hatch, F T; Malfatti, M A

    2006-10-26

    The understanding of mutagenic potency has been primarily approached using ''quantitative structure activity relationships'' (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P-450 1A1 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.

  10. 9 CFR 113.9 - New potency test.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false New potency test. 113.9 Section 113.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Applicability §...

  11. High Potency and Other Alcoholic Beverage Consumption among Adolescents

    ERIC Educational Resources Information Center

    Jobli, Edessa C.; Dore, Heather S.; Werch, Chudley E.; Moore, Michele J.

    2005-01-01

    This study examined the prevalence of high potency (liquor, malt liquor, fortified wine) and other alcoholic beverage consumption (beer, wine/wine coolers) among adolescents, the impact of gender and ethnicity, and the risk and protective factors that predicted consumption. A confidential survey revealed that, among eighth grade students,…

  12. 9 CFR 113.9 - New potency test.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false New potency test. 113.9 Section 113.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Applicability §...

  13. 9 CFR 113.9 - New potency test.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false New potency test. 113.9 Section 113.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Applicability §...

  14. High-potency cannabis and the risk of psychosis

    PubMed Central

    Di Forti, Marta; Morgan, Craig; Dazzan, Paola; Pariante, Carmine; Mondelli, Valeria; Marques, Tiago Reis; Handley, Rowena; Luzi, Sonija; Russo, Manuela; Paparelli, Alessandra; Butt, Alexander; Stilo, Simona A.; Wiffen, Ben; Powell, John; Murray, Robin M.

    2009-01-01

    Background People who use cannabis have an increased risk of psychosis, an effect attributed to the active ingredient Δ9-tetrahydrocannabinol (Δ9-THC). There has recently been concern over an increase in the concentration of Δ9-THC in the cannabis available in many countries. Aims To investigate whether people with a first episode of psychosis were particularly likely to use high-potency cannabis. Method We collected information on cannabis use from 280 cases presenting with a first episode of psychosis to the South London & Maudsley National Health Service (NHS) Foundation Trust, and from 174 healthy controls recruited from the local population. Results There was no significant difference between cases and controls in whether they had ever taken cannabis, or age at first use. However, those in the cases group were more likely to be current daily users (OR = 6.4) and to have smoked cannabis for more than 5 years (OR = 2.1). Among those who used cannabis, 78% of the cases group used high-potency cannabis (sinsemilla, ‘skunk’) compared with 37% of the control group (OR 6.8). Conclusions The finding that people with a first episode of psychosis had smoked higher-potency cannabis, for longer and with greater frequency, than a healthy control group is consistent with the hypothesis that Δ9-THC is the active ingredient increasing risk of psychosis. This has important public health implications, given the increased availability and use of high-potency cannabis. PMID:19949195

  15. 9 CFR 113.9 - New potency test.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false New potency test. 113.9 Section 113.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Applicability §...

  16. Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation

    PubMed Central

    Baroja-Mazo, Alberto; Revilla-Nuin, Beatriz; Ramírez, Pablo; Pons, José A

    2016-01-01

    Mammalian target of rapamycin, also known as mechanistic target of rapamycin (mTOR) is a protein kinase that belongs to the PI3K/AKT/mTOR signaling pathway, which is involved in several fundamental cellular functions such as cell growth, proliferation, and survival. This protein and its associated pathway have been implicated in cancer development and the regulation of immune responses, including the rejection response generated following allograft transplantation. Inhibitors of mTOR (mTORi) such as rapamycin and its derivative everolimus are potent immunosuppressive drugs that both maintain similar rates of efficacy and could optimize the renal function and diminish the side effects compared with calcineurin inhibitors. These drugs are used in solid-organ transplantationtoinduceimmunosuppression while also promoting the expansion of CD4+CD25+FOXP3+ regulatory T-cells that could favor a scenery of immunological tolerance. In this review, we describe the mechanisms by which inhibitors of mTOR induce suppression by regulation of these pathways at different levels of the immune response. In addition, we particularly emphasize about the main methods that are used to assess the potency of immunosuppressive drugs, highlighting the studies carried out about immunosuppressive potency of inhibitors of mTOR. PMID:27011916

  17. Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus

    PubMed Central

    2012-01-01

    Background The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. Methods Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. Results Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. Conclusions Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel. PMID:22950645

  18. Crossing the Vascular Wall: Common and Unique Mechanisms Exploited by Different Leukocyte Subsets during Extravasation

    PubMed Central

    Schnoor, Michael; Alcaide, Pilar; Voisin, Mathieu-Benoit; van Buul, Jaap D.

    2015-01-01

    Leukocyte extravasation is one of the essential and first steps during the initiation of inflammation. Therefore, a better understanding of the key molecules that regulate this process may help to develop novel therapeutics for treatment of inflammation-based diseases such as atherosclerosis or rheumatoid arthritis. The endothelial adhesion molecules ICAM-1 and VCAM-1 are known as the central mediators of leukocyte adhesion to and transmigration across the endothelium. Engagement of these molecules by their leukocyte integrin receptors initiates the activation of several signaling pathways within both leukocytes and endothelium. Several of such events have been described to occur during transendothelial migration of all leukocyte subsets, whereas other mechanisms are known only for a single leukocyte subset. Here, we summarize current knowledge on regulatory mechanisms of leukocyte extravasation from a leukocyte and endothelial point of view, respectively. Specifically, we will focus on highlighting common and unique mechanisms that specific leukocyte subsets exploit to succeed in crossing endothelial monolayers. PMID:26568666

  19. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

    PubMed

    Winsnes, R; Sesardic, D; Daas, A; Behr-Gross, M-E

    2004-01-01

    A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus

  20. Aligning Potency and Pharmacokinetic Properties for Pyridine-Based NCINIs.

    PubMed

    Fader, Lee D; Bailey, Murray; Beaulieu, Eric; Bilodeau, François; Bonneau, Pierre; Bousquet, Yves; Carson, Rebekah J; Chabot, Catherine; Coulombe, René; Duan, Jianmin; Fenwick, Craig; Garneau, Michel; Halmos, Ted; Jakalian, Araz; James, Clint; Kawai, Stephen H; Landry, Serge; LaPlante, Steven R; Mason, Stephen W; Morin, Sebastien; Rioux, Nathalie; Simoneau, Bruno; Surprenant, Simon; Thavonekham, Bounkham; Thibeault, Carl; Trinh, Thao; Tsantrizos, Youla; Tsoung, Jennifer; Yoakim, Christiane; Wernic, Dominik

    2016-08-11

    Optimization of pyridine-based noncatalytic site integrase inhibitors (NCINIs) based on compound 2 has led to the discovery of molecules capable of inhibiting virus harboring N124 variants of HIV integrase (IN) while maintaining minimal contribution of enterohepatic recirculation to clearance in rat. Structure-activity relationships at the C6 position established chemical space where the extent of enterohepatic recirculation in the rat is minimized. Desymmetrization of the C4 substituent allowed for potency optimization against virus having the N124 variant of integrase. Combination of these lessons led to the discovery of compound 20, having balanced serum-shifted antiviral potency and minimized excretion in to the biliary tract in rat, potentially representing a clinically viable starting point for a new treatment option for individuals infected with HIV. PMID:27563405

  1. Fc Receptor-like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue-like Memory B Cells.

    PubMed

    Li, Huifang; Borrego, Francisco; Nagata, Satoshi; Tolnay, Mate

    2016-05-15

    Fc receptor-like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5(+) cells are most enriched among atypical CD21(-/lo)/CD27(-) tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infectious diseases. Using multicolor flow cytometry, FCRL5(+) TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL5(-) TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5(+) subset accumulated significantly more somatic mutations. Furthermore, the FCRL5(+) subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5(+) TLM subset responds poorly to multiple stimuli compared with the FCRL5(-) subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5(+) TLM subset, but not the FCRL5(-) TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases. PMID:27076679

  2. Redesigned HIV antibodies exhibit enhanced neutralizing potency and breadth.

    PubMed

    Willis, Jordan R; Sapparapu, Gopal; Murrell, Sasha; Julien, Jean-Philippe; Singh, Vidisha; King, Hannah G; Xia, Yan; Pickens, Jennifer A; LaBranche, Celia C; Slaughter, James C; Montefiori, David C; Wilson, Ian A; Meiler, Jens; Crowe, James E

    2015-06-01

    Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100(F)Y, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100(F)Y fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100(F)Y, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies. PMID:25985274

  3. Characterization of mesenchymal stromal cells: potency assay development.

    PubMed

    Hematti, Peiman

    2016-04-01

    Based on their many different mechanisms of action, presumed immune-privileged status, and relative ease of production, mesenchymal stromal cells (MSCs) are under intensive clinical investigation for treating a wide range of degenerative, inflammatory, and immunologic disorders. Identification of relevant and robust potency assays is not only a regulatory requirement, but it is also the basis for producing and delivering a product that is consistent, safe, and ultimately an effective therapy. Although development of an appropriate potency assay is one of the most challenging issues in cell-based therapies, it is of paramount importance in the process of developing and testing cellular products. Regardless of the many different tissue sources and methods used in culture expansion of MSCs, they possess many of the same morphologic, cell surface markers, and differentiation characteristics. However, MSC products with similar phenotypic characteristics could still have major differences in their biologic and functional attributes. Understanding the different mechanisms of action and establishment of relevant potency assays is of pivotal importance in allowing investigators and regulatory agencies to compare MSCs used in different clinical trials. PMID:27079322

  4. A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

    PubMed

    Aggarwal, Anshu; Tandon, Simran; Singla, Surinder Kumar; Tandon, Chanderdeep

    2012-08-01

    Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis. PMID:22702898

  5. Rapid infrared determination of the potency of chlorinated bactericides.

    PubMed

    Spagnolo, F; Cestaro, J P

    1971-06-01

    A rapid infrared reflectance method for evaluating the germicidal potency of synthetic materials containing various amounts of two chlorinated bactericides was developed. The dimeric product 2,2'-methylenebis (4,6-dichlorophenol) exhibited a characteristic C=C skeletal inplane stretching infrared absorption band at 1,640 cm(-1). The monomeric 2,4-dichlorophenol precursor showed a characteristic absorption band at 1,579 cm(-1). These characteristic infrared absorptions may be used for analysis of the potency of the manufactured chlorinated bactericide. For a series of samples known to vary in dimer content, the micrograms per milliliter required for a 100% bacterial kill is first determined by a standard American Petroleum Institute method. Then the area ratio of the infrared absorption bands characteristic of the chlorinated bactericides is measured for each sample and plotted versus the microgram per milliliter required for 100% bacterial kill. The potency of subsequent samples is simply and rapidly determined by measuring this ratio from the infrared absorption curve and calculating micrograms per milliliter required for 100% kill from the calibration curve. Analysis time is approximately 1 hr compared to biocidal tests in current use requiring approximately a 1-month incubation period. PMID:5564677

  6. Potency of Transgenic Effectors for Neurogenetic Manipulation in Drosophila Larvae

    PubMed Central

    Pauls, Dennis; von Essen, Alina; Lyutova, Radostina; van Giesen, Lena; Rosner, Ronny; Wegener, Christian; Sprecher, Simon G.

    2015-01-01

    Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies. PMID:25359929

  7. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics.

    PubMed

    Kleinstreuer, N C; Smith, A M; West, P R; Conard, K R; Fontaine, B R; Weir-Hauptman, A M; Palmer, J A; Knudsen, T B; Dix, D J; Donley, E L R; Cezar, G G

    2011-11-15

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. PMID:21925528

  8. International regulatory requirements for Leptospira vaccine potency testing. Roundtable: current requirements and opportunity for harmonization.

    PubMed

    Draayer, Hans A; Bruckner, Lukas; Peña-Moctezuma, Alejandro de la; Srinivas, Geetha

    2013-09-01

    Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests. PMID:23890730

  9. Selective Recruitment of T-Cell Subsets to the Udder during Staphylococcal and Streptococcal Mastitis: Analysis of Lymphocyte Subsets and Adhesion Molecule Expression

    PubMed Central

    Soltys, Jindrich; Quinn, Mark T.

    1999-01-01

    During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct αβ and γδ T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of γδ T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in αβ T-cell levels and a 100% increase in γδ T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in αβ T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4+ T cells, while the increase in αβ T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4+ and CD8+ T-cell numbers. The increased numbers of γδ T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct γδ T-cell subset (GD3.1+), while no change in the numbers of GD197+ γδ T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies

  10. The role of complement in dendritic cell (DC) control of T-cell subsets.

    PubMed

    Levis, William R; Martiniuk, Frank

    2010-11-01

    This section of the Journal of Drugs in Dermatology (JDD) is dedicated to Dendreon's Provenge (Sipuleucel-T), the first therapeutic DC vaccine proven effective and approved by the United States (U.S.) Food and Drug Administration (FDA) for advanced cancer. This editorial will discuss three articles in this issue, their relationship to Provenge and the recent TH17-Treg subsets that are regulated by CD46. PMID:21061758

  11. [Construction of terminology subsets: contributions to clinical nursing practice].

    PubMed

    Clares, Jorge Wilker Bezerra; de Freitas, Maria Célia; Guedes, Maria Vilaní Cavalcante; da Nóbrega, Maria Miriam Lima

    2013-08-01

    The International Classification for Nursing Practice (ICNP®) is a classification system that unifies the elements of nursing practice (diagnoses, interventions and outcomes), enabling elucidation of elements of a specific nursing language through the construction of terminology subsets. In this reflective essay, aspects relevant to the construction of ICNP® terminology subsets are highlighted, as well as their contributions to clinical nursing practice. The development of subsets as a tool that contributes to making nursing language universal, facilitates the communication process, as well as the scientific and technological advancement of the profession, is discussed. Therefore, its use by nurses worldwide is encouraged. PMID:24310697

  12. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    PubMed

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  13. Characterization of a novel subset of T cells from human spleen that lacks L-selectin.

    PubMed Central

    Wallace, D L; Beverley, P C

    1993-01-01

    Human L-selection (LAM-1, Leu-8, TQ1, DREG 56) is a member of the 'selection' family of adhesion molecules. Antibodies to L-selectin have been shown to block the binding of T cells to peripheral lymph node high endothelial venules (HEV). Most unstimulated peripheral blood T cells express high levels of L-selectin whilst it is only weakly expressed on the majority of T cells from secondary lymphoid organs. We show here (a) that T cells from tonsil and lymph node up-regulate L-selectin when released from their microenvironment, (b) that in contrast, spleen contains a stable L-selectin negative subset, (c) that this subset remains surface L-selection negative after stimulation even though the T cells can respond by proliferation, (d) that this subset expresses minimal levels of LAM-1 mRNA and (e) that mucosal lymphocyte antigen (MLA) positive and T-cell receptor (TcR) gamma delta positive T cells found within the L-selectin negative population are similar to subsets of T cells found amongst lamina propria (LP) and intraepithelial lymphocytes (IEL) of the gut. Images Figure 3 PMID:7684357

  14. Establishing criteria for human mesenchymal stem cell potency.

    PubMed

    Samsonraj, Rebekah M; Rai, Bina; Sathiyanathan, Padmapriya; Puan, Kia Joo; Rötzschke, Olaf; Hui, James H; Raghunath, Michael; Stanton, Lawrence W; Nurcombe, Victor; Cool, Simon M

    2015-06-01

    This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application. PMID:25752682

  15. A recent evaluation of the lethal potencies of ammodytoxins.

    PubMed

    Prijatelj-Žnidaršič, Petra; Pungerčar, Jože

    2012-05-01

    Ammodytoxin A (AtxA) is the most toxic secreted phospholipase A(2) of the three isotoxins with presynaptic neurotoxicity, isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes), with an LD(50) of 21 μg/kg in mice. The toxic potencies of two other isoforms have been re-evaluated using highly purified recombinant proteins, with their intraperitoneal LD(50)s determined as 960 μg/kg for AtxB and 310 μg/kg for AtxC. AtxB and AtxC differ from AtxA in only three and two amino acid residues, respectively. PMID:22406514

  16. Cannabis-induced psychosis associated with high potency "wax dabs".

    PubMed

    Pierre, Joseph M; Gandal, Michael; Son, Maya

    2016-04-01

    With mounting evidence that the risk of cannabis-induced psychosis may be related to both dose and potency of tetrahydrocannbinol (THC), increasing reports of psychosis associated with cannabinoids containing greater amounts of THC are anticipated. We report two cases of emergent psychosis after using a concentrated THC extract known as cannabis "wax," "oil," or "dabs" raising serious concerns about its psychotic liability. Although "dabbing" with cannabis wax is becoming increasingly popular in the US for both recreational and "medicinal" intentions, our cases raise serious concerns about its psychotic liability and highlight the importance of understanding this risk by physicians recommending cannabinoids for purported medicinal purposes. PMID:26876313

  17. Bridging the Gap Between Validation and Implementation of Non-Animal Veterinary Vaccine Potency Testing Methods

    PubMed Central

    Dozier, Samantha; Brown, Jeffrey; Currie, Alistair

    2011-01-01

    Simple Summary Many vaccines are tested for quality in experiments that require the use of large numbers of animals in procedures that often cause significant pain and distress. Newer technologies have fostered the development of vaccine quality control tests that reduce or eliminate the use of animals, but the availability of these newer methods has not guaranteed their acceptance by regulators or use by manufacturers. We discuss a strategic approach that has been used to assess and ultimately increase the use of non-animal vaccine quality tests in the U.S. and U.K. Abstract In recent years, technologically advanced high-throughput techniques have been developed that replace, reduce or refine animal use in vaccine quality control tests. Following validation, these tests are slowly being accepted for use by international regulatory authorities. Because regulatory acceptance itself has not guaranteed that approved humane methods are adopted by manufacturers, various organizations have sought to foster the preferential use of validated non-animal methods by interfacing with industry and regulatory authorities. After noticing this gap between regulation and uptake by industry, we began developing a paradigm that seeks to narrow the gap and quicken implementation of new replacement, refinement or reduction guidance. A systematic analysis of our experience in promoting the transparent implementation of validated non-animal vaccine potency assays has led to the refinement of our paradigmatic process, presented here, by which interested parties can assess the local regulatory acceptance of methods that reduce animal use and integrate them into quality control testing protocols, or ensure the elimination of peripheral barriers to their use, particularly for potency and other tests carried out on production batches. PMID:26486625

  18. TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets

    PubMed Central

    Han, Sun Murray; Na, Hye Young; Ham, Onju; Choi, Wanho; Sohn, Moah; Ryu, Seul Hye; In, Hyunju; Hwang, Ki-Chul

    2016-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells that sample their environment and present antigens to naïve T lymphocytes for the subsequent antigen-specific immune responses. DCs exist in a range of distinct subpopulations including plasmacytoid DCs (pDCs) and classical DCs (cDCs), with the latter consisting of the cDC1 and cDC2 lineages. Although the roles of DC-specific transcription factors across the DC subsets have become understood, the posttranscriptional mechanisms that regulate DC development are yet to be elucidated. MicroRNAs (miRNAs) are pivotal posttranscriptional regulators of gene expression in a myriad of biological processes, but their contribution to the immune system is just beginning to surface. In this study, our in-house probe collection was screened to identify miRNAs possibly involved in DC development and function by targeting the transcripts of relevant mouse transcription factors. Examination of DC subsets from the culture of mouse bone marrow with Flt3 ligand identified high expression of miR-124 which was able to target the transcript of TCF4, a transcription factor critical for the development and homeostasis of pDCs. Further expression profiling of mouse DC subsets isolated from in vitro culture as well as via ex vivo purification demonstrated that miR-124 was outstandingly expressed in CD24+ cDC1 cells compared to in pDCs and CD172α+ cDC2 cells. These results imply that miR-124 is likely involved in the processes of DC subset development by posttranscriptional regulation of a transcription factor(s). PMID:26937233

  19. Ineffective oesophageal motility: Manometric subsets exhibit different symptom profiles

    PubMed Central

    Haack, Horst Gunter; Hansen, Ross David; Malcolm, Allison; Kellow, John Edward

    2008-01-01

    AIM: To compare the demographic and clinical features of different manometric subsets of ineffective oesophageal motility (IOM; defined as ≥ 30% wet swallows with distal contractile amplitude < 30 mmHg), and to determine whether the prevalence of gastro-oesophageal reflux differs between IOM subsets. METHODS: Clinical characteristics of manometric subsets were determined in 100 IOM patients (73 female, median age 58 years) and compared to those of 100 age-and gender-matched patient controls with oesophageal symptoms, but normal manometry. Supine oesophageal manometry was performed with an eight-channel DentSleeve water-perfused catheter, and an ambulatory pH study assessed gastro-oesophageal reflux. RESULTS: Patients in the IOM subset featuring a majority of low-amplitude simultaneous contractions (LASC) experienced less heartburn (prevalence 26%), but more dysphagia (57%) than those in the IOM subset featuring low-amplitude propagated contractions (LAP; heartburn 70%, dysphagia 24%; both P ≤ 0.01). LASC patients also experienced less heartburn and more dysphagia than patient controls (heartburn 68%, dysphagia 11%; both P < 0.001). The prevalence of heartburn and dysphagia in IOM patients featuring a majority of non-transmitted sequences (NT) was 54% (P = 0.04 vs LASC) and 36% (P < 0.01 vs controls), respectively. No differences in age and gender distribution, chest pain prevalence, acid exposure time (AET) and symptom/reflux association existed between IOM subsets, or between subsets and controls. CONCLUSION: IOM patients with LASC exhibit a different symptom profile to those with LAP, but do not differ in gastro-oesophageal reflux prevalence. These findings raise the possibility of different pathophysiological mechanisms in IOM subsets, which warrants further investigation. PMID:18595138

  20. Domino: Extracting, Comparing, and Manipulating Subsets across Multiple Tabular Datasets

    PubMed Central

    Gratzl, Samuel; Gehlenborg, Nils; Lex, Alexander; Pfister, Hanspeter; Streit, Marc

    2016-01-01

    Answering questions about complex issues often requires analysts to take into account information contained in multiple interconnected datasets. A common strategy in analyzing and visualizing large and heterogeneous data is dividing it into meaningful subsets. Interesting subsets can then be selected and the associated data and the relationships between the subsets visualized. However, neither the extraction and manipulation nor the comparison of subsets is well supported by state-of-the-art techniques. In this paper we present Domino, a novel multiform visualization technique for effectively representing subsets and the relationships between them. By providing comprehensive tools to arrange, combine, and extract subsets, Domino allows users to create both common visualization techniques and advanced visualizations tailored to specific use cases. In addition to the novel technique, we present an implementation that enables analysts to manage the wide range of options that our approach offers. Innovative interactive features such as placeholders and live previews support rapid creation of complex analysis setups. We introduce the technique and the implementation using a simple example and demonstrate scalability and effectiveness in a use case from the field of cancer genomics. PMID:26356916

  1. Antibody engineering for increased potency, breadth and half-life

    PubMed Central

    Sievers, Stuart A.; Scharf, Louise; West, Anthony P.; Bjorkman, Pamela J.

    2015-01-01

    Purpose of review This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies. Recent findings Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems. Summary Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed. PMID:25760931

  2. SWAP-70 Identifies a Transitional Subset of Actin Filaments in Motile CellsV⃞

    PubMed Central

    Hilpelä, Pirta; Oberbanscheidt, Pia; Hahne, Penelope; Hund, Martin; Kalhammer, Georg; Small, J. Victor; Bähler, Martin

    2003-01-01

    Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3′-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actincytoskeletonasaneffectororadaptorproteininresponsetoagoniststimulatedphosphatidylinositol (3,4)-bisphosphate production and cell protrusion. PMID:12925760

  3. Enhancing OPeNDAP for Multi-Source Subset Selection

    NASA Astrophysics Data System (ADS)

    Fulker, D. W.

    2012-12-01

    For nearly two decades, web services built on OPeNDAP methods have increased the practicality of accessing scientific data from multiple, disparate sources. Data accessible via the Data Access Protocol (DAP) now include public holdings at NOAA, NASA, and many other organizations whose disciplinary foci are diverse. Central to the DAP's utility is its domain-neutral data model, which includes simple mechanisms for retrieving user-specified subsets of the source data. Though DAP allows whole-dataset retrievals in principle, network limitations and other constraints often make subsetting essential. DAP subsetting is particularly efficient where data are organized as multidimensional arrays, common for many kinds of satellite imagery and model outputs. The current version (DAP2) lets users define which array elements are returned by specifying their indices, skipping some (decimation) and/or limiting their ranges. This permits, e.g., rapid selection of a 2-D slice from a dataset organized as a 4-D hypercube, whose axes might correspond to three dimensions of space and one of time. Despite its power, DAP2 subsetting has significant limitations in cases where the subset of interest is not nicely defined by lines or hyperplanes that parallel the axes of the source array. Though DAP2 servers can allow specification of subset boundaries by coordinate values, rather than indices, the resulting subsets nonetheless must take the form of subarrays whose shapes are tightly linked to those of the source arrays. Ways to overcome these limitations are being considered in a project dubbed OPULS (OPeNDAP-Unidata Linked Servers), funded by NOAA. Project goals include DAP4 (the DAP2 successor) and a new, experimental form of subsetting. The latter addresses the case where the source data and the desired subset are irregular meshes (AKA UGRIDS or unstructured grids), whose shapes are defined polygonally. Though the experiment is a special case, where client and server both are aware of

  4. Definition of Drosophila hemocyte subsets by cell-type specific antigens.

    PubMed

    Kurucz, Eva; Váczi, B; Márkus, R; Laurinyecz, Barbara; Vilmos, P; Zsámboki, J; Csorba, Kinga; Gateff, Elisabeth; Hultmark, D; Andó, I

    2007-01-01

    We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes. PMID:18297797

  5. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues.

    PubMed

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  6. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues

    PubMed Central

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  7. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo

    PubMed Central

    Sommermeyer, Daniel; Hudecek, Michael; Kosasih, Paula L.; Gogishvili, Tea; Maloney, David G.; Turtle, Cameron J.; Riddell, Stanley R.

    2016-01-01

    Adoptive T-cell therapy with gene-modified T-cells expressing a tumor-reactive T-cell receptor (TCR) or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprised of random compositions of CD4+ and CD8+ naïve and memory T-cells, meaning that each patient received a different therapeutic agent. This variation might have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T-cells derived from different subsets (CD4+/CD8+ naïve, central memory, effector memory). T-cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T-cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies. PMID:26369987

  8. Subsetting and Formatting Landsat-7 LOR ETM+ and Data Products

    NASA Technical Reports Server (NTRS)

    Reid, Michael R.

    2000-01-01

    The Landsat-7 Processing System (LPS) processes Landsat-7 Enhanced Thematic Mapper (ETM+) instrument data into large, contiguous segments called "subintervals" and stores them in Level OR (LOR) data files. The LPS processed subinterval products must be subsetted and reformatted before the Level I processing systems can ingest them. The initial full subintervals produced by the LPS are stored mainly in HDF Earth Observing System (HDF-EOS) format which is an extension to the Hierarchical Data Format (HDF). The final LOR products are stored in native HDF format. Primarily the EOS Core System (ECS) and alternately the DAAC Emergency System (DES) subset the subinterval data for the operational Landsat-7 data processing systems. The HDF and HDF-EOS application programming interfaces (APIs) can be used for extensive data subsetting and data reorganization. A stand-alone subsetter tool has been developed which is based on some of the DES code. This tool makes use of the HDF and HDFEOS APIs to perform Landsat-7 LOR product subsetting and demonstrates how HDF and HDFEOS can be used for creating various configurations of full LOR products. How these APIs can be used to efficiently subset, format, and organize Landsat-7 LOR data as demonstrated by the subsetter tool and the DES is discussed.

  9. Peripheral blood T-lymphocyte subsets in autoimmune thyroid disease.

    PubMed

    Covas, M I; Esquerda, A; García-Rico, A; Mahy, N

    1992-01-01

    Interest in T-lymphocyte subsets has arisen because of their involvement in the autoimmune process. Contradictory results have been published in the literature about the number of peripheral blood lymphocyte subsets in autoimmune diseases. In order to investigate the number and distribution of peripheral blood lymphocyte subsets in autoimmune thyroid disease, the levels of total T-lymphocytes (CD3), T-helper (CD4) and T-suppressor/cytotoxic (CD8) lymphocytes were determined in 44 patients with Graves' disease (1), multinodular goiter (2) and Hashimoto's thyroiditis (3). All patients had high levels of antithyroglobulin and thyroid antiperoxidase (antimicrosomal) antibodies. The T subset levels were related to the functional thyroid status, measured as serum free thyroxine (FT4) and thyrotropin (TSH). Our data show the existence of a strong influence of functional status on CD3, CD4 and CD8 levels, as reflected in the significant correlations obtained with FT4 (negative) and TSH (positive). A significant decrease in all populations was observed in Graves' disease hyperthyroid patients. A decrease in the CD4/CD8 ratio in Hashimoto's thyroiditis hypothyroid patients was observed, in contrast to an increase in the ratio in autoimmune hyperthyroid patients. This points to the CD4/CD8 ratio as a differential characteristic between the two autoimmune (hypothyroid and hyperthyroid) entities, independent of free thyroxine levels. No significant correlation was found between antithyroid antibody levels and peripheral blood T-lymphocyte subsets or serum levels of FT4 and TSH. PMID:1342892

  10. Potency testing of inactivated rabies vaccines using a serological method.

    PubMed

    Kamphuis, E; Krämer, B; Schildger, H; Duchow, K

    2012-01-01

    Batch potency testing of rabies vaccines could be done by challenge, measurement of serum response or antigen quantification. Here, we show the development of a serological test that was successfully validated for use in batch release. The serological test is based on serum neutralization (SNT). The correlation to the NIH challenge was demonstrated by batches passing respectively failing equivalently in the NIH and SNT. The SNT provides information on immunogenicity and exhibits several advantages to the NIH: 1) SNT uses many fewer animals for batch release. 2) SNT allows quantitative information on the individual serum response, in contrast to the "dead"/"alive" interpretation of the NIH. 3) SNT is quicker than the NIH and needs fewer working hours. 4) SNT avoids the highly disturbing intra-cerebral injection and suffering from rabies for mice and spares the staff the emotional stress of massively harming animals. PMID:22888591

  11. Biosorption potency of Aspergillus niger for removal of chromium (VI).

    PubMed

    Srivastava, Shaili; Thakur, Indu Shekhar

    2006-09-01

    Aspergillus niger isolated from soil and effluent of leather tanning mills had higher activity to remove chromium. The potency of Aspergillus niger was evaluated in shake flask culture by absorption of chromium at pH 6 and temperature 30 degrees C. The results of the study indicated removal of more than 75% chromium by Aspergillus niger determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometry after 7 days. Study of microbial Cr(VI) reduction and identification of reduction intermediates has been hindered by the lack of analytical techniques that can identify the oxidation state with subcellular spatial resolution. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX), which indicated an accumulation of chromium in the fungal mycelium. PMID:16874547

  12. Antibacterial potency of methanol extracts of lower plants

    PubMed Central

    Ojo, O.O.; Ajayi, A.O.; Anibijuwon, I.I.

    2007-01-01

    Antibacterial potency of methanol extracts of three green lower plants, Pneumatopteris afra, Platycerium bifurcatum and Nephrolepsis bisserata was determined using agar dilution method on clinical strains of Escherichia coli, Staphylococcus aureus, Klebsiella spp. and Salmomelia typhi. Antibacterial activities were observed at concentrations of 12.5, 25.0, 50.0 and 100.0 µg/ml. Their minimum inhibitory concentrations ranged from 12.5~100 μg/ml. Extracts of P. afra and P. bifurcatum were most active. Antibacterial activities observed with N. bisserata were less pronounced with no detectable activity at extract concentrations of 12.5 and 25.0 µg/ml. E. coli, together with S. aureus appeared to be the most susceptible of the test bacteria while Klebsiella spp. was least sensitive. The significance of our findings is discussed. PMID:17323431

  13. Antibacterial potency of methanol extracts of lower plants.

    PubMed

    Ojo, O O; Ajayi, A O; Anibijuwon, I I

    2007-03-01

    Antibacterial potency of methanol extracts of three green lower plants, Pneumatopteris afra, Platycerium bifurcatum and Nephrolepsis bisserata was determined using agar dilution method on clinical strains of Escherichia coli, Staphylococcus aureus, Klebsiella spp. and Salmomelia typhi. Antibacterial activities were observed at concentrations of 12.5, 25.0, 50.0 and 100.0 microg/ml. Their minimum inhibitory concentrations ranged from 12.5approximately 100 microg/ml. Extracts of P. afra and P. bifurcatum were most active. Antibacterial activities observed with N. bisserata were less pronounced with no detectable activity at extract concentrations of 12.5 and 25.0 microg/ml. E. coli, together with S. aureus appeared to be the most susceptible of the test bacteria while Klebsiella spp. was least sensitive. The significance of our findings is discussed. PMID:17323431

  14. Biologically Active Cannabinoids from High-Potency Cannabis sativa

    PubMed Central

    Radwan, Mohamed M.; ElSohly, Mahmoud A.; Slade, Desmond; Ahmed, Safwat A.; Khan, Ikhlas A.; Ross, Samir A.

    2016-01-01

    Nine new cannabinoids (1–9) were isolated from a high-potency variety of Cannabis sativa. Their structures were identified as (±)-4-acetoxycannabichromene (1), (±)-3″-hydroxy-Δ(4″,5″)-cannabichromene (2), (−)-7-hydroxycannabichromane (3), (−)-7R-cannabicoumarononic acid A (4), 5-acetyl-4-hydroxycannabigerol (5), 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenol (6), 8-hydroxycannabinol (7), 8-hydroxycannabinolic acid A (8), and 2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone (9) through 1D and 2D NMR spectroscopy, GC-MS, and HRESIMS. The known sterol β-sitosterol-3-O-β-d-glucopyranosyl-6′-acetate was isolated for the first time from cannabis. Compounds 6 and 7 displayed significant antibacterial and antifungal activities, respectively, while 5 displayed strong antileishmanial activity. PMID:19344127

  15. Direct immunomagnetic quantification of lymphocyte subsets in blood.

    PubMed Central

    Brinchmann, J E; Vartdal, F; Gaudernack, G; Markussen, G; Funderud, S; Ugelstad, J; Thorsby, E

    1988-01-01

    A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies (MoAb) are used for the direct and fast quantification of the absolute number of cells of various lymphocyte subsets in blood. Blood samples were incubated with microspheres coated with a subset specific MoAb. Using a magnet the microsphere-rosetted cells were isolated and washed. Following lysis of the cell walls to detach the microspheres, the cell nuclei were stained with acridine orange and counted in a haemocytometer using an immunofluorescence microscope. With MoAb specific for CD2, CD4, CD8 and CD19, reproducible absolute counts of the corresponding lymphocyte subsets were obtained which correlated closely with those obtained by an indirect quantification method. PMID:3349645

  16. Subset-sum phase transitions and data compression

    NASA Astrophysics Data System (ADS)

    Merhav, Neri

    2011-09-01

    We propose a rigorous analysis approach for the subset-sum problem in the context of lossless data compression, where the phase transition of the subset-sum problem is directly related to the passage between ambiguous and non-ambiguous decompression, for a compression scheme that is based on specifying the sequence composition. The proposed analysis lends itself to straightforward extensions in several directions of interest, including non-binary alphabets, incorporation of side information at the decoder (Slepian-Wolf coding), and coding schemes based on multiple subset sums. It is also demonstrated that the proposed technique can be used to analyze the critical behavior in a more involved situation where the sequence composition is not specified by the encoder.

  17. Sexual Function and the Use of Medical Devices or Drugs to Optimize Potency After Prostate Brachytherapy

    SciTech Connect

    Whaley, J. Taylor; Levy, Lawrence B.; Swanson, David A.; Pugh, Thomas J.; Kudchadker, Rajat J.; Bruno, Teresa L.; Frank, Steven J.

    2012-04-01

    Purpose: Prospective evaluation of sexual outcomes after prostate brachytherapy with iodine-125 seeds as monotherapy at a tertiary cancer care center. Methods and Materials: Subjects were 129 men with prostate cancer with I-125 seed implants (prescribed dose, 145 Gy) without supplemental hormonal or external beam radiation therapy. Sexual function, potency, and bother were prospectively assessed at baseline and at 1, 4, 8, and 12 months using validated quality-of-life self-assessment surveys. Postimplant dosimetry values, including dose to 10% of the penile bulb (D10), D20, D33, D50, D75, D90, and penile volume receiving 100% of the prescribed dose (V100) were calculated. Results: At baseline, 56% of patients recorded having optimal erections; at 1 year, 62% of patients with baseline erectile function maintained optimal potency, 58% of whom with medically prescribed sexual aids or drugs. Variables associated with pretreatment-to-posttreatment decline in potency were time after implant (p = 0.04) and age (p = 0.01). Decline in urinary function may have been related to decline in potency. At 1 year, 69% of potent patients younger than 70 years maintained optimal potency, whereas 31% of patients older than 70 maintained optimal potency (p = 0.02). Diabetes was related to a decline in potency (p = 0.05), but neither smoking nor hypertension were. For patients with optimal potency at baseline, mean sexual bother scores had declined significantly at 1 year (p < 0.01). Sexual potency, sexual function, and sexual bother scores failed to correlate with any dosimetric variable tested. Conclusions: Erections firm enough for intercourse can be achieved at 1 year after treatment, but most men will require medical aids to optimize potency. Although younger men were better able to maintain erections firm enough for intercourse than older men, there was no correlation between potency, sexual function, or sexual bother and penile bulb dosimetry.

  18. Short-term study investigating the estrogenic potency of diethylstilbesterol in the fathead minnow (Pimephales promelas).

    PubMed

    Adedeji, Olufemi B; Durhan, Elizabeth J; Garcia-Reyero, Natàlia; Kahl, Michael D; Jensen, Kathleen M; Lalone, Carlie A; Makynen, Elizabeth A; Perkins, Edward J; Thomas, Linnea; Villeneuve, Daniel L; Ankley, Gerald T

    2012-07-17

    Diethylstilbestrol (DES) is a synthetic estrogen that has been banned for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world. Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish. In this study, sexually mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10, or 100 ng of DES/L of water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive end points were measured in the fish at the end of a 4 d exposure and after a 4 d depuration/recovery period in clean water. Accumulation of DES was sex-dependent, with females exhibiting higher tissue residues than males after the 4 d exposure. The observed bioconcentration of DES in the fish was about 1 order of magnitude lower than that predicted on the basis of the octanol-water partition coefficient of the chemical, suggesting relatively efficient metabolic clearance by the fish. Exposure to 1, 10, or 100 ng of DES/L caused decreased testis weight and morphological demasculinization of males (regression of dorsal nuptial tubercles). Diethylstilbesterol induced plasma vitellogenin (VTG) in both sexes at water concentrations ≥10 ng/L; this response (especially in males) persisted through the end of the 4 d recovery period. Hepatic transcripts of VTG and estrogen receptor-α also were affected at DES concentrations ≥10 ng/L. Evaluation of transcript profiles in the liver of females using a 15K-gene fathead minnow microarray revealed a concentration-dependent change in gene expression, with mostly up-regulated transcripts after the exposure and substantial numbers of down-regulated gene products after depuration. Genes previously identified as vitellogenesis-related and regulated by 17β-estradiol were significantly enriched among those

  19. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    SciTech Connect

    Kleinstreuer, N.C.; Smith, A.M.; West, P.R.; Conard, K.R.; Fontaine, B.R.; Weir-Hauptman, A.M.; Palmer, J.A.; Knudsen, T.B.; Dix, D.J.; Donley, E.L.R.; Cezar, G.G.

    2011-11-15

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast Trade-Mark-Sign chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox Registered-Sign model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. -- Highlights: Black-Right-Pointing-Pointer We tested 11 environmental compounds in a hESC metabolomics platform. Black-Right-Pointing-Pointer Significant changes in secreted small molecule metabolites were observed. Black-Right-Pointing-Pointer Perturbed mass features map to pathways critical for normal development and

  20. Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics

    PubMed Central

    Vu Manh, Thien-Phong; Elhmouzi-Younes, Jamila; Urien, Céline; Ruscanu, Suzana; Jouneau, Luc; Bourge, Mickaël; Moroldo, Marco; Foucras, Gilles; Salmon, Henri; Marty, Hélène; Quéré, Pascale; Bertho, Nicolas; Boudinot, Pierre; Dalod, Marc; Schwartz-Cornil, Isabelle

    2015-01-01

    Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey. PMID:26150816

  1. Distinct germline progenitor subsets defined through Tsc2–mTORC1 signaling

    PubMed Central

    Hobbs, Robin M; La, Hue M; Mäkelä, Juho-Antti; Kobayashi, Toshiyuki; Noda, Tetsuo; Pandolfi, Pier Paolo

    2015-01-01

    Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional heterogeneity existing within this self-renewing population is poorly understood. Here, we define distinct subsets of undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation of mTORC1, a key growth-promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 throughout the SPC pool using Vasa-Cre promotes differentiation at the expense of self-renewal and leads to germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8-Cre did not compromise SPC function. SPC activity also appeared unaffected by Amh-Cre-mediated Tsc2 deletion within somatic cells of the niche. Importantly, we find that differentiation-prone SPCs have elevated mTORC1 activity when compared to SPCs with high self-renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with mTORC1-active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and defines phenotypically distinct SPC subpopulations with varying propensities for self-renewal and differentiation. PMID:25700280

  2. Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics.

    PubMed

    Vu Manh, Thien-Phong; Elhmouzi-Younes, Jamila; Urien, Céline; Ruscanu, Suzana; Jouneau, Luc; Bourge, Mickaël; Moroldo, Marco; Foucras, Gilles; Salmon, Henri; Marty, Hélène; Quéré, Pascale; Bertho, Nicolas; Boudinot, Pierre; Dalod, Marc; Schwartz-Cornil, Isabelle

    2015-01-01

    Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey. PMID:26150816

  3. CD16+ monocytes control T-cell subset development in immune thrombocytopenia

    PubMed Central

    Zhong, Hui; Bao, Weili; Li, Xiaojuan; Miller, Allison; Seery, Caroline; Haq, Naznin; Bussel, James

    2012-01-01

    Immune thrombocytopenia (ITP) results from decreased platelet production and accelerated platelet destruction. Impaired CD4+ regulatory T-cell (Treg) compartment and skewed Th1 and possibly Th17 responses have been described in ITP patients. The trigger for aberrant T-cell polarization remains unknown. Because monocytes have a critical role in development and polarization of T-cell subsets, we explored the contribution of monocyte subsets in control of Treg and Th development in patients with ITP. Unlike circulating classic CD14hiCD16− subpopulation, the CD16+ monocyte subset was expanded in ITP patients with low platelet counts on thrombopoietic agents and positively correlated with T-cell CD4+IFN-γ+ levels, but negatively with circulating CD4+CD25hiFoxp3+ and IL-17+ Th cells. Using a coculture model, we found that CD16+ ITP monocytes promoted the expansion of IFN-γ+CD4+ cells and concomitantly inhibited the proliferation of Tregs and IL-17+ Th cells. Th-1–polarizing cytokine IL-12, secreted after direct contact of patient T-cell and CD16+ monocytes, was responsible for the inhibitory effect on Treg and IL-17+CD4+ cell proliferation. Our findings are consistent with ITP CD16+ monocytes promoting Th1 development, which in turn negatively regulates IL-17 and Treg induction. This underscores the critical role of CD16+ monocytes in the generation of potentially pathogenic Th responses in ITP. PMID:22915651

  4. 75 FR 8937 - Development of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-26

    ... AGENCY Development of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH...) Approach for Polycyclic Aromatic Hydrocarbon (PAH) Mixtures'' (EPA/635/R-08/012A). The draft document was... of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH) Mixtures''...

  5. COMPARATIVE POTENCY METHOD FOR CANCER RISK ASSESSMENT: APPLICATION TO DIESEL PARTICULATE EMISSIONS

    EPA Science Inventory

    An estimation of the human lung cancer 'unit risk' from diesel engine particulate emissions has been made using a comparative potency approach. This approach involves evaluating the tumorigenic and mutagenic potencies of the particlates from four diesel and one gasoline engine in...

  6. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-16

    ..., 2008 (73 FR 59635), FDA announced the availability of the draft guidance of the same title. FDA... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Potency Tests for Cellular and Gene... Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance...

  7. [Dynamics of CD4⁺ T cell subsets and their roles in schistosome infections: a review].

    PubMed

    Fan, Xiao-lin; Gao, Qi; Yang, Jun-qi

    2015-12-01

    Schistosomiasis is a chronic helminthic disease that affects about two hundred millions of people in the world. The pathogenesis of schistosome infection is primarily due to hepatic and intestinal granuloma formation around deposited eggs and subsequent fibrosis. It is known that CD4⁺ T cell subsets play critical roles in the host immunity and immunopathogenesis to schistosome infections, in which T helper 1 (Th1) and Th2 cells are major effector T cell subsets, whereas T regulatory (Treg) cells exert immunosuppressive roles in general. The recently discovered Th17 cells are also actively involved in the immune responses to the infection. During the infection, these T cell subsets cross-talk and exhibit different kinetics and roles in the control and regulation of infection progress and fibrosis. This review summarizes current findings of Th1, Th2, Th17 and Treg cells and their effector cytokines in schistosome infection. PMID:27097497

  8. The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus

    PubMed Central

    Tacke, Robert; Hilgendorf, Ingo; Garner, Hannah; Waterborg, Claire; Park, Kiwon; Nowyhed, Heba; Hanna, Richard N.; Wu, Runpei; Swirski, Filip K.; Geissmann, Frederic; Hedrick, Catherine C.

    2015-01-01

    Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1, and Nr4a1-deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset. PMID:26091486

  9. SAGE II v6.20 Binary File Subset Tool

    Atmospheric Science Data Center

    2013-04-01

    ... files. The tool is written in Exelis Visual Information Solutions IDL programming language. It can be run either with a licensed ... with IDL and is available from  Exelis Visual Information Solutions . The subset may be specified by latitude and longitude regions ...

  10. Histone Deacetylation Critically Determines T-cell Subset Radiosensitivity1

    PubMed Central

    Pugh, Jason L.; Sukhina, Alona S.; Seed, Thomas M.; Manley, Nancy R.; Sempowski, Gregory A.; van den Brink, Marcel R.M.; Smithey, Megan J.; Nikolich-Zugich, Janko

    2014-01-01

    Lymphocytes are sensitive to ionizing radiation and naïve lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naïve (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice, and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and : (i) levels of pro- and anti-apoptotic Bcl-2 family members; or (ii) the H2-AX content and maximal γH2-AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2-AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment, improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells, but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences. PMID:24990082

  11. A SOAP Web Service for accessing MODIS land product subsets

    SciTech Connect

    SanthanaVannan, Suresh K; Cook, Robert B; Pan, Jerry Yun; Wilson, Bruce E

    2011-01-01

    Remote sensing data from satellites have provided valuable information on the state of the earth for several decades. Since March 2000, the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor on board NASA s Terra and Aqua satellites have been providing estimates of several land parameters useful in understanding earth system processes at global, continental, and regional scales. However, the HDF-EOS file format, specialized software needed to process the HDF-EOS files, data volume, and the high spatial and temporal resolution of MODIS data make it difficult for users wanting to extract small but valuable amounts of information from the MODIS record. To overcome this usability issue, the NASA-funded Distributed Active Archive Center (DAAC) for Biogeochemical Dynamics at Oak Ridge National Laboratory (ORNL) developed a Web service that provides subsets of MODIS land products using Simple Object Access Protocol (SOAP). The ORNL DAAC MODIS subsetting Web service is a unique way of serving satellite data that exploits a fairly established and popular Internet protocol to allow users access to massive amounts of remote sensing data. The Web service provides MODIS land product subsets up to 201 x 201 km in a non-proprietary comma delimited text file format. Users can programmatically query the Web service to extract MODIS land parameters for real time data integration into models, decision support tools or connect to workflow software. Information regarding the MODIS SOAP subsetting Web service is available on the World Wide Web (WWW) at http://daac.ornl.gov/modiswebservice.

  12. Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements

    ERIC Educational Resources Information Center

    Schafer, Mark; Kell, Holly; Navalta, James; Tibana, Ramires; Lyons, Scott; Arnett, Scott

    2014-01-01

    Tennis is an activity requiring both endurance and anaerobic components, which could have immunosuppressive effects postexercise. Purpose: The purpose of this investigation was to determine the effect of a simulated tennis match on apoptotic and migratory markers on lymphocyte subsets. Method: Male high school (n = 5) and college (n = 3) tennis…

  13. Clustering, Seriation, and Subset Extraction of Confusion Data

    ERIC Educational Resources Information Center

    Brusco, Michael J.; Steinley, Douglas

    2006-01-01

    The study of confusion data is a well established practice in psychology. Although many types of analytical approaches for confusion data are available, among the most common methods are the extraction of 1 or more subsets of stimuli, the partitioning of the complete stimulus set into distinct groups, and the ordering of the stimulus set. Although…

  14. Codes With Parity Conditions on Subsets of Coordinates

    NASA Technical Reports Server (NTRS)

    Posner, E.; Reichstein, Z.

    1986-01-01

    New theorems aid search for efficient code alphabets. Paper discusses theory of finding largest binary codes 2k bits in length, in which all words differ from each other in at least d places and in which words truncated by ignoring certain subsets of bit positions belong to shorter linear codes.

  15. Pathogenic Th cell subsets in chronic inflammatory diseases.

    PubMed

    Kumagai, Jin; Hirahara, Kiyoshi; Nakayama, Toshinori

    2016-01-01

      CD4(+) T cells play central roles to appropriate protection against pathogens. While, they can also be pathogenic driving inflammatory diseases. Besides the classical model of differentiation of T helper 1 (Th1) and Th2 cells, various CD4(+) T cell subsets, including Th17, Th9, T follicular helper (Tfh) and T regulatory (Treg) cells, have been recognized recently. In this review, we will focus on how these various CD4(+) T cell subsets contribute to the pathogenesis of immune-mediated inflammatory diseases. We will also discuss various unique subpopulations of T helper cells that have been identified. Recent advancement of the basic immunological research revealed that T helper cells are plastic than we imagined. So, we will focus on the molecular mechanisms underlying the generation of the plasticity and heterogeneity of T helper cell subsets. These latest finding regarding T helper cell subsets has pushed us to reconsider the etiology of immune-mediated inflammatory diseases beyond the model based on the conventional Th1/Th2 balance. Toward this end, we put forward another model, "the pathogenic Th population disease induction model", as a possible mechanism for the induction and/or persistence of immune-mediated inflammatory diseases. PMID:27212597

  16. Feature Subset Selection, Class Separability, and Genetic Algorithms

    SciTech Connect

    Cantu-Paz, E

    2004-01-21

    The performance of classification algorithms in machine learning is affected by the features used to describe the labeled examples presented to the inducers. Therefore, the problem of feature subset selection has received considerable attention. Genetic approaches to this problem usually follow the wrapper approach: treat the inducer as a black box that is used to evaluate candidate feature subsets. The evaluations might take a considerable time and the traditional approach might be unpractical for large data sets. This paper describes a hybrid of a simple genetic algorithm and a method based on class separability applied to the selection of feature subsets for classification problems. The proposed hybrid was compared against each of its components and two other feature selection wrappers that are used widely. The objective of this paper is to determine if the proposed hybrid presents advantages over the other methods in terms of accuracy or speed in this problem. The experiments used a Naive Bayes classifier and public-domain and artificial data sets. The experiments suggest that the hybrid usually finds compact feature subsets that give the most accurate results, while beating the execution time of the other wrappers.

  17. Ordering Elements and Subsets: Examples for Student Understanding

    ERIC Educational Resources Information Center

    Mellinger, Keith E.

    2004-01-01

    Teaching the art of counting can be quite difficult. Many undergraduate students have difficulty separating the ideas of permutation, combination, repetition, etc. This article develops some examples to help explain some of the underlying theory while looking carefully at the selection of various subsets of objects from a larger collection. The…

  18. Differentiation and diversity of subsets in group 1 innate lymphoid cells.

    PubMed

    Seillet, Cyril; Belz, Gabrielle T

    2016-01-01

    NK cells were first identified in 1975 and represent the prototypical group 1 innate lymphoid cell (ILC). More recently, the discovery of new members of the ILC family has highlighted the complexity of this innate lymphoid lineage. Importantly, it has been recognized that different subsets exist within the group 1 ILC, which have potential roles in mediating immune protection and immunosurveillance, and in regulating tissue homeostasis and inflammation. Here, we review the developmental relationships between the different group 1 ILC, which have been identified to date and discuss how heterogeneity within this expanding family may have arisen. PMID:26346810

  19. Cell fate decision: T-helper 1 and 2 subsets in immune responses

    PubMed Central

    Dong, Chen; Flavell, Richard A

    2000-01-01

    After activation CD4+ helper T cells differentiate into T-helper (Th) 1 or Th2 effector cells. These two subsets are characterized by their distinct cytokine expression pattern and the immune function they mediate. Over the past years, a number of factors have been identified to affect helper T cell lineage determination, including antigen receptor, coreceptors and, most importantly, cytokine environment. In this review, we also summarize recent advancement in understanding of transcriptional and signaling regulation of the differentiation process. This knowledge will become important in the future to develop means in treating immune disorders. PMID:11094427

  20. Peripheral T lymphocyte subset imbalances in children with enterovirus 71-induced hand, foot and mouth disease.

    PubMed

    Li, Shuxian; Cai, Chunyan; Feng, Jinyan; Li, Xuejing; Wang, Yingshuo; Yang, Jun; Chen, Zhimin

    2014-02-13

    Inflammatory mediators (i.e. cytokines) play a pivotal role in the regulation of pathophysiological processes during EV71-induced hand, foot and mouth disease (HFMD). Different T cell subsets have distinct cytokine secretion profiles, and alteration in the T cell subsets frequency (imbalance) during infection leads to changed cytokine patterns. However, the effects of EV71 infection on T cell subsets were not clear. The objective of this study was to determine whether EV71-induced HFMD can be explained by the emergence of particular T-cell subsets (Th1, Th2, Tc1, Tc2, Th17, Tc17 and Treg cells) and the cytokine they produced (IFN-γ, IL-4, IL-17A and TGF-β1), as well as distinct responses to EV71 infection. We found that when compared to the control group, the percentage of Th1 and Tc1 cells was significantly higher in mild and severe HFMD group. Similar results were found in the Th1/Th2 ratio and IFN-γ levels. On the other hand, the percentage of Th17 cells and IL-17A levels were the highest in severe HFMD cases, and lowest in controls. Similar trend was also found for the Th17/Treg cell ratio. An optimal cutoff value of 2.15% for Th17 cell and 6.72 pg/ml for IL-17A provided a discriminatory value for differentiating the severity of HFMD cases by receiver operating characteristic curve analyses. These findings reveal that the Th1/Th2 and Th17/Treg imbalance exist in HFMD patients, suggesting their involvement in the pathogenesis of EV71 infection, which may have potential value as biomarkers. PMID:24316007

  1. Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

    PubMed Central

    Pachynski, Russell K.; Scholz, Alexander; Monnier, Justin; Butcher, Eugene C.; Zabel, Brian A.

    2015-01-01

    Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.  Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune “escape,” including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed. PMID:25938949

  2. Quantitative reconstruction of leukocyte subsets using DNA methylation

    PubMed Central

    2014-01-01

    Background Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach. Results Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods. Conclusions Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells. PMID:24598480

  3. Determination of H5N1 vaccine potency using reference antisera from heterologous strains of influenza

    PubMed Central

    Vodeiko, Galina M.; Weir, Jerry P.

    2011-01-01

    Please cite this paper as: Vodeiko and Weir (2011). Determination of H5N1 vaccine potency using reference antisera from heterologous strains of influenza. Influenza and Other Respiratory Viruses 6(3), 176–187. Background  Standardization of inactivated influenza vaccines by hemagglutinin (HA) content is performed by the single radial immunodiffusion (SRID) method. Regulatory agencies prepare, calibrate, and distribute SRID reagent standards necessary for testing of seasonal influenza vaccines, and a similar process is used to produce potency reagents for candidate pandemic influenza vaccines that are manufactured for emergency stockpiles. Objectives  Because of the concerns in generating a timely strain‐specific potency antiserum for an emerging pandemic virus, we evaluated the feasibility of using heterologous potency reference antiserum as a replacement for a strain‐specific (homologous) antiserum in the SRID potency assay for stockpiled H5N1 vaccines. Results  The results indicate that a heterologous H5N1 antiserum can be used to determine the accurate potency of inactivated H5N1 influenza vaccines. Additionally, when H5N1 vaccine was subjected to an accelerated stability protocol, both homologous and heterologous antisera provided similar measurements of vaccine potency decline. Limitations to the heterologous antiserum approach to potency determination were shown by the inability of antiserum to recent seasonal H1N1 viruses to work in an SRID assay with the 2009 pandemic H1N1 A/California/07/2009 antigen. Conclusions  The data demonstrate the feasibility of using heterologous antiserum for potency determination of at least some candidate vaccines in case of a shortage or delay of homologous antiserum. Further, the results suggest the prudence of stockpiling a broad library of potency reagents including many strains of influenza viruses with pandemic potential to provide an added measure of assurance that reagent production would not be a

  4. Analysis of the In Vivo Turnover of CD4+ T-Cell Subsets in Chronically SIV-Infected Sooty Mangabeys

    PubMed Central

    Ortiz, Alexandra M.; Carnathan, Diane G.; Yu, Joana; Sheehan, Katherine M.; Kim, Peter; Reynaldi, Arnold; Vanderford, Thomas H.; Klatt, Nichole R.; Brenchley, Jason M.; Davenport, Miles P.; Silvestri, Guido

    2016-01-01

    Aberrant turnover of memory CD4+ T-cells is central to Acquired Immunodeficiency Syndrome (AIDS) progression. Understanding the relationship between the turnover of CD4+ subsets and immunological homeostasis during simian immunodeficiency virus (SIV) infection in natural hosts may provide insight into mechanisms of immune regulation that may serve as models for therapeutic intervention in Human Immunodeficiency Virus (HIV)-infected persons. Sooty mangabeys (SMs) have naturally evolved with SIV to avoid AIDS progression while maintaining healthy peripheral CD4+ T-cell counts and thus represent a model by which therapeutic interventions for AIDS progression might be elucidated. To assess the relationship between the turnover of CD4+ subsets and immunological homeostasis during SIV infection in non-progressive hosts, we treated 6 SIV-uninfected and 9 SIV-infected SMs with 2’-bromo-5’-deoxyuridine (BrdU) for 14 days and longitudinally assessed CD4+ T-cell subset turnover by polychromatic flow cytometry. We observed that, in SIV-infected SMs, turnover of CD4+ T-cell naïve and central, transitional, and effector memory subsets is comparable to that in uninfected animals. Comparable turnover of CD4+ T-cell subsets irrespective of SIV-infection status likely contributes to the lack of aberrant immune activation and disease progression observed after infection in non-progressive hosts. PMID:27227993

  5. Analysis of the In Vivo Turnover of CD4+ T-Cell Subsets in Chronically SIV-Infected Sooty Mangabeys.

    PubMed

    Ortiz, Alexandra M; Carnathan, Diane G; Yu, Joana; Sheehan, Katherine M; Kim, Peter; Reynaldi, Arnold; Vanderford, Thomas H; Klatt, Nichole R; Brenchley, Jason M; Davenport, Miles P; Silvestri, Guido

    2016-01-01

    Aberrant turnover of memory CD4+ T-cells is central to Acquired Immunodeficiency Syndrome (AIDS) progression. Understanding the relationship between the turnover of CD4+ subsets and immunological homeostasis during simian immunodeficiency virus (SIV) infection in natural hosts may provide insight into mechanisms of immune regulation that may serve as models for therapeutic intervention in Human Immunodeficiency Virus (HIV)-infected persons. Sooty mangabeys (SMs) have naturally evolved with SIV to avoid AIDS progression while maintaining healthy peripheral CD4+ T-cell counts and thus represent a model by which therapeutic interventions for AIDS progression might be elucidated. To assess the relationship between the turnover of CD4+ subsets and immunological homeostasis during SIV infection in non-progressive hosts, we treated 6 SIV-uninfected and 9 SIV-infected SMs with 2'-bromo-5'-deoxyuridine (BrdU) for 14 days and longitudinally assessed CD4+ T-cell subset turnover by polychromatic flow cytometry. We observed that, in SIV-infected SMs, turnover of CD4+ T-cell naïve and central, transitional, and effector memory subsets is comparable to that in uninfected animals. Comparable turnover of CD4+ T-cell subsets irrespective of SIV-infection status likely contributes to the lack of aberrant immune activation and disease progression observed after infection in non-progressive hosts. PMID:27227993

  6. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity

    PubMed Central

    2015-01-01

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure–activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes. PMID:26555042

  7. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  8. Cannabinoid Ester Constituents from High-Potency Cannabis sativa

    PubMed Central

    Ahmed, Safwat A.; Ross, Samir A.; Slade, Desmond; Radwan, Mohamed M.; Zulfiqar, Fazila; ElSohly, Mahmoud A.

    2016-01-01

    Eleven new cannabinoid esters, together with three known cannabinoid acids and Δ9-tetrahydrocannabinol (Δ9-THC), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be β-fenchyl Δ9-tetrahydrocannabinolate (1), epi-bornyl Δ9-tetrahydrocannabinolate (2), α-terpenyl Δ9-tetrahydrocannabinolate (3), 4-terpenyl Δ9-tetrahydrocannabinolate (4), α-cadinyl Δ9-tetrahydrocannabinolate (5), γ-eudesmyl Δ9-tetrahydrocannabinolate (6), γ-eudesmyl cannabigerolate (7), 4-terpenyl cannabinolate (8), bornyl Δ9-tetrahydrocannabinolate (9), α-fenchyl Δ9-tetrahydrocannabinolate (10), α-cadinyl cannabigerolate (11), Δ9-tetrahydro-cannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA), and cannabigerolic acid (CBGA). Compound 8 showed moderate antimicrobial activity against Candida albicans ATCC 90028 with an IC50 value of 8.5 μg/mL. CB-1 receptor assay indicated that the esters, as well as the parent acids, are not active. PMID:18303850

  9. Minor oxygenated cannabinoids from high potency Cannabis sativa L

    PubMed Central

    Ahmed, Safwat A.; Ross, Samir A.; Slade, Desmond; Radwan, Mohamed M.; Khan, Ikhlas A.; ElSohly, Mahmoud A.

    2016-01-01

    Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC–MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ9-THC aldehyde A, 8-oxo-Δ9-THC, 10aα-hydroxy-10-oxo-Δ8-THC, 9α-hydroxy-10-oxo-Δ6a,10a-THC, and 1′S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0 μg/mL), moderate antileishmanial (IC50 14.0 μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3 μg/mL, respectively. PMID:26093324

  10. Minor oxygenated cannabinoids from high potency Cannabis sativa L.

    PubMed

    Ahmed, Safwat A; Ross, Samir A; Slade, Desmond; Radwan, Mohamed M; Khan, Ikhlas A; ElSohly, Mahmoud A

    2015-09-01

    Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC-MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ(9)-THC aldehyde A, 8-oxo-Δ(9)-THC, 10aα-hydroxy-10-oxo-Δ(8)-THC, 9α-hydroxy-10-oxo-Δ(6a,10a)-THC, and 1'S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0 μg/mL), moderate antileishmanial (IC50 14.0 μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3 μg/mL, respectively. PMID:26093324

  11. Bioluminescence for assessing drug potency against nonreplicating Mycobacterium tuberculosis.

    PubMed

    Vocat, Anthony; Hartkoorn, Ruben C; Lechartier, Benoit; Zhang, Ming; Dhar, Neeraj; Cole, Stewart T; Sala, Claudia

    2015-07-01

    Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications. PMID:25896710

  12. Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    Vocat, Anthony; Hartkoorn, Ruben C.; Lechartier, Benoit; Zhang, Ming; Dhar, Neeraj

    2015-01-01

    Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications. PMID:25896710

  13. Reevaluating the potency of the memory conformity effect.

    PubMed

    Bodner, Glen E; Musch, Elisabeth; Azad, Tanjeem

    2009-12-01

    Witnesses sometimes report event details that are acquired solely from another witness. We reevaluated the potency of this memory conformity effect. After viewing a crime video, some participants learned about nonwitnessed details via discussion (dyad group), reading another participant's report (read group), or watching another version of the video (both-video group). In Experiment 1, these participants often reported nonwitnessed details, but on a source-judgment test most details were attributed primarily to the actual source rather than to the video. In addition, the dyad group was not more likely than the read or both-video groups to report nonwitnessed details. Participants in Experiment 2 were explicitly discouraged from providing details that were remembered from the secondary source only. These postwarning instructions substantially reduced the memory conformity effect, and a dyad group was not more likely than a read group to report nonwitnessed details. Encouraging source monitoring at test can reduce the negative consequences of co-witness collaboration. PMID:19933452

  14. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use.

    PubMed

    Korimbocus, Jehanara; Dehay, Nicolas; Tordo, Noël; Cano, François; Morgeaux, Sylvie

    2016-06-14

    In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice. PMID:27151880

  15. A reassessment of IgM memory subsets in humans

    PubMed Central

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M.; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K.; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-01-01

    From paired blood and spleen samples from three adult donors we performed high-throughput V-h sequencing of human B-cell subsets defined by IgD and CD27 expression: IgD+CD27+ (“MZ”), IgD−CD27+(“memory”, including IgM (“IgM-only”), IgG and IgA) and IgD−CD27− cells (“double-negative”, including IgM, IgG and IgA). 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for Vh-gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin, for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency, and lower Vh4 and higher Jh6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (between MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells, and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  16. A Reassessment of IgM Memory Subsets in Humans.

    PubMed

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-10-15

    From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  17. Cutoff in potency implicates alcohol inhibition of N-methyl-D-aspartate receptors in alcohol intoxication.

    PubMed Central

    Peoples, R W; Weight, F F

    1995-01-01

    As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this "cutoff" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication. PMID:7708732

  18. Chromatographic method for quick estimation of DNA interaction potency of environmental pollutants.

    PubMed

    Feng, Yong-Lai; Lian, Hong-Zhen; Liao, Xiang-Jun; Zhu, Ji-Ping

    2009-10-01

    The DNA interaction potency of a chemical has been defined in the present study as the degree of a chemical's ability to interact with DNA. An estimation method of such a potency has been established based on the peak reduction of an oligonucleotide probe resulting from its interaction with chemicals based on high-performance liquid chromatography. A DNA interaction potency equivalency (PEQ) also has been proposed to evaluate the relative interaction potency of test chemicals against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Five known direct DNA interaction chemicals were employed to demonstrate the method. Two known inactive chemicals were used as negative controls. Both the potency and PEQ(50) values (PEQ of testing chemical at 50% of the probe peak reduction) of these five chemicals were determined as BPDE > phenyl glycidyl ether (PGE) > tetrachlorohydroquinone (Cl4HQ) > methyl methanesulfonate (MMS) > styrene-7,8-oxide (SO). Among the reactive chemicals, MMS was found to break the oligonucleotide into smaller fragments, whereas BPDE, PGE, and SO form covalent adducts with the oligonucleotide. In the latter case, the formation of multi-chemical-oligonucleotide adducts also was observed by mass spectrometry. The method was employed to estimate the DNA interaction potency equivalency of diesel vehicle exhaust gas to demonstrate the applicability of this approach in evaluating the interaction potency of environmental pollutants in both gas and liquid phases. PMID:19432508

  19. Auditing Complex Concepts in Overlapping Subsets of SNOMED

    PubMed Central

    Wang, Yue; Wei, Duo; Xu, Junchuan; Elhanan, Gai; Perl, Yehoshua; Halper, Michael; Chen, Yan; Spackman, Kent A.; Hripcsak, George

    2008-01-01

    Limited resources and the sheer volume of concepts make auditing a large terminology, such as SNOMED CT, a daunting task. It is essential to devise techniques that can aid an auditor by automatically identifying concepts that deserve attention. A methodology for this purpose based on a previously introduced abstraction network (called the p-area taxonomy) for a SNOMED CT hierarchy is presented. The methodology algorithmically gathers concepts appearing in certain overlapping subsets, defined exclusively with respect to the p-area taxonomy, for review. The results of applying the methodology to SNOMED’s Specimen hierarchy are presented. These results are compared against a control sample composed of concepts residing in subsets without the overlaps. With the use of the double bootstrap, the concept group produced by our methodology is shown to yield a statistically significant higher proportion of error discoveries. PMID:18998838

  20. Variable selection with stepwise and best subset approaches

    PubMed Central

    2016-01-01

    While purposeful selection is performed partly by software and partly by hand, the stepwise and best subset approaches are automatically performed by software. Two R functions stepAIC() and bestglm() are well designed for stepwise and best subset regression, respectively. The stepAIC() function begins with a full or null model, and methods for stepwise regression can be specified in the direction argument with character values “forward”, “backward” and “both”. The bestglm() function begins with a data frame containing explanatory variables and response variables. The response variable should be in the last column. Varieties of goodness-of-fit criteria can be specified in the IC argument. The Bayesian information criterion (BIC) usually results in more parsimonious model than the Akaike information criterion. PMID:27162786

  1. Design of a verifiable subset for HAL/S

    NASA Technical Reports Server (NTRS)

    Browne, J. C.; Good, D. I.; Tripathi, A. R.; Young, W. D.

    1979-01-01

    An attempt to evaluate the applicability of program verification techniques to the existing programming language, HAL/S is discussed. HAL/S is a general purpose high level language designed to accommodate the software needs of the NASA Space Shuttle project. A diversity of features for scientific computing, concurrent and real-time programming, and error handling are discussed. The criteria by which features were evaluated for inclusion into the verifiable subset are described. Individual features of HAL/S with respect to these criteria are examined and justification for the omission of various features from the subset is provided. Conclusions drawn from the research are presented along with recommendations made for the use of HAL/S with respect to the area of program verification.

  2. Robust enumeration of cell subsets from tissue expression profiles

    PubMed Central

    Newman, Aaron M.; Liu, Chih Long; Green, Michael R.; Gentles, Andrew J.; Feng, Weiguo; Xu, Yue; Hoang, Chuong D.; Diehn, Maximilian; Alizadeh, Ash A.

    2015-01-01

    We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles. When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen, and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content, and closely related cell types. CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersort.stanford.edu). PMID:25822800

  3. Plasticity of human CD4 T cell subsets.

    PubMed

    Geginat, Jens; Paroni, Moira; Maglie, Stefano; Alfen, Johanna Sophie; Kastirr, Ilko; Gruarin, Paola; De Simone, Marco; Pagani, Massimiliano; Abrignani, Sergio

    2014-01-01

    Human beings are exposed to a variety of different pathogens, which induce tailored immune responses and consequently generate highly diverse populations of pathogen-specific T cells. CD4(+) T cells have a central role in adaptive immunity, since they provide essential help for both cytotoxic T cell- and antibody-mediated responses. In addition, CD4(+) regulatory T cells are required to maintain self-tolerance and to inhibit immune responses that could damage the host. Initially, two subsets of CD4(+) helper T cells were identified that secrete characteristic effector cytokines and mediate responses against different types of pathogens, i.e., IFN-γ secreting Th1 cells that fight intracellular pathogens, and IL-4 producing Th2 cells that target extracellular parasites. It is now well established that this dichotomy is insufficient to describe the complexity of CD4(+) T cell differentiation, and in particular the human CD4 compartment contains a myriad of T cell subsets with characteristic capacities to produce cytokines and to home to involved tissues. Moreover, it has become increasingly clear that these T cell subsets are not all terminally differentiated cells, but that the majority is plastic and that in particular central memory T cells can acquire different properties and functions in secondary immune responses. In addition, there is compelling evidence that helper T cells can acquire regulatory functions upon chronic stimulation in inflamed tissues. The plasticity of antigen-experienced human T cell subsets is highly relevant for translational medicine, since it opens new perspectives for immune-modulatory therapies for chronic infections, autoimmune diseases, and cancer. PMID:25566245

  4. Plasticity of Human CD4 T Cell Subsets

    PubMed Central

    Geginat, Jens; Paroni, Moira; Maglie, Stefano; Alfen, Johanna Sophie; Kastirr, Ilko; Gruarin, Paola; De Simone, Marco; Pagani, Massimiliano; Abrignani, Sergio

    2014-01-01

    Human beings are exposed to a variety of different pathogens, which induce tailored immune responses and consequently generate highly diverse populations of pathogen-specific T cells. CD4+ T cells have a central role in adaptive immunity, since they provide essential help for both cytotoxic T cell- and antibody-mediated responses. In addition, CD4+ regulatory T cells are required to maintain self-tolerance and to inhibit immune responses that could damage the host. Initially, two subsets of CD4+ helper T cells were identified that secrete characteristic effector cytokines and mediate responses against different types of pathogens, i.e., IFN-γ secreting Th1 cells that fight intracellular pathogens, and IL-4 producing Th2 cells that target extracellular parasites. It is now well established that this dichotomy is insufficient to describe the complexity of CD4+ T cell differentiation, and in particular the human CD4 compartment contains a myriad of T cell subsets with characteristic capacities to produce cytokines and to home to involved tissues. Moreover, it has become increasingly clear that these T cell subsets are not all terminally differentiated cells, but that the majority is plastic and that in particular central memory T cells can acquire different properties and functions in secondary immune responses. In addition, there is compelling evidence that helper T cells can acquire regulatory functions upon chronic stimulation in inflamed tissues. The plasticity of antigen-experienced human T cell subsets is highly relevant for translational medicine, since it opens new perspectives for immune-modulatory therapies for chronic infections, autoimmune diseases, and cancer. PMID:25566245

  5. COMPASS identifies T-cell subsets correlated with clinical outcomes

    PubMed Central

    Lin, Lin; Finak, Greg; Ushey, Kevin; Seshadri, Chetan; Hawn, Thomas R.; Frahm, Nicole; Scriba, Thomas J.; Mahomed, Hassan; Hanekom, Willem; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; Tomaras, Georgia D.; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Michael, Nelson L.; Kim, Jerome H.; Robb, Merlin L.; O’Connell, Robert J.; Karasavvas, Nicos; Gilbert, Peter; DeRosa, Stephen; McElrath, M. Juliana

    2015-01-01

    Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells and allowed interrogation of cell population heterogeneity. Computational tools to take full advantage of these technologies are lacking. Here, we present COMPASS, a computational framework for unbiased polyfunctionality analysis of antigen-specific T-cell subsets. COMPASS uses a Bayesian hierarchical framework to model all observed functional cell subsets and select those most likely to exhibit antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, while subject-level responses are quantified by two novel summary statistics that can be correlated directly with clinical outcome, and describe the quality of an individual’s (poly)functional response. Using three clinical datasets of cytokine production we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals novel cellular correlates of protection in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software. PMID:26006008

  6. Diagnostic utility of lymphocyte subset analysis in AIDS case finding.

    PubMed Central

    Boyko, W J; Schechter, M T; MacLeod, A; Douglas, B; Maynard, M; Sharp, R; Wiggs, B

    1986-01-01

    Abnormalities of lymphocyte subsets, especially low absolute number of helper T cells, are characteristically present in acquired immune deficiency syndrome (AIDS). Similar abnormalities can be found in patients with persistent generalized lymphadenopathy (PGL) or AIDS-related complex (ARC) and, to a lesser degree, in asymptomatic people who have been exposed to human T-lymphotropic virus type III (HTLV-III). Nevertheless, there appears to be a widespread perception that lymphocyte subset analysis may be useful in AIDS case finding within high-risk groups. We evaluated the diagnostic utility of absolute number of helper T cells and ratio of helper to suppressor T cells in 33 patients with AIDS, 43 patients with PGL who had been referred for lymph node biopsy, 90 patients with PGL and 195 male homosexual controls. At conventional cutoff levels the tests did not appear to revise the probability of AIDS upward to any clinically significant degree when the pretest probability of AIDS was low. Lymphocyte subset analysis does not appear to be a cost-effective method of AIDS case finding in identified groups at risk in which the prevalence of AIDS is low. PMID:2938707

  7. Preparation of Internal Quality Control Material for Lymphocyte Subset Analysis.

    PubMed

    Roh, Eun Youn; Shin, Sue; Yoon, Jong Hyun; Oh, Sohee; Park, Kyoung Un; Lee, Nuri; Song, Eun Young

    2016-07-01

    Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20°C, -80°C, or -196°C. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80°C'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings. PMID:27139609

  8. COMPASS identifies T-cell subsets correlated with clinical outcomes.

    PubMed

    Lin, Lin; Finak, Greg; Ushey, Kevin; Seshadri, Chetan; Hawn, Thomas R; Frahm, Nicole; Scriba, Thomas J; Mahomed, Hassan; Hanekom, Willem; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; Tomaras, Georgia D; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Michael, Nelson L; Kim, Jerome H; Robb, Merlin L; O'Connell, Robert J; Karasavvas, Nicos; Gilbert, Peter; C De Rosa, Stephen; McElrath, M Juliana; Gottardo, Raphael

    2015-06-01

    Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells as well as the interrogation of cell population heterogeneity. However, in many instances, computational tools to analyze the wealth of data generated by these technologies are lacking. Here, we present a computational framework for unbiased combinatorial polyfunctionality analysis of antigen-specific T-cell subsets (COMPASS). COMPASS uses a Bayesian hierarchical framework to model all observed cell subsets and select those most likely to have antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, and human subject-level responses are quantified by two summary statistics that describe the quality of an individual's polyfunctional response and can be correlated directly with clinical outcome. Using three clinical data sets of cytokine production, we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals cellular 'correlates of protection/immunity' in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software. PMID:26006008

  9. T Helper Cell Subsets in Clinical Manifestations of Psoriasis.

    PubMed

    Diani, Marco; Altomare, Gianfranco; Reali, Eva

    2016-01-01

    Psoriasis is a chronic inflammatory skin disease, which is associated with systemic inflammation and comorbidities, such as psoriatic arthritis and cardiovascular diseases. The autoimmune nature of psoriasis has been established only recently, conferring a central role to epidermal CD8 T cells recognizing self-epitopes in the initial phase of the disease. Different subsets of helper cells have also been reported as key players in the psoriasis pathogenesis. Here, we reviewed the knowledge on the role of each subset in the psoriatic cascade and in the different clinical manifestations of the disease. We will discuss the role of Th1 and Th17 cells in the initiation and in the amplification phase of cutaneous inflammation. Moreover, we will discuss the recently proposed role of tissue resident Th22 cells in disease memory in sites of recurrent psoriasis and the possible involvement of Th9 cells. Finally, we will discuss the hypothesis of a link between T helper cell subsets recirculating from the skin and the systemic manifestations of psoriasis. PMID:27595115

  10. T Helper Cell Subsets in Clinical Manifestations of Psoriasis

    PubMed Central

    Diani, Marco; Altomare, Gianfranco

    2016-01-01

    Psoriasis is a chronic inflammatory skin disease, which is associated with systemic inflammation and comorbidities, such as psoriatic arthritis and cardiovascular diseases. The autoimmune nature of psoriasis has been established only recently, conferring a central role to epidermal CD8 T cells recognizing self-epitopes in the initial phase of the disease. Different subsets of helper cells have also been reported as key players in the psoriasis pathogenesis. Here, we reviewed the knowledge on the role of each subset in the psoriatic cascade and in the different clinical manifestations of the disease. We will discuss the role of Th1 and Th17 cells in the initiation and in the amplification phase of cutaneous inflammation. Moreover, we will discuss the recently proposed role of tissue resident Th22 cells in disease memory in sites of recurrent psoriasis and the possible involvement of Th9 cells. Finally, we will discuss the hypothesis of a link between T helper cell subsets recirculating from the skin and the systemic manifestations of psoriasis. PMID:27595115

  11. Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

    PubMed

    Franco, Francisco M; Jones, Darin E; Harris, Peter K W; Han, Zhenfu; Wildman, Scott A; Jarvis, Cassie M; Janetka, James W

    2015-05-15

    Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA. PMID:25882520

  12. Modeling the emetic potencies of food-borne trichothecenes by benchmark dose methodology.

    PubMed

    Male, Denis; Wu, Wenda; Mitchell, Nicole J; Bursian, Steven; Pestka, James J; Wu, Felicia

    2016-08-01

    Trichothecene mycotoxins commonly co-contaminate cereal products. They cause immunosuppression, anorexia, and emesis in multiple species. Dietary exposure to such toxins often occurs in mixtures. Hence, if it were possible to determine their relative toxicities and assign toxic equivalency factors (TEFs) to each trichothecene, risk management and regulation of these mycotoxins could become more comprehensive and simple. We used a mink emesis model to compare the toxicities of deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, fusarenon-X, HT-2 toxin, and T-2 toxin. These toxins were administered to mink via gavage and intraperitoneal injection. The United States Environmental Protection Agency (EPA) benchmark dose software was used to determine benchmark doses for each trichothecene. The relative potencies of each of these toxins were calculated as the ratios of their benchmark doses to that of DON. Our results showed that mink were more sensitive to orally administered toxins than to toxins administered by IP. T-2 and HT-2 toxins caused the greatest emetic responses, followed by FX, and then by DON, its acetylated derivatives, and NIV. Although these results provide key information on comparative toxicities, there is still a need for more animal based studies focusing on various endpoints and combined effects of trichothecenes before TEFs can be established. PMID:27292944

  13. A General Strategy to Enhance the Potency of Chimeric Transcriptional Activators

    NASA Astrophysics Data System (ADS)

    Natesan, Sridaran; Molinari, Elizabeth; Rivera, Victor M.; Rickles, Richard J.; Gilman, Michael

    1999-11-01

    Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.

  14. Antileukemic potency of CD19-specific T cells against chemoresistant pediatric acute lymphoblastic leukemia.

    PubMed

    Dolnikov, Alla; Shen, Sylvie; Klamer, Guy; Joshi, Swapna; Xu, Ning; Yang, Lu; Micklethwaite, Kenneth; O'Brien, Tracey A

    2015-12-01

    Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy. PMID:26384559

  15. The dynamic lives of macrophage and dendritic cell subsets in atherosclerosis

    PubMed Central

    Taghavie-Moghadam, Paresa L.; Butcher, Matthew J.; Galkina, Elena V.

    2014-01-01

    Atherosclerosis, the major pathological process through which arterial plaques are formed, is a dynamic chronic inflammatory disease of large and medium sized arteries in which the vasculature, lipid metabolism, and the immune system all play integral roles. Both the innate and adaptive immune systems are involved in the development and progression of atherosclerosis but myeloid cells represent the major component of the burgeoning atherosclerotic plaque. Various myeloid cells, including monocytes, macrophages, and dendritic cells can be found within the healthy and atherosclerotic arterial wall, where they can contribute to or regulate inflammation. However, the precise behaviors and functions of these cells in situ are still active areas of investigation that continue to yield exciting and surprising new data. Here, we review recent progress in understanding of the complex biology of macrophages and dendritic cells, focusing particularly on the dynamic regulation of these subsets in the arterial wall and novel, emerging functions of these cells during atherogenesis. PMID:24628328

  16. Emergency planning and the acute toxic potency of inhaled ammonia.

    PubMed Central

    Michaels, R A

    1999-01-01

    Ammonia is present in agriculture and commerce in many if not most communities. This report evaluates the toxic potency of ammonia, based on three types of data: anecdotal data, in some cases predating World War 1, reconstructions of contemporary industrial accidents, and animal bioassays. Standards and guidelines for human exposure have been driven largely by the anecdotal data, suggesting that ammonia at 5,000-10,000 parts per million, volume/volume (ppm-v), might be lethal within 5-10 min. However, contemporary accident reconstructions suggest that ammonia lethality requires higher concentrations. For example, 33,737 ppm-v was a 5-min zero-mortality value in a major ammonia release in 1973 in South Africa. Comparisons of secondary reports of ammonia lethality with original sources revealed discrepancies in contemporary sources, apparently resulting from failure to examine old documents or accurately translate foreign documents. The present investigation revealed that contemporary accident reconstructions yield ammonia lethality levels comparable to those in dozens of reports of animal bioassays, after adjustment of concentrations to human equivalent concentrations via U.S. Environmental Protection Agency (EPA) procedures. Ammonia levels potentially causing irreversible injury or impairing the ability of exposed people to escape from further exposure or from coincident perils similarly have been biased downwardly in contemporary sources. The EPA has identified ammonia as one of 366 extremely hazardous substances subject to community right-to-know provisions of the Superfund Act and emergency planning provisions of the Clean Air Act. The Clean Air Act defines emergency planning zones (EPZs) around industrial facilities exceeding a threshold quantity of ammonia on-site. This study suggests that EPZ areas around ammonia facilities can be reduced, thereby also reducing emergency planning costs, which will vary roughly with the EPZ radius squared. Images Figure 1

  17. Relative antidipsogenic potencies of six homologous natriuretic peptides in eels.

    PubMed

    Miyanishi, Hiroshi; Nobata, Shigenori; Takei, Yoshio

    2011-10-01

    Atrial natriuretic peptide (ANP) exhibits a potent antidipsogenic effect in seawater (SW) eels to limit excess Na(+) uptake, thereby effectively promoting SW adaptation. Recently, cardiac ANP, BNP and VNP and brain CNP1, 3 and 4, have been identified in eels. We examined the antidipsogenic effect of all homologous NPs using conscious, cannulated eels in both FW and SW together with parameters that affect drinking. A dose-response study (0.01-1 nmol/kg) in SW eels showed the relative potency of the antidipsogenic effect was in the order ANP ≥ VNP > BNP = CNP3 > CNP1 ≥ CNP4, while the order was ANP = VNP = BNP > CNP3 = CNP1 = CNP4 for the vasodepressor effect. The minimum effective dose of ANP for the antidipsogenic effect is much lower than that in mammals. ANP, BNP and VNP at 0.3 nmol/kg decreased drinking, plasma Na(+) concentration and aortic pressure and increased hematocrit in SW eels. The cardiac NPs induced similar changes in drinking, aortic pressure and hematocrit in FW eels, but aside from BNP no change in plasma Na(+) concentration. CNPs had no effect on drinking, plasma Na(+) concentration and hematocrit but induced mild hypotension in both FW and SW eels, except for CNP3 that inhibited drinking in SW eels. These results show that ANP, BNP and VNP are potent antidipsogenic hormones in eels in spite of other regulatory factors working to induce drinking, and that CNPs are without effects on drinking except for the ancestor of the cardiac NPs, CNP3. PMID:21967218

  18. Pyridalyl, a novel insecticide: potency and insecticidal selectivity.

    PubMed

    Isayama, S; Saito, S; Kuroda, K; Umeda, K; Kasamatsu, K

    2005-04-01

    Pyridalyl is an insecticide of a novel chemical class (unclassified insecticides). Toxicity of pyridalyl to two insect pest species, Spodoptera litura and Frankliniella occidentalis, an insect predator, Orius stringicollis, and a pollinator, Bombus terrestris, was evaluated in the laboratory. The insecticidal activity of pyridalyl against S. litura was evaluated using the leaf-dipping method. The potency of pyridalyl was highly effective against all development stages (2nd to 6th instar larvae) of S. litura. This compound was also evaluated against F. occidentalis using the direct spray method. For F. occidentalis, toxicity of pyridalyl was almost similar to that of acrinathrin, but greater than acrinathrin for adults. Then the toxicity of this product to the natural enemies, Orius stringicollis and the pollinating insect Bombus terrestris, was evaluated using the body-dipping method or direct spray method. No acute toxicity of this product was observed on these non-target insects. Moreover, the influence of pyridalyl to the nest of Bombus terrestris was evaluated using the direct spray to the inside of the nest. No apparent influence of this compound was observed by 21 days after treatment. The cytotoxicity of pyridalyl to the Sf9 insect cell line and the CHO-K1 mammalian cell line was evaluated using the trypan-blue exclusion method. High toxicity to the insect cell line, but almost no toxicity to the mammalian cell line, was observed. Thus, pyridalyl exhibited high selectivity in cytotoxicity between the insect and mammalian cell line as well as in insecticidal activity among insect species. We infer pyridalyl may be useful for IPM programs of greenhouse cultivation system. PMID:15756699

  19. Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

    PubMed Central

    Green, David W.; Kwon, Hyuk-Jae; Jung, Han-Sung

    2015-01-01

    Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s. PMID:25666352

  20. Persistent Pain Model Reveals Sex Difference in Morphine Potency

    PubMed Central

    Wang, Xiaoya; Traub, Richard J.; Murphy, Anne Z.

    2010-01-01

    Central or systemic administration of agonists directed at the mu or delta opiate receptors generally produce a greater degree of analgesia in males than in females. To date, the majority of studies examining sex based differences in opioid analgesia have employed acute noxious stimuli (i.e. tail-flick and hot plate test); thus, the potential dimorphic response of centrally acting opiates in the alleviation of persistent inflammatory pain is not well established. In the present study, right hindpaw withdrawal latency (PWL) to radiant thermal stimuli was measured in intact male and cycling female Sprague-Dawley rats before and after unilateral hindpaw injection of the inflammatory agent complete Freund’s adjuvant (CFA). Control animals received intraplantar injection of saline. Twenty four hours after CFA or saline injection, animals received either saline or morphine bisulfate (0.5 – 15 mg/kg; s.c.). Separate groups of control or inflamed animals were tested on their responsiveness to morphine at 7, 14 and 21 days post-CFA or saline. No sex differences were noted for baseline PWLs, and females displayed slightly less thermal hyperalgesia at 24 hrs post-CFA. At all morphine doses administered, both the antihyperalgesic effects of morphine in the inflamed animals, and the antinociceptive effects of morphine in control animals, were significantly greater in males in comparison to females. Similarly, in males, the antihyperalgesic effects of morphine increased significantly at 7–21 days post-CFA; no significant shift in morphine potency was noted for females. These studies demonstrate sex-based differences in the effects of morphine on thermal hyperalgesia in a model of persistent inflammatory pain. PMID:16497818

  1. A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets

    PubMed Central

    Gren, Susanne T.; Rasmussen, Thomas B.; Janciauskiene, Sabina; Håkansson, Katarina; Gerwien, Jens G.; Grip, Olof

    2015-01-01

    Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies. PMID:26650546

  2. The Incidence of Kidney Injury for Patients Treated With a High‐Potency Versus Moderate‐Potency Statin Regimen After an Acute Coronary Syndrome

    PubMed Central

    Sarma, Amy; Cannon, Christopher P.; de Lemos, James; Rouleau, Jean L.; Lewis, Eldrin F.; Guo, Jianping; Mega, Jessica L.; Sabatine, Marc S.; O'Donoghue, Michelle L.

    2014-01-01

    Background Observational studies have raised concerns that high‐potency statins increase the risk of acute kidney injury. We therefore examined the incidence of kidney injury across 2 randomized trials of statin therapy. Methods and Results PROVE IT‐TIMI 22 enrolled 4162 subjects after an acute coronary syndrome (ACS) and randomized them to atorvastatin 80 mg/day versus pravastatin 40 mg/day. A‐to‐Z enrolled 4497 subjects after ACS and randomized them to a high‐potency (simvastatin 40 mg/day×1 months, then simvastatin 80 mg/day) versus a delayed moderate‐potency statin strategy (placebo×4 months, then simvastatin 20 mg/day). Serum creatinine was assessed centrally at serial time points. Adverse events (AEs) relating to kidney injury were identified through database review. Across both trials, mean serum creatinine was similar between treatment arms at baseline and throughout follow‐up. In A‐to‐Z, the incidence of a 1.5‐fold or ≥0.3 mg/dL rise in serum creatinine was 11.4% for subjects randomized to a high‐potency statin regimen versus 12.4% for those on a delayed moderate‐potency regimen (odds ratio [OR], 0.91; 95% confidence interval [CI], 0.76 to 1.10; P=0.33). In PROVE IT‐TIMI 22, the incidence was 9.4% for subjects randomized to atorvastatin 80 mg/day and 10.6% for subjects randomized to pravastatin 40 mg/day (OR, 0.88; 95% CI, 0.71 to 1.09; P=0.25). Consistent results were observed for different kidney injury thresholds and in individuals with diabetes mellitus or with moderate renal dysfunction. The incidence of kidney injury‐related adverse events (AEs) was not statistically different for patients on a high‐potency versus moderate‐potency statin regimen (OR, 1.06; 95% CI, 0.68 to 1.67; P=0.78). Conclusions For patients enrolled in 2 large randomized trials of statin therapy after ACS, the use of a high‐potency statin regimen did not increase the risk of kidney injury. PMID:24786143

  3. Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

    SciTech Connect

    Warburton, P.E.; Gosden, J.; Lawson, D.

    1996-04-15

    Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.

  4. THE VALENCE AND METHYLATION STATE OF ARSENIC DETERMINES ITS POTENCY IN INTERACTION WITH THE MITOTIC APPARATUS

    EPA Science Inventory

    We have previously shown that the cytotoxic and genotoxic potency of arsenicals is dependent upon their valence and methylation state. Trivalent methylated arsenicals are much more potent DNA damaging agents than are their inorganic and pentavalent counterparts. Furthermore, thei...

  5. Quantitative structure - mesothelioma Potency Model Optimization for Complex Mixtures of Elongated Particles in Rat Pleura

    EPA Science Inventory

    Cancer potencies of mineral and synthetic elongated particle (EP) mixtures, including fibers from asbestos, are influenced by changes in fiber dose composition, bioavailability and biodurability in combination with relevant cytotoxic dose-response relationships. A unique and com...

  6. Comptational comparison of asbestos fibers: Dosimetry model simulations to characterize variabilty and potency (Presentation poster)

    EPA Science Inventory

    Inhaled asbestos fibers result in respiratory diseases such as asbestosis, lung cancer and mesothelioma, but different asbestos fibers exhibit different potency. We applied a recently developed dosimetry model (Asgharian et al., Poster # 104) that describes th...

  7. Toxico-Cheminformatics and QSPR Modeling of the Carcinogenic Potency Database

    EPA Science Inventory

    Report on the development of a tiered, confirmatory scheme for prediction of chemical carcinogenicity based on QSAR studies of compounds with available mutagenic and carcinogenic data. For 693 such compounds from the Carcinogenic Potency Database characterized molecular topologic...

  8. A compilation of ab-initio calculations of embrittling potencies in binary metallic alloys.

    PubMed

    Gibson, Michael A; Schuh, Christopher A

    2016-03-01

    Segregation-induced changes in interfacial cohesion often control the mechanical properties of metals. The change in the work of separation of an interface upon segregation of a solute to the interface, termed the embrittling potency, is an atomic-level quantity used to predict and understand embrittlement phenomena. We present a compilation of calculations of embrittling potencies, along with references for these calculations. A discussion of this data is made in a separate article (Gibson and Schuh, 2016 [1]). PMID:26858979

  9. A compilation of ab-initio calculations of embrittling potencies in binary metallic alloys

    PubMed Central

    Gibson, Michael A.; Schuh, Christopher A.

    2015-01-01

    Segregation-induced changes in interfacial cohesion often control the mechanical properties of metals. The change in the work of separation of an interface upon segregation of a solute to the interface, termed the embrittling potency, is an atomic-level quantity used to predict and understand embrittlement phenomena. We present a compilation of calculations of embrittling potencies, along with references for these calculations. A discussion of this data is made in a separate article (Gibson and Schuh, 2016 [1]). PMID:26858979

  10. Potency of Massoia Bark in Combating Immunosuppressed-related Infection

    PubMed Central

    Hertiani, Triana; Pratiwi, Sylvia Utami Tunjung; Yuswanto, Agustinus; Permanasari, Prisci

    2016-01-01

    Background: As part of our search for new potential natural resources to eradicate infection, we have revealed the prominent potency of massoia bark (Massoia aromatica Becc, Lauraceae) in combating immunosuppressed-related infection. Materials and Methods: The extract was prepared by macerating the pulverized dried bark in ethanol 95%, followed by solvent evaporation. The oil was extracted from the dried bark by steam-hydrodistillation of which preparative thin-layer chromatography was performed on the oil to isolate the active constituent, C-10 massoia lactone (ML). Anti-biofilm assay against Candida albicans was conducted on polystyrene 96 wells microtiter plates, followed by a confocal laser scanning microscope observation to get three-dimensional profiles of the affected biofilms. Effects on the hyphae development inoculated on RPMI-1640 agar plates were observed for 7 days. Influences of samples on mice macrophage phagocytosis were examined by an in vitro technique. Samples concentration tested were in the range of 2.0–0.0625 mg/mL and done in triplicate. Results: Massoia bark extracts (oil and solid phase) and ML exhibited promising activities as anti-biofilm against C. albicans at IC50 0.074% v/v, 271 μg/mL and 0.026 μg/mL, respectively. The ML did not inhibit the hyphae development at the concentration tested; however, the extracts showed inhibition at 62.5 μg/mL. Macrophage phagocytosis stimulation was correlated to the ML content. Conclusion: Massoia bark is potential to be developed as anti-infective in immunosuppressed condition of which the C10 ML (C10H16O2) plays a major role in exerting activity. SUMMARY Massoia bark extracts (oily and solid phase) and C-10 Massoia lactone exhibited promising activities as antibiofilm against Candida albicans at IC50 are 0.074 %v/v, 271 μg/mL and 0.026 μg/mL respectively. The major constituent, C-10 Massoia lactone (C10H16O2) plays major role in exerting anticandida activity and potentially acts as an

  11. The octave potencies convention: a mathematical model of dilution and succussion.

    PubMed

    Anick, David J

    2007-07-01

    Several hypothesized explanations for homeopathy posit that remedies contain a concentration of discrete information-carrying units, such as water clusters, nano-bubbles, or silicates. For any such explanation to be sustainable, dilution must reduce and succussion must restore the concentration of these units. Succussion can be modeled by a logistic equation, which leads to mathematical relationships involving the maximum concentration, the average growth of information-carrying units rate per succussion stroke, the number of succussion strokes, and the dilution factor (x, c, or LM). When multiple species of information-carrying units are present, the fastest-growing species will eventually come to dominate, as the potency is increased. An analogy is explored between iterated cycles dilution and succussion, in making homeopathic remedies, and iterated cycles of reseeding and growth, in bacterial cultures. Drawing on this analogy, the active ingredients in low and medium potency remedies may be present at early dilutions but only gradually come to 'dominate', while high potencies may develop from the occurrence of low-probability but faster-growing 'mutations.' Conclusions from this model include: 'x' and 'c' potencies are best compared by the amount of dilution, not the amount of succussion; the minimum number of succussion strokes needed per cycle is proportional to the logarithm of the dilution factor; and a plausible interpretation of why potencies at approximately regular ratios are traditionally used (the octave potencies convention). PMID:17678818

  12. Kinetic microplate bioassays for relative potency of antibiotics improved by partial Least Square (PLS) regression.

    PubMed

    Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Almeida, Túlia de Souza Botelho; Lourenço, Felipe Rebello

    2016-05-01

    Microbiological assays are widely used to estimate the relative potencies of antibiotics in order to guarantee the efficacy, safety, and quality of drug products. Despite of the advantages of turbidimetric bioassays when compared to other methods, it has limitations concerning the linearity and range of the dose-response curve determination. Here, we proposed to use partial least squares (PLS) regression to solve these limitations and to improve the prediction of relative potencies of antibiotics. Kinetic-reading microplate turbidimetric bioassays for apramacyin and vancomycin were performed using Escherichia coli (ATCC 8739) and Bacillus subtilis (ATCC 6633), respectively. Microbial growths were measured as absorbance up to 180 and 300min for apramycin and vancomycin turbidimetric bioassays, respectively. Conventional dose-response curves (absorbances or area under the microbial growth curve vs. log of antibiotic concentration) showed significant regression, however there were significant deviation of linearity. Thus, they could not be used for relative potency estimations. PLS regression allowed us to construct a predictive model for estimating the relative potencies of apramycin and vancomycin without over-fitting and it improved the linear range of turbidimetric bioassay. In addition, PLS regression provided predictions of relative potencies equivalent to those obtained from agar diffusion official methods. Therefore, we conclude that PLS regression may be used to estimate the relative potencies of antibiotics with significant advantages when compared to conventional dose-response curve determination. PMID:26971814

  13. Cytokine profile and lymphocyte subsets in type 2 diabetes

    PubMed Central

    Francisco, C.O.; Catai, A.M.; Moura-Tonello, S.C.G.; Arruda, L.C.M.; Lopes, S.L.B.; Benze, B.G.; Del Vale, A.M.; Malmegrim, K.C.R.; Leal, A.M.O.

    2016-01-01

    Type 2 diabetes mellitus (T2D) is a metabolic disease with inflammation as an important pathogenic background. However, the pattern of immune cell subsets and the cytokine profile associated with development of T2D are unclear. The objective of this study was to evaluate different components of the immune system in T2D patients' peripheral blood by quantifying the frequency of lymphocyte subsets and intracellular pro- and anti-inflammatory cytokine production by T cells. Clinical data and blood samples were collected from 22 men (51.6±6.3 years old) with T2D and 20 nonsmoking men (49.4±7.6 years old) who were matched for age and sex as control subjects. Glycated hemoglobin, high-sensitivity C-reactive protein concentrations, and the lipid profile were measured by a commercially available automated system. Frequencies of lymphocyte subsets in peripheral blood and intracellular production of interleukin (IL)-4, IL-10, IL-17, tumor necrosis factor-α, and interferon-γ cytokines by CD3+ T cells were assessed by flow cytometry. No differences were observed in the frequency of CD19+ B cells, CD3+CD8+ and CD3+CD4+ T cells, CD16+56+ NK cells, and CD4+CD25+Foxp3+ T regulatory cells in patients with T2D compared with controls. The numbers of IL-10- and IL-17-producing CD3+ T cells were significantly higher in patients with T2D than in controls (P<0.05). The frequency of interferon-γ-producing CD3+ T cells was positively correlated with body mass index (r=0.59; P=0.01). In conclusion, this study shows increased numbers of circulating IL-10- and IL-17-producing CD3+ T cells in patients with T2D, suggesting that these cytokines are involved in the immune pathology of this disease. PMID:27007651

  14. DBA-Lectin Reactivity Defines Mouse Uterine Natural Killer Cell Subsets with Biased Gene Expression 1

    PubMed Central

    Chen, Zhilin; Zhang, Jianhong; Hatta, Kota; Lima, Patricia D. A.; Yadi, Hakim; Colucci, Francesco; Yamada, Aureo T.; Croy, B. Anne

    2012-01-01

    Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of Natural Killer (NK) cells. Mouse uterine (u)NK cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity and by histochemical reactivity with Periodic Acid Schiff’s (PAS) reagent, with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA− and DBA+ mouse uNK cells were equivalent using quantitative (q)RT-PCR analyses of flow separated, midpregnancy (gestation day (gd)10) cells and using immunohistochemistry. CD3E (CD3)-IL2RB (CD122)+DBA− cells were identified as the dominant Ifng transcript source. Skewed IFNG production by uNK cell subsets was confirmed by analysis of uNK cells from eYFP-tagged IFNG-reporter mice. In contrast, CD3E-IL2RB+DBA+ uNK cells expressed genes compatible with significantly greater potential for IL22 synthesis, angiogenesis and participation in regulation mediated by the renin-angiotensin system (RAS). CD3E-IL2RB+DBA+ cells were further divided into VEGFA+ and VEGFA− subsets. CD3E-IL2RB+DBA+ uNK cells but not CD3E-IL2RB+DBA− uNK cells arose from circulating, bone marrow-derived progenitor cells by gd6. These findings indicate the heterogeneous nature of mouse uNK cells and suggest that studies using only DBA + uNK cells will give biased data that does not fully represent the uNK cell population. PMID:22875907

  15. Efficient feature subset selection with probabilistic distance criteria. [pattern recognition

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    Recursive expressions are derived for efficiently computing the commonly used probabilistic distance measures as a change in the criteria both when a feature is added to and when a feature is deleted from the current feature subset. A combinatorial algorithm for generating all possible r feature combinations from a given set of s features in (s/r) steps with a change of a single feature at each step is presented. These expressions can also be used for both forward and backward sequential feature selection.

  16. Feature Subset Selection by Estimation of Distribution Algorithms

    SciTech Connect

    Cantu-Paz, E

    2002-01-17

    This paper describes the application of four evolutionary algorithms to the identification of feature subsets for classification problems. Besides a simple GA, the paper considers three estimation of distribution algorithms (EDAs): a compact GA, an extended compact GA, and the Bayesian Optimization Algorithm. The objective is to determine if the EDAs present advantages over the simple GA in terms of accuracy or speed in this problem. The experiments used a Naive Bayes classifier and public-domain and artificial data sets. In contrast with previous studies, we did not find evidence to support or reject the use of EDAs for this problem.

  17. ECETOC Florence workshop on risk assessment of endocrine substances, including the potency concept.

    PubMed

    Fegert, Ivana

    2013-12-16

    The European regulation on plant protection products (1107/2009) and the Biocidal Products Regulation (EC Regulation 528/2012) only support the marketing and use of chemicals if they do not cause endocrine disruption in humans or wildlife species. Also, substances with endocrine properties are subject to authorization under the European regulation on the registration, evaluation, authorization and restriction of chemicals (REACH; 1907/2006). Therefore, the regulatory consequences of identifying a substance as an endocrine disrupting chemical are severe. In contrast to that, basic scientific criteria, necessary to define endocrine disrupting properties, are not described in any of these legislative documents. Thus, the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) established a task force to provide scientific criteria for the identification and assessment of chemicals with endocrine disrupting properties that may be used within the context of these three legislative texts (ECETOC, 2009a). In 2009, ECETOC introduced a scientific framework as a possible concept for identifying endocrine disrupting properties within a regulatory context (ECETOC, 2009b; Bars et al., 2011a,b). The proposed scientific criteria integrated, in a weight of evidence approach, information from regulatory (eco)toxicity studies and mechanistic/screening studies by combining evidence for adverse effects detected in apical whole-organism studies with an understanding of the mode of action (MoA) of endocrine toxicity. However, since not all chemicals with endocrine disrupting properties are of equal hazard, an adequate concept should also be able to differentiate between chemicals with endocrine properties of low concern from those of higher concern (for regulatory purposes). For this purpose, the task force refined this part of their concept. Following an investigation of the key factors at a second workshop of invited regulatory, academic and industry scientists, the

  18. Estimation of Discriminative Feature Subset Using Community Modularity

    NASA Astrophysics Data System (ADS)

    Zhao, Guodong; Liu, Sanming

    2016-04-01

    Feature selection (FS) is an important preprocessing step in machine learning and data mining. In this paper, a new feature subset evaluation method is proposed by constructing a sample graph (SG) in different k-features and applying community modularity to select highly informative features as a group. However, these features may not be relevant as an individual. Furthermore, relevant in-dependency rather than irrelevant redundancy among the selected features is effectively measured with the community modularity Q value of the sample graph in the k-features. An efficient FS method called k-features sample graph feature selection is presented. A key property of this approach is that the discriminative cues of a feature subset with the maximum relevant in-dependency among features can be accurately determined. This community modularity-based method is then verified with the theory of k-means cluster. Compared with other state-of-the-art methods, the proposed approach is more effective, as verified by the results of several experiments.

  19. Estimation of Discriminative Feature Subset Using Community Modularity

    PubMed Central

    Zhao, Guodong; Liu, Sanming

    2016-01-01

    Feature selection (FS) is an important preprocessing step in machine learning and data mining. In this paper, a new feature subset evaluation method is proposed by constructing a sample graph (SG) in different k-features and applying community modularity to select highly informative features as a group. However, these features may not be relevant as an individual. Furthermore, relevant in-dependency rather than irrelevant redundancy among the selected features is effectively measured with the community modularity Q value of the sample graph in the k-features. An efficient FS method called k-features sample graph feature selection is presented. A key property of this approach is that the discriminative cues of a feature subset with the maximum relevant in-dependency among features can be accurately determined. This community modularity-based method is then verified with the theory of k-means cluster. Compared with other state-of-the-art methods, the proposed approach is more effective, as verified by the results of several experiments. PMID:27121171

  20. Database of Ligand-Receptor Partners, a DIP subset

    DOE Data Explorer

    Graeber, Thomas G.; Eisenberg, David

    The Database of Ligand-Receptor Partners (DLRP) is a subset of DIP (Database of Interacting Proteins). The DLRP is a database of protein ligand and protein receptor pairs that are known to interact with each other. By interact we mean that the ligand and receptor are members of a ligand-receptor complex and, unless otherwise noted, transduce a signal. In some instances the ligand and/or receptor may form a heterocomplex with other ligands/receptors in order to be functional. We have entered the majority of interactions in DLRP as full DIP entries, with links to references and additional information (see the DIP User's Guide). DLRP is a web supplement for: Thomas G. Graeber and David Eisenberg. Bioinformatic identification of potential autocrine signaling loops in cancers from gene expression profiles. Nature Genetics, 29(3):295-300 (November 2001). [Quoted from the DLRP homepage at http://dip.doe-mbi.ucla.edu/dip/DLRP.cgi] Also available from this page is the DLRP chemokine subset.

  1. Estimation of Discriminative Feature Subset Using Community Modularity.

    PubMed

    Zhao, Guodong; Liu, Sanming

    2016-01-01

    Feature selection (FS) is an important preprocessing step in machine learning and data mining. In this paper, a new feature subset evaluation method is proposed by constructing a sample graph (SG) in different k-features and applying community modularity to select highly informative features as a group. However, these features may not be relevant as an individual. Furthermore, relevant in-dependency rather than irrelevant redundancy among the selected features is effectively measured with the community modularity Q value of the sample graph in the k-features. An efficient FS method called k-features sample graph feature selection is presented. A key property of this approach is that the discriminative cues of a feature subset with the maximum relevant in-dependency among features can be accurately determined. This community modularity-based method is then verified with the theory of k-means cluster. Compared with other state-of-the-art methods, the proposed approach is more effective, as verified by the results of several experiments. PMID:27121171

  2. Bovine γδ T Cells Are a Major Regulatory T Cell Subset

    PubMed Central

    Hope, Jayne; Taylor, Geraldine; Smith, Adrian L.; Cubillos-Zapata, Carolina; Charleston, Bryan

    2014-01-01

    In humans and mice, γδ T cells represent <5% of the total circulating lymphocytes. In contrast, the γδ T cell compartment in ruminants accounts for 15–60% of the total circulating mononuclear lymphocytes. Despite the existence of CD4+CD25high Foxp3+ T cells in the bovine system, these are neither anergic nor suppressive. We present evidence showing that bovine γδ T cells are the major regulatory T cell subset in peripheral blood. These γδ T cells spontaneously secrete IL-10 and proliferate in response to IL-10, TGF-β, and contact with APCs. IL-10–expressing γδ T cells inhibit Ag-specific and nonspecific proliferation of CD4+ and CD8+ T cells in vitro. APC subsets expressing IL-10 and TFG-β regulate proliferation of γδ T cells producing IL-10. We propose that γδ T cells are a major regulatory T cell population in the bovine system. PMID:24890724

  3. Different subsets of natural killer T cells may vary in their roles in health and disease

    PubMed Central

    Kumar, Vipin; Delovitch, Terry L

    2014-01-01

    Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class-I-like molecule. Most type I NKT cells express a semi-invariant T-cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR-α dominates CD1d-lipid recognition by type I NKT cells, TCR-α and TCR-β contribute equally to CD1d-lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine-dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen-presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes. PMID:24428389

  4. Effects of human immunodeficiency virus type 1 on CD4 lymphocyte subset activation.

    PubMed

    Masci, A M; Paz, F L; Borriello, A; Cassano, S; Della Pietra, V; Stoiber, H; Matarese, G; Della Ragione, F; Zappacosta, S; Racioppi, L

    1999-06-01

    The pathogenesis of the decline of CD4 lymphocyte counts accompanying the typical course of HIV-1 infection is not completely defined and might be related to a differential susceptibility of naive and memory cells to HIV-1 exposure. Here, we examined the effects induced by heat-inactivated HIV-1 virions on these lymphocyte populations. Exposure of CD45RA naive T cells to inactivated viral particles induced a marked decrease of both mitogenic responses and activation-induced apoptosis. Conversely, the growth of CD45RO cells was less severely restrained. Analysis of intracellular levels of cell cycle regulatory proteins revealed an arrest at the G1/S restriction point of the naive but not memory subset. This effect was associated with alterations in phosphotyrosine profile and with a marked decrease of ERK and NJK kinase activation. Finally, up-regulation of the cAMP-dependent protein kinase A (PKA) activity induced by mitogens was not affected by virus. Altogether, these findings show that interaction of HIV-1 with the T cell surface is sufficient to inhibit the proliferative response of the CD4CD45RA subset by disturbing proximal TCR signaling. This mechanism would affect renewal of naive lymphocytes, contributing in such a way to the impairment of T cell turnover during the course of HIV-1 infection. PMID:10382750

  5. Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs

    PubMed Central

    Kim, Kee K.; Yang, Yanqin; Zhu, Jun; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-01-01

    RNA-binding proteins (RBPs) regulate numerous aspects of gene expression, thus identification of endogenous targets of RBPs is important for understanding their functions in cells. Here we identified transcriptome-wide targets of Rbfox3 in neuronally differentiated P19 cells and mouse brain using Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP). Although Rbfox3 is known to regulate pre-mRNA splicing through binding to the UGCAUG motif, PAR-CLIP analysis revealed diverse Rbfox3 targets including primary-microRNAs (pri-miRNAs) which lack the UGCAUG motif. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs. In vitro analyses revealed that Rbfox3 functions as a positive and a negative regulator at the stage of pri-miRNA processing to precursor-miRNA. Rbfox3 binds directly to pri-miRNAs and regulates the recruitment of the microprocessor complex to pri-miRNAs. Our study proposes a novel function for Rbfox3 in miRNA biogenesis. PMID:25240799

  6. Detergents profoundly affect inhibitor potencies against both cyclo-oxygenase isoforms.

    PubMed

    Ouellet, Marc; Falgueyret, Jean-Pierre; Percival, M David

    2004-02-01

    The sensitivity of Coxs (cyclo-oxygenases) to inhibition is known to be highly dependent on assay conditions. In the present study, the inhibitor sensitivities of purified Cox-1 and -2 were determined in a colorimetric assay using the reducing agent N, N, N ', N '-tetramethyl- p -phenylenediamine. With the detergent genapol X-100 (2 mM) present, the potencies of nimesulide, ibuprofen, flufenamic acid, niflumic acid and naproxen were increased over 100-fold against Cox-2 and titration curve shapes changed, so that maximal inhibition now approached 100%. Indomethacin, diclofenac and flosulide were not changed in potency. Similar effects of genapol were observed with inhibitors of Cox-1. DuP-697 and two analogues became more than 10-fold less potent against Cox-2 with genapol present. Tween-20, Triton X-100 and phosphatidylcholine, but not octylglucoside, gave qualitatively similar effects as genapol. Similar detergent-dependent changes in inhibitor potency were also observed using a [(14)C]arachidonic acid HPLC assay. The increases in potency of ibuprofen, flufenamic acid, isoxicam and niflumic acid towards Cox-2 and ibuprofen towards Cox-1 were accompanied by a change from time-independent to time-dependent inhibition. The interactions of Cox inhibitors has been described in terms of multiple binding step mechanisms. The genapol-dependent increase in inhibitor potency for ketoprofen was associated with an increase in the rate constant for the conversion of the initial enzyme-inhibitor complex to a second, more tightly bound form. The loss of potency for some inhibitors is probably due to inhibitor partitioning into detergent micelles. The present study identifies detergents as another factor that must be considered when determining inhibitor potencies against both Cox isoforms. PMID:14510637

  7. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants.

    PubMed

    Hamers, Timo; Kamstra, Jorke H; Sonneveld, Edwin; Murk, Albertinka J; Kester, Monique H A; Andersson, Patrik L; Legler, Juliette; Brouwer, Abraham

    2006-07-01

    Over the last few years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine-disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action of BFRs in man and wildlife and (2) to classify BFRs with similar profiles of ED potencies. A test set of 27 individual BFRs were selected, consisting of 19 polybrominated diphenyl ether congeners, tetrabromobisphenol-A, hexabromocyclododecane, 2,4,6-tribromophenol, ortho-hydroxylated brominated diphenyl ether 47, and tetrabromobisphenol-A-bis(2,3)dibromopropyl ether. All BFRs were tested for their potency to interact with the arylhydrocarbon receptor, androgen receptor (AR), progesterone receptor (PR), and estrogen receptor. In addition, all BFRs were tested for their potency to inhibit estradiol (sulfation by estradiol sulfotransferase (E2SULT), to interfere with thyroid hormone 3,3',5-triiodothyronine (T3)-mediated cell proliferation, and to compete with T3-precursor thyroxine for binding to the plasma transport protein transthyretin (TTR). The results of the in vitro screening indicated that BFRs have ED potencies, some of which had not or only marginally been described before (AR antagonism, PR antagonism, E2SULT inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR antagonism, E2SULT inhibition, and TTR competition was higher than for natural ligands or clinical drugs used as positive controls. Based on their similarity in ED profiles, BFRs were classified into five different clusters. These findings support further investigation of the potential ED effects of these environmentally relevant BFRs in man and wildlife. PMID:16601080

  8. Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals

    SciTech Connect

    Venkatapathy, Raghuraman Wang Chingyi; Bruce, Robert Mark; Moudgal, Chandrika

    2009-01-15

    Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD{sub 50}]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD{sub 50}) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD{sub 50} and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD{sub 50}s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD{sub 50} for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.

  9. Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry.

    PubMed

    Bravery, Christopher A; Carmen, Jessica; Fong, Timothy; Oprea, Wanda; Hoogendoorn, Karin H; Woda, Juliana; Burger, Scott R; Rowley, Jon A; Bonyhadi, Mark L; Van't Hof, Wouter

    2013-01-01

    The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development. PMID:23260082

  10. New and Improved Version of the ASDC MOPITT Search and Subset Web Application

    Atmospheric Science Data Center

    2016-07-06

    ... and Improved Version of the ASDC MOPITT Search and Subset Web Application Friday, June 24, 2016 A new and improved version of the ASDC MOPITT Search and Subset Web Application has been released. New features include: Versions 5 and 6 ...

  11. Cytokine Networks and T-Cell Subsets in Inflammatory Bowel Diseases.

    PubMed

    Chen, Mei Lan; Sundrud, Mark S

    2016-05-01

    Pathogenesis of the inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC) and Crohn's disease (CD), involve proinflammatory changes within the microbiota, chronic immune-mediated inflammatory responses, and epithelial dysfunction. Converging data from genome-wide association studies, mouse models of IBD, and clinical trials indicate that cytokines are key effectors of both normal homeostasis and chronic inflammation in the gut. Yet many questions remain concerning the role of specific cytokines in different IBDs within distinct regions of the gut, and regarding cellular mechanisms of action. In this article, we review current and emerging concepts concerning the role of cytokines in IBD with a focus on immune regulation, T cell subsets, and potential clinical applications. PMID:26863267

  12. Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun.

    PubMed

    Kim, Daejin; Hoory, Talia; Monie, Archana; Ting, Jenny Pan-Yun; Hung, Chien-Fu; Wu, T-C

    2008-05-15

    Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting. PMID:18453624

  13. Efficient Simulation Budget Allocation for Selecting an Optimal Subset

    NASA Technical Reports Server (NTRS)

    Chen, Chun-Hung; He, Donghai; Fu, Michael; Lee, Loo Hay

    2008-01-01

    We consider a class of the subset selection problem in ranking and selection. The objective is to identify the top m out of k designs based on simulated output. Traditional procedures are conservative and inefficient. Using the optimal computing budget allocation framework, we formulate the problem as that of maximizing the probability of correc tly selecting all of the top-m designs subject to a constraint on the total number of samples available. For an approximation of this corre ct selection probability, we derive an asymptotically optimal allocat ion and propose an easy-to-implement heuristic sequential allocation procedure. Numerical experiments indicate that the resulting allocatio ns are superior to other methods in the literature that we tested, and the relative efficiency increases for larger problems. In addition, preliminary numerical results indicate that the proposed new procedur e has the potential to enhance computational efficiency for simulation optimization.

  14. Codes with Parity Conditions on Subsets of Coordinates

    NASA Technical Reports Server (NTRS)

    Posner, E. C.; Reichstein, Z.

    1985-01-01

    Binary codes with the constraint that the codes restricted to certain subsets of columns must be contained in particular codes of the shorter lengths are considered. In particular, codes of even length 2k, and of minimum distance approximately greater than d, where in the code obtained by restricting to the first k positions has even weight and at the same time the code obtained by restricting to the last k positions also has even weight are considered. If k = 2n, n odd, and d = 2n, it is proved that the code has at most 8n - 4 codewords, and 8n - 4 is attainable for n = 3. This permits a file-transfer protocol control function assignment for personal computers to be chosen for 20 control functions using essentially just pairs of upper-case alphabetic ASCII characters where the Hamming distance between the binary forms of every two different control functions is at least six.

  15. Tachykinins stimulate a subset of mouse taste cells.

    PubMed

    Grant, Jeff

    2012-01-01

    The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods. PMID:22363709

  16. Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

    PubMed

    Fine, N; Hassanpour, S; Borenstein, A; Sima, C; Oveisi, M; Scholey, J; Cherney, D; Glogauer, M

    2016-07-01

    Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state. PMID:27270666

  17. Causes of endocrine disrupting potencies in surface water in East China.

    PubMed

    Shi, Wei; Deng, Dongyang; Wang, Yuting; Hu, Guanjiu; Guo, Jing; Zhang, Xiaowei; Wang, Xinru; Giesy, John P; Yu, Hongxia; Wang, Ziheng

    2016-02-01

    Surface water is essential for human health and ecological diversity, but some endocrine disrupting chemicals are detectable. Both thyroid receptor (TR) and androgen receptor (AR) agonistic/antagonistic potencies in grade II surface water in East China were investigated using reporter gene assays. While none of the water exhibited agonistic potency, significant AR and TR antagonistic potencies were detectable. TR antagonistic equivalents (TR-AntEQ) and AR antagonistic equivalents (AR-AntEQ) ranged from 3.6 to 76.1 μg dibutyl phthalate/L and from 2.3 to 242.6 μg flutamide/L, respectively. The TR and AR antagonistic potencies in the Yangtze River watershed were highlighted, with equivalents greater than the lowest observable effect concentration (LOEC) of dibutyl phthalate and flutamide, respectively. Phthalate esters (PAEs) being the most abundant explained most of the TR antagonistic potency, contributing more than 65% of the TR-AntEQ and diisobutyl phthalate (DiBP) was the major contributor. In most surface waters studied, PAEs contributed little of the AR-AntEQ, but the frequently detected octylphenol, nonylphenol and benzo[a]pyrene might be responsible. PMID:26495828

  18. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity.

    PubMed

    Teunis, Marc A T; Spiekstra, Sander W; Smits, Mieke; Adriaens, Els; Eltze, Tobias; Galbiati, Valentina; Krul, Cyrille; Landsiedel, Robert; Pieters, Raymond; Reinders, Judith; Roggen, Erwin; Corsini, Emanuela; Gibbs, Susan

    2014-01-01

    This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential. PMID:24535238

  19. Relative potencies of aroclor mixtures derived from avian in vitro bioassays: comparisons with calculated toxic equivalents.

    PubMed

    Zhang, Rui; Manning, Gillian E; Farmahin, Reza; Crump, Doug; Zhang, Xiaowei; Kennedy, Sean W

    2013-08-01

    The World Health Organization toxic equivalency factors (WHO-TEFs) for birds were developed to simplify risk assessments of environmental mixtures of dioxin-like compounds (DLCs). Under this framework, toxic equivalents (TEQs) are used to represent the toxic potency of DLC mixtures as an equivalent concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Recently, a luciferase reporter gene (LRG) assay, measuring aryl hydrocarbon receptor 1 (AHR1)-mediated gene expression, accurately predicted the relative potency of individual polychlorinated biphenyl (PCB) congeners in different avian species. The study presented here used the LRG assay to predict the relative potency of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 on induction of LRG activity in cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 constructs. LRG assay results were compared to (1) results of ethoxyresorufin-O-deethylase (EROD) assays conducted in chicken hepatocytes and (2) calculated TEQs from the literature. The relative potencies of Aroclors were similar between the LRG and EROD assays, and bioassay-derived TEQs for the chicken closely resembled calculated TEQs. However, LRG assay-derived TEQs for the Japanese quail construct were 1-2 orders of magnitude higher than calculated TEQs for Aroclors 1254 and 1016. These results suggest that the WHO-TEFs are not representative of relative PCB potency for all avian species. PMID:23815337

  20. A new lymphocyte proliferation assay for potency determination of bovine tuberculin PPDs.

    PubMed

    Spohr, Christina; Kaufmann, Eva; Battenfeld, Sibylle; Duchow, Karin; Cussler, Klaus; Balks, Elisabeth; Bastian, Max

    2015-01-01

    The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. Aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future. To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test. PMID:25935213

  1. Evaluation of an in vitro cell culture assay for the potency assessment of recombinant human erythropoietin.

    PubMed

    Machado, Francine T; Maldaner, Fernanda P S; Perobelli, Rafaela F; Xavier, Bruna; da Silva, Francielle S; de Freitas, Guilherme W; Bartolini, Paolo; Ribela, M Tereza C P; Dalmora, Sérgio L

    2016-05-01

    Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines. PMID:27256453

  2. USDA regulatory guidelines and practices for veterinary Leptospira vaccine potency testing.

    PubMed

    Srinivas, G B; Walker, A; Rippke, B

    2013-09-01

    Batch-release potency testing of leptospiral vaccines licensed by the United States Department of Agriculture (USDA) historically was conducted through animal vaccination-challenge models. The hamster vaccination-challenge assay was Codified in 1974 for bacterins containing Leptospira pomona, Leptospira icterohaemorrhagiae, and Leptospira canicola, and in 1975 for bacterins containing Leptospira grippotyphosa. In brief, 10 hamsters are vaccinated with a specified dilution of bacterin. After a holding period, the vaccinated hamsters, as well as nonvaccinated controls, are challenged with virulent Leptospira and observed for mortality. Eighty percent of vaccinated hamsters must survive in the face of a valid challenge. The high cost of the Codified tests, in terms of monetary expense and animal welfare, prompted the Center for Veterinary Biologics (CVB) to develop ELISA alternatives for them. Potency tests for other serogroups, such as Leptospira hardjo-bovis, that do not have Codified requirements for potency testing continue to be examined on a case-by-case basis. PMID:23838570

  3. Potency prediction of β-secretase (BACE-1) inhibitors using density functional methods.

    PubMed

    Roos, Katarina; Viklund, Jenny; Meuller, Johan; Kaspersson, Karin; Svensson, Mats

    2014-03-24

    Scoring potency is a main challenge for structure based drug design. Inductive effects of subtle variations in the ligand are not possible to accurately predict by classical computational chemistry methods. In this study, the problem of predicting potency of ligands with electronic variations participating in key interactions with the protein was addressed. The potency was predicted for a large set of cyclic amidine and guanidine cores extracted from β-secretase (BACE-1) inhibitors. All cores were of similar size and had equal interaction motifs but were diverse with respect to electronic substitutions. A density functional theory approach, in combination with a representation of the active site of a protein using only key residues, was shown to be predictive. This computational approach was used to guide and support drug design, within the time frame of a normal drug discovery design cycle. PMID:24456077

  4. Augmentation by calcium channel antagonists of general anaesthetic potency in mice.

    PubMed

    Dolin, S J; Little, H J

    1986-08-01

    The effects of three kinds of calcium channel antagonists on the anaesthetic potencies of ethanol, pentobarbitone and argon were examined in mice. Ethanol and pentobarbitone anaesthetic potencies in mice were significantly increased by verapamil 10 mg kg-1, flunarizine 40 mg kg-1 and nitrendipine 100 mg kg-1. Argon anaesthetic potency was significantly increased by nitrendipine 50 mg kg-1 and 100 mg kg-1 in a dose-related fashion. Even at very high doses the calcium channel antagonists did not produce anaesthesia by themselves. At the doses used the calcium channel antagonists did not affect the blood concentrations of ethanol, 2 h, or pentobarbitone, 15 min, after anaesthetic administration. PMID:2943355

  5. Peptide length and prime-side sterics influence potency of peptide phosphonate protease inhibitors

    PubMed Central

    Brown, Christopher M.; Ray, Manisha; Eroy-Reveles, Aura A.; Egea, Pascal; Tajon, Cheryl; Craik, Charles S.

    2010-01-01

    Summary The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells. PMID:21276938

  6. Hydraphiles enhance antimicrobial potency against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis.

    PubMed

    Patel, Mohit B; Garrad, Evan C; Stavri, Ariel; Gokel, Michael R; Negin, Saeedeh; Meisel, Joseph W; Cusumano, Zachary; Gokel, George W

    2016-06-15

    Hydraphiles are synthetic amphiphiles that form ion-conducting pores in liposomal membranes. These pores exhibit open-close behavior when studied by planar bilayer conductance techniques. In previous work, we showed that when co-administered with various antibiotics to the DH5α strain of Escherichia coli, they enhanced the drug's potency. We report here potency enhancements at low concentrations of hydraphiles for the structurally and mechanistically unrelated antibiotics erythromycin, kanamycin, rifampicin, and tetracycline against Gram negative E. coli (DH5α and K-12) and Pseudomonas aeruginosa, as well as Gram positive Bacillus subtilis. Earlier work suggested that potency increases correlated to ion transport function. The data presented here comport with the function of hydraphiles to enhance membrane permeability in addition to, or instead of, their known function as ion conductors. PMID:27166575

  7. Effector and regulatory T cell subsets in diabetes-associated inflammation. Is there a connection with ST2/IL-33 axis? Perspective.

    PubMed

    Ryba-Stanisławowska, Monika; Stanisławowski, Marcin; Myśliwska, Jolanta

    2014-09-01

    Type 1 diabetes (DM1) is a chronic inflammatory disease, which when progresses leads to the development of late vascular complications. The disease involves impairments in regulatory and effector subsets of T lymphocytes, which suppress and maintain inflammatory response, respectively. ST2/IL-33 pathway is involved in T-cell-mediated immune response and might regulate the inflammatory process in several diseases. This review presents the latest research findings regarding effector and regulatory T cell subsets in the context of inflammation accompanying DM1 with particular focus on the ST2/IL-33 network and its possible association with T cell-mediated immunity. PMID:24547981

  8. Lymphocyte subset ratios and factor VIII usage in haemophilia.

    PubMed Central

    Beddall, A C; Al-Rubei, K; Williams, M D; Hill, F G

    1985-01-01

    Type and quantity of replacement treatment, together with haematological and immunological parameters were determined in 37 boys with severe haemophilia A and 41 children with other bleeding disorders. The quantity of factor VIII concentrate given to boys with severe haemophilia A (mean U/year) showed a significant inverse correlation with total white cell counts, lymphocyte counts, platelet counts, and the ratio of monoclonal antibody defined T lymphocyte subsets, T4 and T8 (T4:T8). Of the boys with severe haemophilia A, 49% had inversed T4:T8 ratios and 24% had thrombocytopenia. Treatment with high dose factor VIII concentrate (more than 25 000 U/year) was associated with low platelet counts, low lymphocyte counts, low T4:T8 ratios, and hypergammaglobulinaemia. In addition, six patients with severe haemophilia A and factor VIII inhibitors had inversed T4:T8 ratios. Patients treated exclusively with cryoprecipitate or prothrombin complex concentrates had normal T4:T8 ratios and platelet counts. The severity of the haematological and immunological abnormalities observed seems to be associated with high usage of factor VIII concentrates. Similar abnormalities have been described in patients with the acquired immune deficiency syndrome (AIDS). Prospective study of haemophiliacs is required to assess long term sequelae of factor concentrate usage, including the possible development of AIDS. PMID:3925894

  9. Leukocyte subsets and neutrophil function after short-term spaceflight

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Sams, C. F.; Mehta, S. K.; Kaur, I.; Jones, M. L.; Feeback, D. L.; Pierson, D. L.

    1999-01-01

    Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

  10. Probabilistic dynamics of mistuned bladed disc systems using subset simulation

    NASA Astrophysics Data System (ADS)

    Yuan, Jie; Allegri, Giuliano; Scarpa, Fabrizio; Rajasekaran, Ramesh; Patsias, Sophoclis

    2015-08-01

    The work describes an assessment of subset simulation (SubSim) techniques to increase the computational efficiency for the predictions of probabilistic dynamic behaviour in mistuned bladed disc systems. SubSim is an adaptive stochastic procedure to efficiently compute small failure probabilities, which are expressed as a product of large conditional failures probabilities by introducing intermediate failure events. The original version of SubSim with a classical modified Markov chain Monte Carlo (MCMC) method is used in this work to generate samples related to intermediate failure events. A 2-DOFs model with lumped parameters identified from a high-fidelity finite element model is used to represent a bladed disc. The statistics associated to the maximum forced frequency response amplitudes are evaluated from different levels of the blade mistuning using stiffness perturbations of the blades. Direct Monte Carlo simulations (MCS) are used to benchmark the results from the SubSim. The proposed methodology is shown to capture efficiently the statistical properties of the mistuned blades with less than 5% samples compared to the direct MCS method. Trade-off parametric studies of the SubSim method indicate that 2000 samples at each level yield an overall good computational efficiency and accuracy for the bladed disk system considered in this work. The study confirms that SubSim techniques can be effectively used in stochastic analysis of bladed disc systems with uncertainty related to the blade configurations.

  11. Allergic reactions, "spillover' reactions, and T-cell subsets.

    PubMed

    Bruynzeel, D P; Nieboer, C; Boorsma, D M; Scheper, R J; van Ketel, W G

    1983-01-01

    A strong positive, allergic patch-test reaction was elicited in 15 patients with an established allergy for a particular allergen. Patches with a marginally irritating concentration of sodium lauryl sulfate (SLS) were applied at fixed distances. The SLS patch situated adjacent to the allergic reaction was significantly enhanced in 12 of 15 patients (P less than 0.01) compared to more distant SLS reactions ("spillover'). Only quantitative differences were observed in the histologic pictures of the different types of reaction. The infiltrate consisted of lymphocytes and histiocytes, mainly located perivascular in the upper dermis. T-cell subsets were assessed with monoclonal antibodies using an immunoperoxidase technique. The distribution of the different T cells was the same for both reaction types. T cells located outside the perivascular infiltrates (e.g., in the epidermal vesicles) were OKT-8-positive (cytotoxic/suppressor T lymphocytes). Immunofluorescence examination did not show different patterns for the allergic or "enhanced toxic' reactions with regard to the presence of immunoglobulins and complement. The "spillover' phenomenon may cause false-positive patch-test reactions. PMID:6223603

  12. NK Cell Subset Redistribution during the Course of Viral Infections

    PubMed Central

    Lugli, Enrico; Marcenaro, Emanuela; Mavilio, Domenico

    2014-01-01

    Natural killer (NK) cells are important effectors of innate immunity that play a critical role in the control of human viral infections. Indeed, given their capability to directly recognize virally infected cells without the need of specific antigen presentation, NK cells are on the first line of defense against these invading pathogens. By establishing cellular networks with a variety of cell types such as dendritic cells, NK cells can also amplify anti-viral adaptive immune responses. In turn, viruses evolved and developed several mechanisms to evade NK cell-mediated immune activity. It has been reported that certain viral diseases, including human immunodeficiency virus-1 as well as human cytomegalovirus infections, are associated with a pathologic redistribution of NK cell subsets in the peripheral blood. In particular, it has been observed the expansion of unconventional CD56neg NK cells, whose effector functions are significantly impaired as compared to that of conventional CD56pos NK cells. In this review, we address the impact of these two chronic viral infections on the functional and phenotypic perturbations of human NK cell compartment. PMID:25177322

  13. Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

    PubMed

    Moogk, Duane; Zhong, Shi; Yu, Zhiya; Liadi, Ivan; Rittase, William; Fang, Victoria; Dougherty, Janna; Perez-Garcia, Arianne; Osman, Iman; Zhu, Cheng; Varadarajan, Navin; Restifo, Nicholas P; Frey, Alan B; Krogsgaard, Michelle

    2016-07-15

    CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. PMID:27271569

  14. IL7Rα expression and upregulation by IFNβ in dendritic cell subsets is haplotype-dependent.

    PubMed

    McKay, Fiona C; Hoe, Edwin; Parnell, Grant; Gatt, Prudence; Schibeci, Stephen D; Stewart, Graeme J; Booth, David R

    2013-01-01

    The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response. PMID:24147013

  15. Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

    PubMed Central

    2013-01-01

    Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c+ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets. PMID:20552278

  16. Human Monocyte Subsets at Homeostasis and Their Perturbation in Numbers and Function in Filarial Infection

    PubMed Central

    Moore, Vanessa; Bennuru, Sasisekhar; McDonald-Fleming, Renee; Ganesan, Sundar; Cotton, Rachel; Anuradha, Rajamanickam; Babu, Subash; Nutman, Thomas B.

    2014-01-01

    To characterize the function and plasticity of the major human circulating monocyte populations and to explore their role in systemic helminth infection, highly purified (by flow-based sorting) human monocyte subsets (CD14hi/CD16neg [classical], CD14+ or hi/CD16med [intermediate], and CD14neg/CD16hi [nonclassical]) were examined at homeostasis and after activation. Among these three subsets the classical and intermediate subsets were found to be the major sources of inflammatory and regulatory cytokines, as well as cytokines/chemokines associated with alternative activation, whereas the nonclassical and classical populations demonstrated an ability to transmigrate through endothelial monolayers. Moreover, it was primarily the classical subset that was the most efficient in promoting autologous T cell proliferation. The distribution of these subsets changed in the context of a systemic helminth (Wuchereria bancrofti) infection such that patent infection altered the frequency and distribution of these monocyte subsets with the nonclassical monocytes being expanded (almost 2-fold) in filarial infection. To understand further the filarial/monocyte interface, in vitro modeling demonstrated that the classical subset internalized filarial antigens more efficiently than the other two subsets but that the parasite-driven regulatory cytokine interleukin-10 was exclusively coming from the intermediate subset. Our data suggest that monocyte subsets have a differential function at homeostasis and in response to helminth parasites. PMID:25114121

  17. CXCR6 marks a novel subset of T-bet(lo)Eomes(hi) natural killer cells residing in human liver.

    PubMed

    Stegmann, Kerstin A; Robertson, Francis; Hansi, Navjyot; Gill, Upkar; Pallant, Celeste; Christophides, Theodoros; Pallett, Laura J; Peppa, Dimitra; Dunn, Claire; Fusai, Giuseppe; Male, Victoria; Davidson, Brian R; Kennedy, Patrick; Maini, Mala K

    2016-01-01

    Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56(bright)CD16-CD57-), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6- fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bet(hi)Eomes(lo)(CXCR6-) and T-bet(lo)Eomes(hi)(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bet(hi)Eomes(lo), suggesting its lineage was closer to CXCR6- peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-bet(lo)Eomes(hi) NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity. PMID:27210614

  18. CXCR6 marks a novel subset of T-betloEomeshi natural killer cells residing in human liver

    PubMed Central

    Stegmann, Kerstin A.; Robertson, Francis; Hansi, Navjyot; Gill, Upkar; Pallant, Celeste; Christophides, Theodoros; Pallett, Laura J.; Peppa, Dimitra; Dunn, Claire; Fusai, Giuseppe; Male, Victoria; Davidson, Brian R.; Kennedy, Patrick; Maini, Mala K.

    2016-01-01

    Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56brightCD16−CD57−), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6− fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bethiEomeslo(CXCR6−) and T-betloEomeshi(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bethiEomeslo, suggesting its lineage was closer to CXCR6− peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-betloEomeshi NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity. PMID:27210614

  19. The transcription factor NRSF contributes to epileptogenesis by selective repression of a subset of target genes

    PubMed Central

    McClelland, Shawn; Brennan, Gary P; Dubé, Celine; Rajpara, Seeta; Iyer, Shruti; Richichi, Cristina; Bernard, Christophe; Baram, Tallie Z

    2014-01-01

    The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (∼10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts. DOI: http://dx.doi.org/10.7554/eLife.01267.001 PMID:25117540

  20. Development of Quantitative Structure-Activity Relationship (QSAR) Models to Predict the Carcinogenic Potency of Chemicals

    EPA Science Inventory

    Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to either: (1) identify alternative toxicity measures (shorter duration) that may be used as...

  1. Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

    PubMed

    Webb, Nicholas E; Montefiori, David C; Lee, Benhur

    2015-01-01

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose-response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120-gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations. PMID:26416571

  2. Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products.

    PubMed

    Hansen, Martin; Krogh, Kristine A; Brandt, Asbjørn; Christensen, Jan H; Halling-Sørensen, Bent

    2009-02-01

    The antibacterial potency of eight anticoccidial drugs was tested in a soil bacteria bioassay (pour plate method), EC(50)-values between 2.4 and 19.6 microM were obtained; however, one compound, nicarbazin exhibited an EC(50)-value above the maximum tested concentration (21 microM, 9.1 mg L(-1)). The potency of mixtures of two of the compounds, narasin and nicarbazin, was synergistic (more than additive) with 10-fold greater antibacterial potency of the mixture than can be explained by their individual EC(50)-values. The influence of pH, temperature, oxygen concentration and light on the transformation of robenidine and salinomycin was investigated. Robenidine was transformed by photolysis (DT(50) of 4.1 days) and was unstable at low pH (DT(50) of approximately 4 days); salinomycin was merely transformed at low pH, the latter into an unknown number of products. The antibacterial potency of the mixtures of transformation products of robenidine after photolysis and at low pH was comparable with that of the parent compound. Finally five photo-transformation products of robenidine were structural elucidated by accurate mass measurements, i-FIT values (isotopic pattern fit) and MS/MS fragmentation patterns. PMID:18976841

  3. Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

    PubMed Central

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B.; Jacky, Birgitte P. S.; Aoki, K. Roger

    2012-01-01

    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease. PMID:23185348

  4. Stereoselective potencies and relative toxicities of y-Coniceine and N-Methylconiine enantiomers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    '-Coniceine, coniine, and N-methylconiine are toxic alkaloids present in poison hemlock (Conium maculatum). We previously reported the comparison of the relative potencies of (+)- and (-)-coniine enantiomers. In this study, we synthesized '-coniceine and the enantiomers of N-methylconiine and dete...

  5. A Luciferase-Based Quick Potency Assay to Predict Chondrogenic Differentiation.

    PubMed

    Oberbauer, Eleni; Steffenhagen, Carolin; Feichtinger, Georg; Hildner, Florian; Hacobian, Ara; Danzer, Martin; Gabriel, Christian; Redl, Heinz; Wolbank, Susanne

    2016-05-01

    Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs. PMID:27019357

  6. Relative potency of culture supernatants of Xenorhabdus and Photorhabdus spp. on growth of some fungal phytopathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated the potency of 10% v/v cell-free culture supernatants of cultures of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), Fusicladium effusum (pecan scab), Moni...

  7. Beyond arousal: valence and potency/control cues in the vocal expression of emotion.

    PubMed

    Goudbeek, Martijn; Scherer, Klaus

    2010-09-01

    The important role of arousal in determining vocal parameters in the expression of emotion is well established. There is less evidence for the contribution of emotion dimensions such as valence and potency/control to vocal emotion expression. Here, an acoustic analysis of the newly developed Geneva Multimodal Emotional Portrayals corpus, is presented to examine the role of dimensions other than arousal. This corpus contains twelve emotions that systematically vary with respect to valence, arousal, and potency/control. The emotions were portrayed by professional actors coached by a stage director. The extracted acoustic parameters were first compared with those obtained from a similar corpus [Banse and Scherer (1996). J. Pers. Soc. Psychol. 70, 614-636] and shown to largely replicate the earlier findings. Based on a principal component analysis, seven composite scores were calculated and were used to determine the relative contribution of the respective vocal parameters to the emotional dimensions arousal, valence, and potency/control. The results show that although arousal dominates for many vocal parameters, it is possible to identify parameters, in particular spectral balance and spectral noise, that are specifically related to valence and potency/control. PMID:20815467

  8. A carcinogenic potency database of the standardized results of animal bioassays

    PubMed Central

    Gold, Lois Swirsky; Sawyer, Charles B.; Magaw, Renae; Backman, Georganne M.; De Veciana, Margarita; Levinson, Robert; Hooper, N. Kim; Havender, William R.; Bernstein, Leslie; Peto, Richard; Pike, Malcolm C.; Ames, Bruce N.

    1984-01-01

    The preceding paper described our numerical index of carcinogenic potency, the TD50 and the statistical procedures adopted for estimating it from experimental data. This paper presents the Carcinogenic Potency Database, which includes results of about 3000 long-term, chronic experiments of 770 test compounds. Part II is a discussion of the sources of our data, the rationale for the inclusion of particular experiments and particular target sites, and the conventions adopted in summarizing the literature. Part III is a guide to the plot of results presented in Part IV. A number of appendices are provided to facilitate use of the database. The plot includes information about chronic cancer tests in mammals, such as dose and other aspects of experimental protocol, histopathology and tumor incidence, TD50 and its statistical significance, dose response, author's opinion and literature reference. The plot readily permits comparisons of carcinogenic potency and many other aspects of cancer tests; it also provides quantitative information about negative tests. The range of carcinogenic potency is over 10 million-fold. PMID:6525996

  9. A CONCEPTUAL MODEL FOR EVALUATING RELATIVE POTENCY DATA FOR USE IN ECOLOGICAL RISK ASSESSMENTS

    EPA Science Inventory

    For chemicals with a common mechanism of toxicity, relative potency factors (RPFs) allow dose and exposure measures to be normalized to an equivalent toxicity amount of a model chemical... In ecological risk assessments the large number of possible target species, variety of expo...

  10. The botulinum toxin LD50 potency assay - another chapter, another mystery.

    PubMed

    Pickett, Andy

    2012-09-01

    Observers of the potency assay used for botulinum toxin were greeted last year with the news that one company had an alternative, non-animal alternative in place. But all was not as it seemed from the press release, and over a year later, information is still lacking. PMID:23067303

  11. Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

    SciTech Connect

    Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.; Caspers, Nicole; Poda, Gennadiy I.; Long, Scott A.; Pierce, Betsy S.; Mahoney, Matthew W.; Mourey, Robert J.; Parikh, Mihir D.; Pfizer

    2010-10-01

    Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

  12. Development of Leptospira in vitro potency assays: EU/industry experience and perspectives.

    PubMed

    Klaasen, H L B M; van der Veen, M; Molkenboer, M J C H; Bruderer, U

    2013-09-01

    Nobivac® Lepto (MSD Animal Health) is a non-adjuvanted canine leptospirosis vaccine containing inactivated whole cells of Leptospira interrogans serogroup Canicola serovar Portlandvere and L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. The current standard in vivo potency test is a hamster challenge test associated with major drawbacks such as animal suffering and poor reproducibility. Here, the quantification of antigenic mass by ELISA as a new in vitro potency test is described, supporting the 3Rs concept (replacement, reduction, and refinement of animal tests) and in accordance with European Pharmacopoeia Monograph 0447 (Canine Leptospirosis Vaccine [Inactivated]). The two corresponding sandwich ELISAs are based on monoclonal antibodies specific for immunodominant leptospiral lipopolysaccharide epitopes. Protection in passive immunization experiments demonstrate that these monoclonal antibodies recognize key protective antigens in currently licensed human and veterinary whole cell Leptospira vaccines. The high precision and robustness renders the two ELISAs much more reliable correlates of potency in dogs than the hamster potency test. The recent approval of these assays for a new canine leptospirosis vaccine is an important contribution to the 3Rs in quality control testing of Leptospira vaccines. PMID:23867758

  13. Social Capital, Team Efficacy and Team Potency: The Mediating Role of Team Learning Behaviors

    ERIC Educational Resources Information Center

    van Emmerik, Hetty; Jawahar, I. M.; Schreurs, Bert; de Cuyper, Nele

    2011-01-01

    Purpose: Drawing on social capital theory and self-identification theory, this study aims to examine the associations of two indicators of social capital, personal networks and deep-level similarity, with team capability measures of team efficacy and team potency. The central focus of the study is to be the hypothesized mediating role of team…

  14. Dose–response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies

    PubMed Central

    Webb, Nicholas E.; Montefiori, David C.; Lee, Benhur

    2015-01-01

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose–response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120–gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations. PMID:26416571

  15. Comparative Elongated Mineral Particle Toxicology & Erionite’s Apparent  High Potency for Inducing Mesothelioma

    EPA Science Inventory

    Recent NHEERL research under EPA's Libby Action Plan has determined that elongated particle relative potency for rat pleural mesothelioma is best predicted on the basis of total external surface area (TSA) of slightly acid leached test samples which simulate particle bio-durabili...

  16. Use of pulsed ultraviolet light to reduce the allergenic potency of soybean extracts.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pulsed ultraviolet light (PUV), a non-thermal food processing technology, is reported to be able to inactivate enzymes and reduce allergen levels from peanut extracts. The objective of this study was to determine if PUV would reduce the allergen levels and allergenic potency of soy extracts. Soy ext...

  17. RELTIVE POTENCIES OF SELECTED DIHALOACETATES AND THEIR MAJOR METABOLITES IN RODENT WHOLE EMBRYO CULTURE

    EPA Science Inventory

    Relative potencies of selected dihaloacetic acids and their major metabolites in rodent whole embryo culture.

    S. Hunter, M. Blanton, E. Rogers
    RTD, NHEERL, ORD, US EPA, RTP, NC, 27711

    Haloacetic acids (HAA) are produced by disinfection and present in tap water. S...

  18. MULTI-FACTOR POTENCY SCHEME FOR COMPARING THE CARCINOGENIC ACTIVITY OF CHEMICALS

    EPA Science Inventory

    A scheme for ranking the quantitative activity `of chemical carcinogens is described. his activity scheme uses as its base, dose potency measured as TD50, which after conversion into an inverse log scale, a decile scale, is adjusted by weighing factors that describe other paramet...

  19. Functional potencies of dopamine agonists and antagonists at human dopamine D₂ and D₃ receptors.

    PubMed

    Tadori, Yoshihiro; Forbes, Robert A; McQuade, Robert D; Kikuchi, Tetsuro

    2011-09-01

    We measured the functional agonist potencies of dopamine agonists including antiparkinson drugs, and functional antagonist potencies of antipsychotics at human dopamine D(2) and D(3) receptors. In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cells expressing low and high densities of human dopamine D(2L) and D(2S) receptors (hD(2L)-Low, hD(2L)-High, hD(2S)-Low and hD(2S)-High, respectively) and human dopamine D(3) Ser-9 and D(3) Gly-9 receptors (hD(3)-Ser-9 and hD(3)-Gly-9, respectively). Cabergoline, bromocriptine, pergolide, (±)-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), talipexole, pramipexole, R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-olhydrochloride (PD128907) and ropinirole behaved as dopamine D(2) and D(3) receptor full agonists and showed higher potencies in hD(2L)-High and hD(2S)-High compared to hD(2L)-Low and hD(2S)-Low. In hD(3)-Ser-9 and hD(3)-Gly-9 compared to hD(2L)-Low and hD(2S)-Low, dopamine, ropinirole, PD128907, and pramipexole potencies were clearly higher; talipexole and 7-OH-DPAT showed slightly higher potencies; pergolide showed slightly lower potency; and, cabergoline and bromocriptine potencies were lower. Aripiprazole acted as an antagonist in hD(2L)-Low; a low intrinsic activity partial agonist in hD(2S)-Low; a moderate partial agonist in hD(3)-Ser-9 and hD(3)-Gly-9; a robust partial agonist in hD(2L)-High; and a full agonist in hD(2S)-High. Amisulpride, sulpiride and perphenazine behaved as preferential antagonists; and chlorpromazine and asenapine behaved as modest preferential antagonists; whereas fluphenazine, haloperidol, and blonanserin behaved as non-preferential antagonists in hD(2S)-Low and hD(2S)-High compared to hD(3)-Ser-9 and hD(3)-Gly-9. These findings may help to elucidate the basis of therapeutic benefit observed with these drugs, with

  20. A QSAR for the Mutagenic Potencies of Twelve 2-Amino-trimethylimidazopyridine Isomers: Structural, Quantum Chemical,and Hydropathic Factors

    SciTech Connect

    Knize, M G; Hatch, F T; Tanga, M J; Lau, E V; Colvin, M E

    2005-04-23

    An isomeric series of heterocyclic amines related to one found in heated muscle meats was investigated for properties that predict their measured mutagenic potency. Eleven of the 12 possible 2-amino-trimethylimidazopyridine (TMIP) isomers were tested for mutagenic potency in the Ames/Salmonella test with bacterial strain TA98, and resulted in a 600-fold range in potency. Structural, quantum chemical and hydropathic data were calculated on the parent molecules and the corresponding nitrenium ions of all of the tested isomers to establish models for predicting the potency of the unknown isomer. The regression model accounting for the largest fraction of the total variance in mutagenic potency contains four predictor variables: dipole moment, a measure of the gap between amine LUMO and HOMO energies, percent hydrophilic surface, and energy of amine LUMO. The most important determinants of high mutagenic potency in these amines are: (1) a small dipole moment, (2) the combination of b-face ring fusion and N3-methyl group, and (3) a lower calculated energy of the {pi} electron system. Based on predicted potency from the average of five models, the isomer not yet synthesized and tested is expected to have a mutagenic potency of 0.84 revertants/{micro}g in test strain TA98.

  1. RELATIVE POTENCY OF FUNGAL EXTRACTS IN INDUCING ALLERGIC ASTHMA-LIKE RESPONSES IN BALB/C MICE

    EPA Science Inventory

    Indoor mold has been associated with the development of allergic asthma. However, relative potency of molds in the induction of allergic asthma is not clear. In this study, we tested the relative potency of fungal extracts (Metarizium anisophilae [MACA], Stachybotrys ...

  2. Determination of relative potencies for chemical inhibition of spontaneous neuronal activity using a four amplifier MEA system.

    EPA Science Inventory

    Potency determination is important to identify the most promising drug candidates as well as identification of and ranking of compound toxicity. In our laboratory, we have utilized MEA recording techniques to determine the relative potency of 11 insecticidal compounds and rank th...

  3. Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity

    PubMed Central

    Chen, Ronald JY; Chung, Tse-yu; Li, Feng-yin; Lin, Nan-hei; Tzen, Jason TC

    2009-01-01

    Aim: To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Methods: The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase. Results: Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure. Conclusion: The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure. PMID:19060914

  4. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

    PubMed Central

    Khurana, Taruna; Dobrovolskaia, Ekaterina; Shartouny, Jessica R.; Slater, Jay E.

    2015-01-01

    Background German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. Objective Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. Methods Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. Results Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. Conclusions An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts. PMID:26444288

  5. Estimating the carcinogenic potency of chemicals from the in vivo micronucleus test.

    PubMed

    Soeteman-Hernández, Lya G; Johnson, George E; Slob, Wout

    2016-05-01

    In this study, we investigated the applicability of using in vivo mouse micronucleus (MN) data to derive cancer potency information. We also present a new statistical methodology for correlating estimated potencies between in vivo MN tests and cancer studies, which could similarly be used for other systems (e.g. in vitro vs. in vivo genotoxicity tests). The dose-response modelling program PROAST was used to calculate benchmark doses (BMDs) for estimating the genotoxic and carcinogenic potency for 48 compounds in mice; most of the data were retrieved from the National Toxicology Program (NTP) database, while some additional data were retrieved from the Carcinogenic Potency Database and published studies. BMD05s (doses with 5% increase in MN frequency) were derived from MN data, and BMD10s (doses with 10% extra cancer risk) were derived from carcinogenicity data, along with their respective lower (BMDL) and upper (BMDU) confidence bounds. A clear correlation between the in vivo MN BMD05s and the cancer BMD10s was observed when the lowest BMD05 from the in vivo MN was plotted against the lowest BMD10 from the carcinogenicity data for each individual compound. By making a further selection of BMDs related to more or less equally severe cancer lesions, the correlation was considerably improved. Getting a general scientific consensus on how we can quantitatively compare different tumour lesion types and investigating the impact of MN study duration are needed to refine this correlation analysis. Nevertheless, our results suggest that a BMD derived from genotoxicity data might provide a prediction of the tumour potency (BMD10) with an uncertainty range spanning roughly a factor of 100. PMID:26163673

  6. Evaluation of residual antibacterial potency in antibiotic production wastewater using a real-time quantitative method.

    PubMed

    Zhang, Hong; Zhang, Yu; Yang, Min; Liu, Miaomiao

    2015-11-01

    While antibiotic pollution has attracted considerable attention due to its potential in promoting the dissemination of antibiotic resistance genes in the environment, the antibiotic activity of their related substances has been neglected, which may underestimate the environmental impacts of antibiotic wastewater discharge. In this study, a real-time quantitative approach was established to evaluate the residual antibacterial potency of antibiotics and related substances in antibiotic production wastewater (APW) by comparing the growth of a standard bacterial strain (Staphylococcus aureus) in tested water samples with a standard reference substance (e.g. oxytetracycline). Antibiotic equivalent quantity (EQ) was used to express antibacterial potency, which made it possible to assess the contribution of each compound to the antibiotic activity in APW. The real-time quantitative method showed better repeatability (Relative Standard Deviation, RSD 1.08%) compared with the conventional fixed growth time method (RSD 5.62-11.29%). And its quantification limits ranged from 0.20 to 24.00 μg L(-1), depending on the antibiotic. We applied the developed method to analyze the residual potency of water samples from four APW treatment systems, and confirmed a significant contribution from antibiotic transformation products to potent antibacterial activity. Specifically, neospiramycin, a major transformation product of spiramycin, was found to contribute 13.15-22.89% of residual potency in spiramycin production wastewater. In addition, some unknown related substances with antimicrobial activity were indicated in the effluent. This developed approach will be effective for the management of antibacterial potency discharge from antibiotic wastewater and other waste streams. PMID:26395288

  7. Differential influence of dopamine transport rate on the potencies of cocaine, amphetamine, and methylphenidate.

    PubMed

    Calipari, Erin S; Ferris, Mark J; Siciliano, Cody A; Jones, Sara R

    2015-01-21

    Dopamine transporter (DAT) levels vary across brain regions and individuals, and are altered by drug history and disease states; however, the impact of altered DAT expression on psychostimulant effects in brain has not been systematically explored. Using fast scan cyclic voltammetry, we measured the effects of elevated DAT levels on presynaptic dopamine parameters as well as the uptake inhibition potency of the blockers cocaine and methylphenidate (MPH) and the releaser amphetamine (AMPH) in the nucleus accumbens core. Here we found that increases in DAT levels, resulting from either genetic overexpression or MPH self-administration, caused markedly increased maximal rates of uptake (Vmax) that were positively correlated with the uptake inhibition potency of AMPH and MPH, but not cocaine. AMPH and MPH were particularly sensitive to DAT changes, with a 100% increase in Vmax resulting in a 200% increase in potency. The relationship between Vmax and MPH potency was the same as that for AMPH, but was different from that for cocaine, indicating that MPH more closely resembles a releaser with regard to uptake inhibition. Conversely, the effects of MPH on stimulated dopamine release were similar to those of cocaine, with inverted U-shaped increases in release over a concentration-response curve. This was strikingly different from the release profile of AMPH, which showed only reductions at high concentrations, indicating that MPH is not a pure releaser. These data indicate that although MPH is a DAT blocker, its uptake-inhibitory actions are affected by DAT changes in a similar manner to releasers. Together, these data show that fluctuations in DAT levels alter the potency of releasers and MPH but not blockers and suggest an integral role of the DAT in the addictive potential of AMPH and related compounds. PMID:25474655

  8. Neural circuits: Interacting interneurons regulate fear learning.

    PubMed

    Ozawa, Takaaki; Johansen, Joshua P

    2014-08-01

    A recent study has found that, during associative fear learning, different sensory stimuli activate subsets of inhibitory interneurons in distinct ways to dynamically regulate glutamatergic neural activity and behavioral memory formation. PMID:25093560

  9. On the reliable and flexible solution of practical subset regression problems

    NASA Technical Reports Server (NTRS)

    Verhaegen, M.

    1987-01-01

    A new algorithm for solving subset regression problems is described. The algorithm performs a QR decomposition with a new column-pivoting strategy, which permits subset selection directly from the originally defined regression parameters. This, in combination with a number of extensions of the new technique, makes the method a very flexible tool for analyzing subset regression problems in which the parameters have a physical meaning.

  10. Detection of TCD4+ subsets in human carotid atheroma.

    PubMed

    Oliveira, Rômulo Tadeu Dias; Silva, Rosiane Maria; Teo, Fabio Haach; Mineiro, Marcela Franco; Ferreira, Maria Carolina; Altemani, Albina; Mamoni, Ronei Luciano; Menezes, Fábio Husseman; Blotta, Maria Heloisa Souza Lima

    2013-04-01

    Activated TCD4(+) cells are detected in human atherosclerotic plaques which indicate their participation in disease progression and destabilization. Among these cells, IFN-γ-producing T cells (TH1) are recognized as having a pro-atherogenic role. Recently, the IL-17-producing T helper lineage of cells (TH17) has been identified in atherosclerotic lesions. They have been linked to atheroma development through the production of pro-inflammatory mediators present in these lesions. Furthermore, IL-22 producing TCD4(+) cells (TH22) have been identified in the atheromatous environment, but their presence and function has not been investigated. The aim of this study was to analyze the immune response mediated by pro-inflammatory subtypes of TCD4(+) cells in atheromatous lesions. Atherosclerotic plaques of 57 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Three carotid fragments from organ donors were used as control. mRNA analysis showed expression of TH1 (IFN-γ, T-bet, IL-2, IL-12p35, TNF-α and IL-18); TH2 (GATA-3); TH17 (IL-17A, IL-17RA, Rorγt, TGF-β, IL-6, IL-1β, IL-23p19, CCL20, CCR4 and CCR6) and TH22 (IL-22 and Ahr) related markers. Asymptomatic patients showed higher expression of mRNA of IL-10, TGF-β, CCR4 and GATA-3 when compared to symptomatic ones. Immunohistochemistry analysis showed higher levels of IL-23, TGF-β, IL-1β and IL-18 in macrophages and foam cells in unstable lesions compared to stable and control ones. In vitro stimulation of atheroma cells induced IL-17 and IFN-γ production. Finally we were able to detect, the following subpopulations of TCD3(+) cells: TCD4(+) IFN-γ(+), TCD4(+)IL-17(+), TCD4(+)IL-4(+), TCD4(+)IL-22(+) and double positive cells (IFN-γ/IL-17(+), IFN-γ/IL-22(+) or IL-17/IL-22(+)). Our results showed the presence of distinct TCD4(+) cells subsets in human carotid lesions and suggest that interactions among them may contribute to the atheroma progression and destabilization. PMID

  11. Survival and signaling changes in antigen presenting cell subsets after radiation

    NASA Astrophysics Data System (ADS)

    Parker, Jennifer Janell

    Radiation therapy is a widely used cancer treatment that has the potential to influence anti-tumor immune responses. Both myeloablative and non-myeloablative radiation are often used as part of preparatory regimens for hematopoetic stem cell transplantation, in combination with other chemotherapy or immuno-modulatory (e.g. Anti-thymocyte globulin (ATG)) therapies for both cytotoxic and immune modulatory purposes. However, the mechanisms responsible for the effect of radiation on antigen presenting cell (APC) responsiveness and radioresistance are poorly understood. The first studies described in this thesis were designed to identify and characterize early radiation-induced signaling changes in antigen presenting cells and to determine the effects of these signaling changes on APC receptor expression and function. The NFkappaB pathway in antigen presenting cells was chosen for study because it is activated by radiation in a wide range of other cell types and plays a vital role in the maintenance and regulation of the immune system. The effects of therapeutically relevant doses radiation (2 and 20 Gy) were compared at various timepoints in the human monocytic cell line (U937) using phospho-flow cytometry staining methods and cytometric analysis. These studies demonstrated that radiation-induced changes in the phosphorylation state of NFkappaB family members that were p53 independent. However, these changes were dependent upon activation of ATM in response to single or double-stranded breaks in DNA, as shown in experiments using an inhibitor of ATM and ATM siRNA knockdown U937 cells. In addition, studies examining the effect of radiation on co-stimulatory receptors with and without inhibition of the NFkappaB pathway via phospho-flow cytometry revealed that radiation-induced phosphorylation of NEMO promoted the activation and functional maturation of U937 cells. Furthermore, functional studies using both phospho-flow cytometry and/or mixed lymphocyte reactions to

  12. Potency trends of Δ9-THC and other cannabinoids in confiscated cannabis preparations from 1993 to 2008.

    PubMed

    Mehmedic, Zlatko; Chandra, Suman; Slade, Desmond; Denham, Heather; Foster, Susan; Patel, Amit S; Ross, Samir A; Khan, Ikhlas A; ElSohly, Mahmoud A

    2010-09-01

    The University of Mississippi has a contract with the National Institute on Drug Abuse (NIDA) to carry out a variety of research activities dealing with cannabis, including the Potency Monitoring (PM) program, which provides analytical potency data on cannabis preparations confiscated in the United States. This report provides data on 46,211 samples seized and analyzed by gas chromatography-flame ionization detection (GC-FID) during 1993-2008. The data showed an upward trend in the mean Δ(9)-tetrahydrocannabinol (Δ(9)-THC) content of all confiscated cannabis preparations, which increased from 3.4% in 1993 to 8.8% in 2008. Hashish potencies did not increase consistently during this period; however, the mean yearly potency varied from 2.5-9.2% (1993-2003) to 12.0-29.3% (2004-2008). Hash oil potencies also varied considerably during this period (16.8 ± 16.3%). The increase in cannabis preparation potency is mainly due to the increase in the potency of nondomestic versus domestic samples. PMID:20487147

  13. Estimating Potency in High-Throughput Screening Experiments by Maximizing the Rate of Change in Weighted Shannon Entropy

    PubMed Central

    Shockley, Keith R.

    2016-01-01

    High-throughput in vitro screening experiments can be used to generate concentration-response data for large chemical libraries. It is often desirable to estimate the concentration needed to achieve a particular effect, or potency, for each chemical tested in an assay. Potency estimates can be used to directly compare chemical profiles and prioritize compounds for confirmation studies, or employed as input data for prediction modeling and association mapping. The concentration for half-maximal activity derived from the Hill equation model (i.e., AC50) is the most common potency measure applied in pharmacological research and toxicity testing. However, the AC50 parameter is subject to large uncertainty for many concentration-response relationships. In this study we introduce a new measure of potency based on a weighted Shannon entropy measure termed the weighted entropy score (WES). Our potency estimator (Point of Departure, PODWES) is defined as the concentration producing the maximum rate of change in weighted entropy along a concentration-response profile. This approach provides a new tool for potency estimation that does not depend on the assumption of monotonicity or any other pre-specified concentration-response relationship. PODWES estimates potency with greater precision and less bias compared to the conventional AC50 assessed across a range of simulated conditions. PMID:27302286

  14. Estimating Potency in High-Throughput Screening Experiments by Maximizing the Rate of Change in Weighted Shannon Entropy.

    PubMed

    Shockley, Keith R

    2016-01-01

    High-throughput in vitro screening experiments can be used to generate concentration-response data for large chemical libraries. It is often desirable to estimate the concentration needed to achieve a particular effect, or potency, for each chemical tested in an assay. Potency estimates can be used to directly compare chemical profiles and prioritize compounds for confirmation studies, or employed as input data for prediction modeling and association mapping. The concentration for half-maximal activity derived from the Hill equation model (i.e., AC50) is the most common potency measure applied in pharmacological research and toxicity testing. However, the AC50 parameter is subject to large uncertainty for many concentration-response relationships. In this study we introduce a new measure of potency based on a weighted Shannon entropy measure termed the weighted entropy score (WES). Our potency estimator (Point of Departure, PODWES) is defined as the concentration producing the maximum rate of change in weighted entropy along a concentration-response profile. This approach provides a new tool for potency estimation that does not depend on the assumption of monotonicity or any other pre-specified concentration-response relationship. PODWES estimates potency with greater precision and less bias compared to the conventional AC50 assessed across a range of simulated conditions. PMID:27302286

  15. The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression.

    PubMed Central

    Lehner, T; Avery, J; Jones, T

    1985-01-01

    Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and

  16. Precipitated silica as flow regulator.

    PubMed

    Müller, Anne-Kathrin; Ruppel, Joanna; Drexel, Claus-Peter; Zimmermann, Ingfried

    2008-08-01

    Flow regulators are added to solid pharmaceutical formulations to improve the flow properties of the powder mixtures. The primary particles of the flow regulators exist in the form of huge agglomerates which are broken down into smaller aggregates during the blending process. These smaller aggregates adsorb at the surface of the solid's grains and thus diminish attractive Van-der-Waals-forces by increasing the roughness of the host's surface. In most cases amorphous silica is used as flow additive but material properties like particle size or bond strength influence the desagglomeration tendency of the agglomerates and thus the flow regulating potency of each silica. For some silica types we will show that the differences in their flow regulating potency are due to the rate and extent by which they are able to cover the surface of the host particles. Binary powder mixtures consisting of a pharmaceutical excipient and an added flow regulator were blended in a Turbula mixer for a defined period of time. As pharmaceutical excipient corn starch was used. The flow regulators were represented by a selection of amorphous silicon dioxide types like a commercial fumed silica and various types of SIPERNAT precipitated silica provided by Evonik-Degussa GmbH, Hanau, Germany. Flowability parameters of the mixtures were characterized by means of a tensile strength tester. The reduction of tensile strength with the blending time can be correlated with an increase in fragmentation of the flow regulator. PMID:18595668

  17. Variable Neighborhood Search Heuristics for Selecting a Subset of Variables in Principal Component Analysis

    ERIC Educational Resources Information Center

    Brusco, Michael J.; Singh, Renu; Steinley, Douglas

    2009-01-01

    The selection of a subset of variables from a pool of candidates is an important problem in several areas of multivariate statistics. Within the context of principal component analysis (PCA), a number of authors have argued that subset selection is crucial for identifying those variables that are required for correct interpretation of the…

  18. Phenotypic, ultra-structural and functional characterization of bovine peripheral blood dendritic cell subsets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dendritic cells (DC) are multifunctional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets...

  19. Successful development and validation of an in vitro replacement assay for Leptospira vaccine potency tests.

    PubMed

    Kulpa-Eddy, J

    2012-01-01

    The standard requirement for serial release potency testing of Leptospira bacterins in the United States is the hamster vaccination challenge test. It is a test that uses a large number of animals experiencing pain or distress, takes weeks to conduct, can be expensive and requires that laboratory personnel handle a viable zoonotic pathogen. In an effort to address these concerns, the United States Department of Agriculture (USDA) developed an in vitro method for potency testing of four Leptospira serovars. This enzyme-linked immunosorbent assay (ELISA) was subsequently validated in the target species. USDA informed their biologics licensees, permittees and applicants of the availability of reference bacterins and the regulatory acceptance regarding this alternative test method in notices issued in 2007 and 2009. This presentation describes how the initial research and subsequent development and validation work were accomplished. PMID:22888601

  20. An evolved oxazolidinone with selective potency against Mycobacterium tuberculosis and gram positive bacteria.

    PubMed

    Kaushik, Amit; Heuer, Abigail M; Bell, Drew T; Culhane, Jeffrey C; Ebner, David C; Parrish, Nicole; Ippoliti, J Thomas; Lamichhane, Gyanu

    2016-08-01

    Innovation of new antibacterials that are effective against strains that have developed resistance to existing drugs would strengthen our ability to treat and subsequently control spread of pathogenic bacteria. Increasing incidence of infections with drug resistant bacteria has become a common occurrence in recent times. We have developed an evolved oxazolidinone, T145, which inhibits growth of Enterococcus faecalis, Staphylococcus aureus and Mycobacterium tuberculosis (Mtb) with sub μg/ml potencies that are potentially therapeutically valuable. The oxazolidinone is bactericidal against Mtb but bacteriostatic against E. faecalis and S. aureus. In addition to therapeutically valuable potency and bactericidal activity against Mtb, T145 minimizes selection of spontaneous resistant mutants, a trait that prolongs longevity of a drug in clinical use. PMID:27329794

  1. Assessment of mosquito larvicidal potency of cyclic lipopeptides produced by Bacillus subtilis strains.

    PubMed

    Das, Kishore; Mukherjee, Ashis K

    2006-02-01

    In this study, mosquito larvicidal potency of cyclic lipopeptides (CLPs) secreted by two Bacillus subtilis strains were determined. LC50 of the crude CLPs secreted by B. subtilis DM-03 and DM-04 strains against third instar larvae of Culex quinquefasciatus was 120.0+/-5.0 and 300.0+/-8.0mg/l respectively post 24 h of treatment. Physico-chemical factors such as pH of water, incubation temperature, heating and exposure to sunlight hardly influenced the larvicidal potency of these CLPs. Present study provided the evidence that B. subtilis lipopeptides were safe to Indian major carp Labeo rohita, a non-target aquatic organism. These properties of B. subtilis CLPs can be exploited for the formulation of a safer, novel biopesticide for effective control of mosquito larvae. PMID:16316617

  2. A Pentacyclic Aurora Kinase Inhibitor (AKI-001) With High in Vivo Potency And Oral Bioavailability

    SciTech Connect

    Rawson, T.E.; Ruth, M.; Blackwood, E.; Burdick, D.; Corson, L.; Dotson, J.; Drummond, J.; Fields, C.; Georges, G.J.; Goller, B.; Halladay, J.; Hunsaker, T.; Kleinheinz, T.; Krell, H.-W.; Li, J.; Liang, J.; Limberg, A.; McNutt, A.; Moffat, J.; Phillips, G.; Ran, Y.

    2009-05-21

    Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC{sub 50} < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

  3. Mixed halogenated dioxins/furans (PXDD/Fs) and biphenyls (PXBs) in food: occurrence and toxic equivalent exposure using specific relative potencies.

    PubMed

    Fernandes, Alwyn R; Mortimer, David; Wall, Richard J; Bell, David R; Rose, Martin; Carr, Melanie; Panton, Sean; Smith, Frankie

    2014-12-01

    The occurrence of nineteen mixed halogenated (bromo-chloro) dibenzo-p-dioxins, dibenzofurans (PXDD/Fs) and biphenyls (PXBs) in a range of foods (n>100) was investigated. The analytical methodology used dual activated carbon column fractionation with high resolution mass spectrometric measurement (13,500-15,000 res). Occurrence was observed in most commonly consumed foods but the most frequent detections of these environmental contaminants were made in shellfish and offal. The concentrations of the individual compounds were condensed into toxic equivalents (TEQs) using recently reported relative potency values. Although representing only a small subset of the full range of toxic PXDD/Fs and PXBs, the TEQs estimated for these compounds ranged from 0.2% to approximately 15% (depending on the food matrix) of the corresponding TEQ for the fully chlorinated analogues. This finding is of great toxicological importance as it implies that a potentially greater magnitude of TEQ could be associated with the full range of toxic PXDD/Fs and PXBs, thus making a significant contribution to dioxin-like toxicity from the diet, to human exposure. PMID:25108070

  4. CDCP1 Identifies a CD146 Negative Subset of Marrow Fibroblasts Involved with Cytokine Production

    PubMed Central

    Iwata, Mineo; Torok-Storb, Beverly; Wayner, Elizabeth A.; Carter, William G.

    2014-01-01

    In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1)/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs) Protein Kinase C delta (PKC-δ). When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression. PMID:25275584

  5. Distinct Cytokine Profiles of Neonatal Natural Killer T Cells after Expansion with Subsets of Dendritic Cells

    PubMed Central

    Kadowaki, Norimitsu; Antonenko, Svetlana; Ho, Stephen; Rissoan, Marie-Clotilde; Soumelis, Vassili; Porcelli, Steven A.; Lanier, Lewis L.; Liu, Yong-Jun

    2001-01-01

    Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1–mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4–dependent manner. Thus, it is important to understand how the development of IL-4– versus interferon (IFN)-γ–producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500–70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-γ, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4+IFN-γ− NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4−IFN-γ+ NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4+IFN-γ− NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-γ. PMID:11369793

  6. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

    PubMed

    Engel, Isaac; Seumois, Grégory; Chavez, Lukas; Samaniego-Castruita, Daniela; White, Brandie; Chawla, Ashu; Mock, Dennis; Vijayanand, Pandurangan; Kronenberg, Mitchell

    2016-06-01

    Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions. PMID:27089380

  7. T Helper Lymphocyte Subsets and Plasticity in Autoimmunity and Cancer: An Overview

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2015-01-01

    In response to cytokine signalling and other factors, CD4-positive T lymphocytes differentiate into distinct populations that are characterized by the production of certain cytokines and are controlled by different master transcription factors. The spectrum of such populations, which was initially limited to Th1 and Th2 subsets, is currently broadened to include Th17 and Treg subsets, as well as a number of less studied subtypes, such as Tfh, Th9, and Th22. Although these subsets appear to be relatively stable, certain plasticity exists that allows for transition between the subsets and formation of hybrid transition forms. This provides the immune system flexibility needed for adequate response to pathogens but, at the same time, can play a role in the pathogenic processes in cases of deregulation. In this review, we will discuss the properties of T lymphocyte subsets and their plasticity, as well as its implications for cancer and autoimmune diseases. PMID:26583100

  8. N-Aryl-oxazolidin-2-imine Muscle Selective Androgen Receptor Modulators Enhance Potency through Pharmacophore Reorientation

    SciTech Connect

    Nirschl, Alexandra A.; Zou, Yan; Krystek, Jr., Stanley R.; Sutton, James C.; Simpkins, Ligaya M.; Lupisella, John A.; Kuhns, Joyce E.; Seethala, Ramakrishna; Golla, Rajasree; Sleph, Paul G.; Beehler, Blake C.; Grover, Gary J.; Egan, Donald; Fura, Aberra; Vyas, Viral P.; Li, Yi-Xin; Sack, John S.; Kish, Kevin F.; An, Yongmi; Bryson, James A.; Gougoutas, Jack Z.; DiMarco, John; Zahler, Robert; Ostrowski, Jacek; Hamann, Lawrence G.

    2010-11-09

    A novel selective androgen receptor modulator (SARM) scaffold was discovered as a byproduct obtained during synthesis of our earlier series of imidazolidin-2-ones. The resulting oxazolidin-2-imines are among the most potent SARMs known, with many analogues exhibiting sub-nM in vitro potency in binding and functional assays. Despite the potential for hydrolytic instability at gut pH, compounds of the present class showed good oral bioavailability and were highly active in a standard rodent pharmacological model.

  9. In vivo potency of different ligands on voltage-gated sodium channels.

    PubMed

    Safrany-Fark, Arpad; Petrovszki, Zita; Kekesi, Gabriella; Liszli, Peter; Benedek, Gyorgy; Keresztes, Csilla; Horvath, Gyongyi

    2015-09-01

    The Ranvier nodes of thick myelinated nerve fibers contain almost exclusively voltage-gated sodium channels (Navs), while the unmyelinated fibers have several receptors (e.g., cannabinoid, transient receptor potential vanilloid receptor 1), too. Therefore, a nerve which contains only motor fibers can be an appropriate in vivo model for selective influence of Navs. The goals were to evaluate the potency of local anesthetic drugs on such a nerve in vivo; furthermore, to investigate the effects of ligands with different structures (arachidonic acid, anandamide, capsaicin and nisoxetine) that were proved to inhibit Navs in vitro with antinociceptive properties. The marginal mandibular branch of the facial nerve was explored in anesthetized Wistar rats; after its stimulation, the electrical activity of the vibrissae muscles was registered following the perineural injection of different drugs. Lidocaine, bupivacaine and ropivacaine evoked dose-dependent decrease in electromyographic activity, i.e., lidocaine had lower potency than bupivacaine or ropivacaine. QX-314 did not cause any effect by itself, but its co-application with lidocaine produced a prolonged inhibition. Nisoxetine had a very low potency. While anandamide and capsaicin in high doses caused about 50% decrease in the amplitude of action potential, arachidonic acid did not influence the responses. We proved that the classical local anesthetics have high potency on motor nerves, suggesting that this method might be a reliable model for selective targeting of Navs in vivo circumstances. It is proposed that the effects of these endogenous lipids and capsaicin on sensory fibers are not primarily mediated by Navs. PMID:26033207

  10. Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

    PubMed Central

    Alifimoff, J. K.; Firestone, L. L.; Miller, K. W.

    1989-01-01

    1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles. PMID:2784337

  11. Mechanism of multivalent nanoparticle encounter with HIV-1 for potency enhancement of peptide triazole virus inactivation.

    PubMed

    Rosemary Bastian, Arangassery; Nangarlia, Aakansha; Bailey, Lauren D; Holmes, Andrew; Kalyana Sundaram, R Venkat; Ang, Charles; Moreira, Diogo R M; Freedman, Kevin; Duffy, Caitlin; Contarino, Mark; Abrams, Cameron; Root, Michael; Chaiken, Irwin

    2015-01-01

    Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally. PMID:25371202

  12. Naturally-Occurring Glucosinolates, Glucoraphanin and Glucoerucin, are Antagonists to Aryl Hydrocarbon Receptor as Their Chemopreventive Potency.

    PubMed

    Abdull Razis, Ahmad Faizal; Noor, Noramaliza Mohd

    2015-01-01

    As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency. PMID:26320454

  13. Factors Affecting the Inclusion Potency for Acicular Ferrite Nucleation in High-Strength Steel Welds

    NASA Astrophysics Data System (ADS)

    Kang, Yongjoon; Jeong, Seonghoon; Kang, Joo-Hee; Lee, Changhee

    2016-06-01

    Factors affecting the inclusion potency for acicular ferrite nucleation in high-strength weld metals were investigated and the contribution of each factor was qualitatively evaluated. Two kinds of weld metals with different hardenabilities were prepared, in both, MnTi2O4-rich spinel formed as the predominant inclusion phase. To evaluate the factors determining the inclusion potency, the inclusion characteristics of size, phase distribution in the multiphase inclusion, orientation relationship with ferrite, and Mn distribution near the inclusion were analyzed. Three factors affecting the ferrite nucleation potency of inclusions were evaluated: the Baker-Nutting (B-N) orientation relationship between ferrite and the inclusion; the formation of an Mn-depleted zone (MDZ) near the inclusion; and the strain energy around the inclusion. Among these, the first two factors were found to be the most important. In addition, it was concluded that the increased chemical driving force brought about by the formation of an MDZ contributed more to the formation of acicular ferrite in higher-strength weld metals, because the B-N orientation relationship between ferrite and the inclusion was less likely to form as the transformation temperature decreased.

  14. Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom.

    PubMed

    Rungsiwongse, J; Ratanabanangkoon, K

    1991-01-24

    An ELISA for the quantitation of antibodies against Naja naja siamensis venom proteins has been developed for use as a possible replacement for the in vivo neutralization assay of antivenom potency. Comparison was made with three preparations of venom proteins as antigens of ELISA: these were the crude venom, a toxin fraction and the purified principle postsynaptic neurotoxin of the Thai cobra. Eight batches of horse monovalent therapeutic anti-cobra antivenom, one of which served as positive reference, were assayed by the ELISAs and also by the in vivo neutralization assay using mice. When crude venom, the toxin fraction and the pure neurotoxin were used as antigens in the ELISAs, the correlation coefficients between the ELISA antibody titers and in vivo neutralization of the antivenoms were 0.82 (P less than 0.005), 0.94 (P less than 0.001) and 0.95 (P less than 0.001), respectively. Thus, the ELISA which measures only the antibody against the principle toxin of the snake venom should be most suitable for use as an in vitro assay of antivenom potency. The ELISA should also be useful for potency assessment and standardization of antivenoms against other elapid snake venoms whose lethal components are small, poorly immunogenic peptides. PMID:1995711

  15. In silico approaches to predicting cancer potency for risk assessment of genotoxic impurities in drug substances.

    PubMed

    Bercu, Joel P; Morton, Stuart M; Deahl, J Thom; Gombar, Vijay K; Callis, Courtney M; van Lier, Robert B L

    2010-01-01

    The current risk assessment approach for addressing the safety of very small concentrations of genotoxic impurities (GTIs) in drug substances is the threshold of toxicological concern (TTC). The TTC is based on several conservative assumptions because of the uncertainty associated with deriving an excess cancer risk when no carcinogenicity data are available for the impurity. It is a default approach derived from a distribution of carcinogens and does not take into account the properties of a specific chemical. The purpose of the study was to use in silico tools to predict the cancer potency (TD(50)) of a compound based on its structure. Structure activity relationship (SAR) models (classification/regression) were developed from the carcinogenicity potency database using MultiCASE and VISDOM. The MultiCASE classification models allowed the prediction of carcinogenic potency class, while the VISDOM regression models predicted a numerical TD(50). A step-wise approach is proposed to calculate predicted numerical TD(50) values for compounds categorized as not potent. This approach for non-potent compounds can be used to establish safe levels greater than the TTC for GTIs in a drug substance. PMID:20363275

  16. Performance and potency of tetanus toxoid: implications for eliminating neonatal tetanus.

    PubMed Central

    Dietz, V.; Milstien, J. B.; van Loon, F.; Cochi, S.; Bennett, J.

    1996-01-01

    Neonatal tetanus (NT) is a major cause of mortality in developing countries, with over 400,000 deaths estimated to occur annually. WHO has adopted the goal of eliminating NT worldwide, and a major strategy for its prevention is the administration of at least two properly spaced doses of tetanus toxoid (TT) to women of childbearing age in high-risk areas to protect passively their newborns at birth. In certain countries the locally produced TT vaccine has been shown to be subpotent, while other countries have reported NT among infants born to vaccinated women. An extensive review of production and quality control procedures was carried out between 1993 and 1995 in 8 of 22 TT-producing countries that also report NT cases, with a more superficial assessment being carried out in the remaining 14 countries. Only 4 of the 22 countries have a functioning national control authority to monitor TT production and vaccine quality. A total of 80 TT lots from 21 manufacturers in 14 of the 22 NT-reporting countries were tested for potency. Of these, 15 lots from eight manufacturers in seven countries had potency values below WHO requirements. TT potency can also be compromised by improper vaccine handling. To eliminate neonatal tetanus worldwide requires assurance that all doses of TT meet WHO production and quality requirements and that the field effectiveness of TT is monitored through systematic NT case investigations and assessment of coverage. PMID:9060223

  17. Rainbow trout cell bioassay-derived relative potencies for halogenated aromatic hydrocarbons: Comparison and sensitivity analysis

    SciTech Connect

    Villeneuve, D.L.; Blankenship, A.L.; Giesy, J.P.; Richter, C.A.

    1999-05-01

    Rainbow trout hepatoma cells, stably transfected with a luciferase reporter gene under control of dioxin-responsive elements (RLT 2.0 cells) were used to derive relative potencies (RPs) for a variety of halogenated aromatic hydrocarbons (HAHs) that are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This in vitro bioassay utilizes 96-well microplates, which provide high sample throughput and assay efficiency without affecting sensitivity. The RLT 2.0-derived potencies for dioxin and furan congeners, relative to 2,3,7,8-TCDD, ranged from 0.917 for 1,2,3,4,7,8-hexachlorodibenzofuran to 0.208 or 1,2,3,7,8-pentachlorodibenzofuran. All mono- and di-ortho polychlorinated biphenyls (PCBs) tested had RPs that were orders of magnitude less than TCDD, but point estimates could not be determined. The RLT 2.0-derived RPs were found to be comparable to both other rainbow trout-specific RPs and RPs based on mammalian bioassays. Sensitivity analysis suggested that the range of uncertainty associated with TCDD equivalent (TEQ) estimates based on RLT 2.0-derived RPs is approximately 10-fold. Within this degree of uncertainty and the context of this study, the RLT 2.0 bioassay showed no definitive biases or inaccuracies relative to similar mammalian- or fish-specific in vitro bioassays. Thus, the RLT 2.0 bioassay appears to be a useful tool for evaluating dioxin-like potency of HAHs to fish.

  18. A common genetic influence on human intensity ratings of sugars and high-potency sweeteners.

    PubMed

    Hwang, Liang-Dar; Zhu, Gu; Breslin, Paul A S; Reed, Danielle R; Martin, Nicholas G; Wright, Margaret J

    2015-08-01

    The perception of sweetness varies among individuals but the sources of this variation are not fully understood. Here, in a sample of 1,901 adolescent and young adults (53.8% female; 243 MZ and 452 DZ twin pairs, 511 unpaired individuals; mean age 16.2±2.8, range 12–26 years), we studied the variation in the perception of sweetness intensity of two monosaccharides and two high-potency sweeteners: glucose, fructose, neohesperidine dihydrochalcone (NHDC), and aspartame. Perceived intensity for all sweeteners decreased with age (2–5% per year) and increased with the history of otitis media (6–9%). Males rated aspartame slightly stronger than females (7%). We found similar heritabilities for sugars (glucose: h2=0.31, fructose: h2=0.34) and high-potency sweeteners (NHDC: h2=0.31, aspartame: h2=0.30); all were in the modest range. Multivariate modeling showed that a common genetic factor accounted for >75% of the genetic variance in the four sweeteners, suggesting that individual differences in perceived sweet intensity, which are partly due to genetic factors, may be attributed to a single set of genes. This study provided evidence of the shared genetic pathways between the perception of sugars and high-potency sweeteners. PMID:26181574

  19. The potency-insolubility conundrum in pharmaceuticals: Mechanism and solution for hepatitis C protease inhibitors.

    PubMed

    Connelly, Patrick R; Snyder, Phillip W; Zhang, Yuegang; McClain, Brian; Quinn, Brian P; Johnston, Steve; Medek, Ales; Tanoury, Jerry; Griffith, James; Patrick Walters, W; Dokou, Eleni; Knezic, Dragutin; Bransford, Philip

    2015-01-01

    As compounds are optimized for greater potency during pharmaceutical discovery, their aqueous solubility often decreases, making them less viable as orally-administered drugs. To investigate whether potency and insolubility share a common origin, we examined the structural and thermodynamic properties of telaprevir, a sparingly soluble inhibitor of hepatitis C virus protease. Comparison of the hydrogen bond motifs in crystalline telaprevir with those present in the protease-telaprevir complex revealed striking similarities. Additionally, the thermodynamics of telaprevir dissolution closely resembles those of protein-ligand dissociation. Together, these findings point to a common origin of potency and insolubility rooted in particular amide-amide hydrogen bond patterns. The insolubility of telaprevir is shown by computational analysis to be caused by interactions in the crystal, not unfavorable hydrophobic hydration. Accordingly, competing out the particular amide-amide hydrogen bond motifs in crystalline telaprevir with 4-hydroxybenzoic acid yielded a co-crystalline solid with excellent aqueous dissolution and oral absorption. The analysis suggests a generalizable approach for identifying drug candidate compounds that either can or cannot be rendered orally bioavailable by alteration of their crystalline solid phases, in an approach that provides a pragmatic way to attain substantial enhancements in the success rate of drug discovery and development. PMID:25451684

  20. α-Conotoxin dendrimers have enhanced potency and selectivity for homomeric nicotinic acetylcholine receptors.

    PubMed

    Wan, Jingjing; Huang, Johnny X; Vetter, Irina; Mobli, Mehdi; Lawson, Joshua; Tae, Han-Shen; Abraham, Nikita; Paul, Blessy; Cooper, Matthew A; Adams, David J; Lewis, Richard J; Alewood, Paul F

    2015-03-11

    Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels. PMID:25710197

  1. Modulating potency: Physicochemical characteristics are a determining factor of TLR4-agonist nanosuspension activity.

    PubMed

    Dowling, Quinton M; Sivananthan, Sandra J; Guderian, Jeff A; Moutaftsi, Magdalini; Chesko, James D; Fox, Christopher B; Vedvick, Thomas S; Kramer, Ryan M

    2014-03-01

    Activity of adjuvanted vaccines is difficult to predict in vitro and in vivo. The wide compositional and conformational range of formulated adjuvants, from aluminum salts to oil-in-water emulsions, makes comparisons between physicochemical and immunological properties difficult. Even within a formulated adjuvant class, excipient selection and concentration can alter potency and physicochemical properties of the mixture. Complete characterization of physicochemical properties of adjuvanted vaccine formulations and relationship to biological response is necessary to move beyond a guess-and-check paradigm toward directed development. Here we present a careful physicochemical characterization of a two-component nanosuspension containing synthetic TLR-4 agonist glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at various molar ratios. Physicochemical properties were compared with potency, as measured by stimulation of cytokine production in human whole blood. We found a surprising, nonlinear relationship between physicochemical properties and GLA-DPPC ratios that corresponded well with changes in biological activity. We discuss these data in light of the current understanding of TLR4 activation and the conformation-potency relationship in development of adjuvanted vaccines. PMID:24464844

  2. Factors Affecting the Inclusion Potency for Acicular Ferrite Nucleation in High-Strength Steel Welds

    NASA Astrophysics Data System (ADS)

    Kang, Yongjoon; Jeong, Seonghoon; Kang, Joo-Hee; Lee, Changhee

    2016-03-01

    Factors affecting the inclusion potency for acicular ferrite nucleation in high-strength weld metals were investigated and the contribution of each factor was qualitatively evaluated. Two kinds of weld metals with different hardenabilities were prepared, in both, MnTi2O4-rich spinel formed as the predominant inclusion phase. To evaluate the factors determining the inclusion potency, the inclusion characteristics of size, phase distribution in the multiphase inclusion, orientation relationship with ferrite, and Mn distribution near the inclusion were analyzed. Three factors affecting the ferrite nucleation potency of inclusions were evaluated: the Baker-Nutting (B-N) orientation relationship between ferrite and the inclusion; the formation of an Mn-depleted zone (MDZ) near the inclusion; and the strain energy around the inclusion. Among these, the first two factors were found to be the most important. In addition, it was concluded that the increased chemical driving force brought about by the formation of an MDZ contributed more to the formation of acicular ferrite in higher-strength weld metals, because the B-N orientation relationship between ferrite and the inclusion was less likely to form as the transformation temperature decreased.

  3. Combined targeting of EGFR/HER promotes anti-tumor efficacy in subsets of KRAS mutant lung cancer resistant to single EGFR blockade

    PubMed Central

    Umelo, Ijeoma Adaku; De Wever, Olivier; Kronenberger, Peter; Van Deun, Jan; Noor, Alfiah; Singh, Kshitiz; Teugels, Erik; Chen, Gang; Bracke, Marc; De Grève, Jacques

    2015-01-01

    KRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers. PMID:25992771

  4. Pertussis serological potency test as an alternatively to the intracerebral mouse protection test.

    PubMed

    van der Ark, A; van Straaten-van de Kappelle, I; Hendriksen, C; van de Donk, H

    1996-01-01

    The current potency test for pertussis vaccines, the intracerebral protection test (MPT), is still the only mandatory laboratory model available. This test, however, is a valid, but inhumane and imprecise test and therefore a good candidate for replacement. Recently we have developed the Pertussis Serological Potency Test (PSPT) as an alternative for the MPT. The PSPT is based on in vitro assessment of the humoral immune response against the whole range of surface -antigens of B. pertussis in mice after immunisation with Whole Cell Vaccine (WCV). We have demonstrated a relationship between the mean pertussis antibody concentration at the day of challenge and the proportion of surviving mice at each vaccine dose in the MPT (R = 0.91). The PSPT is a model in which mice (20-24 g) are immunised i.p. with graded doses of vaccine and bled after four weeks. Sera are titrated in a whole cell ELISA and potency based on the vaccine dose-dependent antibody response is estimated by means of a parallel line analysis. In an in-house validation study 13 WCVs were tested in the PSPT and MPT. Homogeneity of both tests was proven by means of the chi-square test; potencies were significantly similar (p = 0.95). Compared to the MPT, the PSPT is more reproducible as is indicated by its smaller 95% confidence intervals. Moreover, by using the PSPT the animal distress can be reduced to an acceptable level and the PSPT also results in a reduction of more than 25% in use of mice. Additional experiments showed that estimation of WCV-potency in the PSPT based on specific antibody responses against protective antigens (PT, FHA, 69- and 92-kDa OMPS) was not possible or did not correlate with protection in MPT. Sera obtained from the PSPT showed a correlation between pertussis antibody levels and complement-mediated killing by pertussis antibodies in in vitro assays. In conclusion, the PSPT is a promising substitute for the MPT though further validation and additional studies on functional

  5. Relative embryotoxic potency of p-substituted phenols in the embryonic stem cell test (EST) and comparison to their toxic potency in vivo and in the whole embryo culture (WEC) assay.

    PubMed

    Strikwold, Marije; Woutersen, Ruud A; Spenkelink, Bert; Punt, Ans; Rietjens, Ivonne M C M

    2012-09-01

    The applicability of the embryonic stem cell test (EST) as an alternative for in vivo embryotoxicity testing was evaluated for a series of five p-substituted phenols. To this purpose, the potency ranking for this class of compounds derived from the inhibition of cardiomyocyte differentiation in the EST was compared to in vivo embryotoxic potency data obtained from literature and to the potency ranking defined in the in vitro whole embryo culture (WEC) assay. From the results obtained it appears that the EST was able to identify the embryotoxic potential for p-substituted phenols, providing an identical potency ranking compared to the WEC assay. However, the EST was not able to predict an accurate ranking for the phenols compared to their potency observed in vivo. Only phenol, the least potent compound within this series, was correctly ranked. Furthermore, p-mercaptophenol was correctly identified as a relative potent congener of the phenols tested, but its ranking was distorted by p-heptyloxyphenol, of which the toxicity was overestimated in the EST. It is concluded that when attempting to explain the observed disparity in potency rankings between in vitro and in vivo embryotoxicity, the in vitro models should be combined with a kinetic model describing in vivo absorption, distribution, metabolism and excretion processes of the compounds. PMID:22820428

  6. Comparative carcinogenic potencies of particulates from diesel engine exhausts, coke oven emissions, roofing tar aerosols and cigarette smoke.

    PubMed Central

    Albert, R E

    1983-01-01

    Mammalian cell mutagenesis, transformation and skin tumorigenesis assays show similar results in comparing the potencies of diesel, coke oven, roofing tar and cigarette smoke particulates. These assay results are reasonably consistent with the comparative carcinogenic potencies of coke oven and roofing tar emissions as determined by epidemiological studies. The bacterial mutagenesis assay tends to show disproportionately high potencies, particularly with diesel particulates. Results to date encourage the approach to the assessment for carcinogenic risks from diesel emissions based on the use of epidemiological data on cancer induced by coke oven emissions, roofing tar particulates and cigarette smoke with the comparative potencies of these materials determined by in vivo and in vitro bioassays. PMID:6186481

  7. Developmental Progression and Interrelationship of Central and Effector Regulatory T Cell Subsets.

    PubMed

    Toomer, Kevin H; Yuan, Xiaomei; Yang, Jing; Dee, Michael J; Yu, Aixin; Malek, Thomas R

    2016-05-01

    Resting central Tregs (cTregs) and activated effector Tregs (eTregs) are required for self-tolerance, but the heterogeneity and relationships within and between phenotypically distinct subsets of cTregs and eTregs are poorly understood. By extensive immune profiling and deep sequencing of TCR-β V regions, two subsets of cTregs, based on expression of Ly-6C, and three subsets of eTregs, based on distinctive expression of CD62L, CD69, and CD103, were identified. Ly-6C(+) cTregs exhibited lower basal activation, expressed on average lower affinity TCRs, and less efficiently developed into eTregs when compared with Ly-6C(-) cTregs. The dominant TCR Vβs of Ly-6C(+) cTregs were shared by eTregs at a low frequency. A single TCR clonotype was also identified that was largely restricted to Ly-6C(+) cTregs, even under conditions that promoted the development of eTregs. Collectively, these findings indicate that some Ly-6C(+) cTregs may persist as a lymphoid-specific subset, with minimal potential to develop into highly activated eTregs, whereas other cTregs readily develop into eTregs. In contrast, subsets of CD62L(lo) eTregs showed higher clonal expansion and were more highly interrelated than cTreg subsets based on their TCR-β repertoires, but exhibited varied immune profiles. The CD62L(lo) CD69(-) CD103(-) eTreg subset displayed properties of a transitional intermediate between cTregs and more activated eTreg subsets. Thus, eTreg subsets appear to exhibit substantial flexibility, most likely in response to environmental cues, to adopt defined immune profiles that are expected to optimize suppression of autoreactive T cells. PMID:27009492

  8. Ultraconservation identifies a small subset of extremely constrained developmental enhancers

    SciTech Connect

    Pennacchio, Len A.; Visel, Axel; Prabhakar, Shyam; Akiyama, Jennifer A.; Shoukry, Malak; Lewis, Keith D.; Holt, Amy; Plajzer-Frick, Ingrid; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len A.

    2007-10-01

    While experimental studies have suggested that non-coding ultraconserved DNA elements are central nodes in the regulatory circuitry that specifies mammalian embryonic development, the possible functional relevance of their>200bp of perfect sequence conservation between human-mouse-rat remains obscure 1,2. Here we have compared the in vivo enhancer activity of a genome-wide set of 231 non-exonic sequences with ultraconserved cores to that of 206 sequences that are under equivalently severe human-rodent constraint (ultra-like), but lack perfect sequence conservation. In transgenic mouse assays, 50percent of the ultraconserved and 50percent of the ultra-like conserved elements reproducibly functioned as tissue-specific enhancers at embryonic day 11.5. In this in vivo assay, we observed that ultraconserved enhancers and constrained non-ultraconserved enhancers targeted expression to a similar spectrum of tissues with a particular enrichment in the developing central nervous system. A human genome-wide comparative screen uncovered ~;;2,600 non-coding elements that evolved under ultra-like human-rodent constraint and are similarly enriched near transcriptional regulators and developmental genes as the much smaller number of ultraconserved elements. These data indicate that ultraconserved elements possessing absolute human-rodent sequence conservation are not distinct from other non-coding elements that are under comparable purifying selection in mammals and suggest they are principal constituents of the cis-regulatory framework of mammalian development.

  9. Helios Controls a Limited Subset of Regulatory T Cell Functions.

    PubMed

    Sebastian, Mathew; Lopez-Ocasio, Maria; Metidji, Amina; Rieder, Sadiye Amcaoglu; Shevach, Ethan M; Thornton, Angela M

    2016-01-01

    A subpopulation (60-70%) of Foxp3(+) regulatory T cells (Tregs) in both mouse and man expresses the transcription factor Helios, but its role in Treg function is still unknown. We generated Treg-specific Helios-deficient mice to examine the function of Helios in Tregs. We show that the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of organ-specific autoimmunity. Helios-deficient Treg suppressor function was normal in vitro, as well as in an in vivo inflammatory bowel disease model. However, Helios-deficient Tregs failed to control the expansion of pathogenic T cells derived from scurfy mice, failed to mediate T follicular regulatory cell function, and failed to control both T follicular helper cell and Th1 effector cell responses. In competitive settings, Helios-deficient Tregs, particularly effector Tregs, were at a disadvantage, indicating that Helios regulates effector Treg fitness. Thus, we demonstrate that Helios controls certain aspects of Treg-suppressive function, differentiation, and survival. PMID:26582951

  10. A monoclonal antibody-based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines

    PubMed Central

    Schmeisser, Falko; Vasudevan, Anupama; Soto, Jackeline; Kumar, Arunima; Williams, Ollie; Weir, Jerry P

    2014-01-01

    Background The potency of inactivated influenza vaccines is determined using a single radial immunodiffusion (SRID) assay. This assay is relatively easy to standardize, it is not technically demanding, and it is capable of measuring the potency of several vaccine strain subtypes in a multivalent vaccine. Nevertheless, alternative methods that retain the major advantages of the SRID, but with a greater dynamic range of measurement and with reduced reagent requirements, are needed. Objectives The feasibility of an ELISA-based assay format was explored as an alternative potency assay for inactivated influenza vaccines. Methods Several murine monoclonal antibodies (mAbs), specific for the 2009 pandemic H1N1 influenza virus hemagglutinin (HA), were evaluated for their potential to capture and quantify HA antigen. Vaccine samples, obtained from four licensed influenza vaccine manufacturers, included monovalent bulk vaccine, monovalent vaccine, and trivalent vaccine. Traditional SRID potency assays were run in parallel with the mAb–ELISA potency assay using the reference antigen standard appropriate for the vaccine samples being tested. Results The results indicated that the ELISA potency assay can quantify HA over a wide range of concentrations, including vaccine at subpotent doses, and the ELISA and SRID potency values correlated well for most vaccine samples. Importantly, the assay was capable of quantifying A/California HA in a trivalent formulation. Conclusions This study demonstrates the general feasibility of the mAb approach and strongly suggests that such ELISAs have potential for continued development as an alternative method to assay the potency of inactivated influenza vaccines. PMID:25087462

  11. Development and validation of a serological potency test for the release of Leptospira vaccines--requirements in the European Union.

    PubMed

    Balks, Elisabeth; Gyra, Heike; Kobe, Babett; Cussler, Klaus; Werner, Esther

    2013-09-01

    Both European Pharmacopoeia Monograph 01/2008:0447 "Canine Leptospirosis vaccine (inactivated)" and the more recent Monograph 01/2008:1939 "Bovine Leptospirosis vaccine (inactivated)" explicitly allow for a sero-response test to assess batch potency. Test setup and requirements for in vivo and in vitro validation are described. Furthermore, the two main strategies to assess batch potency and their specific demands are addressed. PMID:23911253

  12. CD4+ T-cell subsets and host defense in the lung

    PubMed Central

    Kolls, Jay K.

    2012-01-01

    Summary CD4+ T-helper subsets are lineages of T cells that have effector function in the lung and control critical aspects of lung immunity. Depletion of these cells experimentally or by drugs or human immunodeficiency virus (HIV) infection in humans leads to the development of opportunistic infections as well as increased rates of bacteremia with certain bacterial pneumonias. Recently, it has been proposed that CD4+ T-cell subsets may also be excellent targets for mucosal vaccination to prevent pulmonary infections in susceptible hosts. Here we review recent findings that increase our understanding of T-cell subsets and their effector cytokines in the context of pulmonary infection. PMID:23405903

  13. CXCR3 expression defines a novel subset of innate CD8+ T cells that enhance immunity against bacterial infection and cancer upon stimulation with IL-15.

    PubMed

    Oghumu, Steve; Terrazas, Cesar A; Varikuti, Sanjay; Kimble, Jennifer; Vadia, Stephen; Yu, Lianbo; Seveau, Stephanie; Satoskar, Abhay R

    2015-03-01

    Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from conventional CD8(+) T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C-X-C motif) receptor 3 (CXCR3)-positive innate CD8(+) T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL-15 activated CXCR3(+) innate CD8(+) T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN-γ(-/-) mice compared with similarly activated CXCR3(-) subset. This was associated with enhanced proliferation and IFN-γ production in CXCR3(+) cells. Further, CXCR3(+) innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3(+) subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a, IL-2Rβ, Atp5e, and Ly6c but reduced IFN-γR2 and Art2b. Ingenuity pathway analysis revealed an up-regulation of genes associated with T-cell activation, proliferation, cytotoxicity, and translational initiation in CXCR3(+) populations. Our results demonstrate that CXCR3 expression in innate CD8(+) T cells defines a subset with enhanced cytotoxic potential and protective antibacterial immune functions. Immunotherapeutic approaches against infectious disease and cancer could utilize CXCR3(+) innate CD8(+) T-cell populations as novel clinical intervention strategies. PMID:25466888

  14. Siglec-1-positive plasmacytoid dendritic cells (pDCs) in human peripheral blood: A semi-mature and myeloid-like subset imbalanced during protective and autoimmune responses.

    PubMed

    Wilhelm, Theresa R; Taddeo, Adriano; Winter, Oliver; Schulz, Axel Ronald; Mälzer, Julia-Nora; Domingo, Cristina; Biesen, Robert; Alexander, Tobias; Thiel, Andreas; Radbruch, Andreas; Hiepe, Falk; Gerl, Velia

    2016-02-01

    Plasmacytoid dendritic cells (pDCs) play a central role in the pathogenesis of systemic lupus erythematosus (SLE) as IFN-α producers and promoters of T-cell activation or tolerance. Here, we demonstrated by flow-cytometry and confocal microscopy that Siglec-1, a molecule involved in the regulation of adaptive immunoresponses, is expressed in a subset of semi-mature, myeloid-like pDCs in human blood. These pDCs express lower BDCA-2 and CD123 and higher HLA-DR and CD11c than Siglec-1-negative pDCs and do not produce IFN-α via TLR7/TLR9 engagement. In vitro, Siglec-1 expression was induced in Siglec-1-negative pDCs by influenza virus. Proportions of Siglec-1-positive/Siglec-1-negative pDCs were higher in SLE than in healthy controls and correlated with disease activity. Healthy donors immunized with yellow fever vaccine YFV-17D displayed different kinetics of the two pDC subsets during protective immune response. PDCs can be subdivided into two subsets according to Siglec-1 expression. These subsets may play specific roles in (auto)immune responses. PMID:26674280

  15. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

    PubMed

    Sloman, David L; Noucti, Njamkou; Altman, Michael D; Chen, Dapeng; Mislak, Andrea C; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility. PMID:27491711

  16. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

    PubMed

    Trautwein-Weidner, Kerstin; Gladiator, André; Kirchner, Florian R; Becattini, Simone; Rülicke, Thomas; Sallusto, Federica; LeibundGut-Landmann, Salomé

    2015-10-01

    Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity. PMID:26431538

  17. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis

    PubMed Central

    Kirchner, Florian R.; Becattini, Simone; Rülicke, Thomas; Sallusto, Federica; LeibundGut-Landmann, Salomé

    2015-01-01

    Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity. PMID:26431538

  18. The Role of Different Monocyte Subsets in the Pathogenesis of Atherosclerosis and Acute Coronary Syndromes.

    PubMed

    Idzkowska, E; Eljaszewicz, A; Miklasz, P; Musial, W J; Tycinska, A M; Moniuszko, M

    2015-09-01

    The inflammation underlying both atherosclerosis and acute coronary syndromes is strongly related to monocyte-related actions. However, different monocyte subsets can play differential roles in the formation and destabilization of atherosclerotic plaque as well as healing of damaged myocardial tissue. Monocytes are currently being divided into three functionally distinct subsets with different levels of CD14 (cluster of differentiation 14) and CD16 expression. Thus, there are classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. Here, we summarize the current knowledge on complex activities of different monocyte subsets in atherosclerosis and acute coronary syndromes. Moreover, we discuss which monocyte subsets can serve either as predictive biomarkers of cardiovascular risk or as potential targets used in atherosclerosis and its complications. PMID:25997925

  19. SCI model structure determination program (OSR) user's guide. [optimal subset regression

    NASA Technical Reports Server (NTRS)

    1979-01-01

    The computer program, OSR (Optimal Subset Regression) which estimates models for rotorcraft body and rotor force and moment coefficients is described. The technique used is based on the subset regression algorithm. Given time histories of aerodynamic coefficients, aerodynamic variables, and control inputs, the program computes correlation between various time histories. The model structure determination is based on these correlations. Inputs and outputs of the program are given.

  20. Delineation of a novel dendritic-like subset in human spleen

    PubMed Central

    Petvises, Sawang; Talaulikar, Dipti; O'Neill, Helen C

    2016-01-01

    Dendritic cells (DCs) and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c+hCD11b+HLA-DR−hCD86+ subset in the spleen, along with the previously described subsets of hCD1c+ DCs, hCD123+ plasmacytoid DCs (pDCs), hCD16+ DCs and hCD141+ DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c+HLA-DR+ and hCD11c+HLA-DR− cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCD80 and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors. PMID:25891217

  1. Delineation of a novel dendritic-like subset in human spleen.

    PubMed

    Petvises, Sawang; Talaulikar, Dipti; O'Neill, Helen C

    2016-07-01

    Dendritic cells (DCs) and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c(+)hCD11b(+)HLA-DR(-)hCD86(+) subset in the spleen, along with the previously described subsets of hCD1c(+) DCs, hCD123(+) plasmacytoid DCs (pDCs), hCD16(+) DCs and hCD141(+) DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c(+)HLA-DR(+) and hCD11c(+)HLA-DR(-) cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCD80 and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors. PMID:25891217

  2. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

    PubMed Central

    Zeitlin, Larry; Pettitt, James; Scully, Corinne; Bohorova, Natasha; Kim, Do; Pauly, Michael; Hiatt, Andrew; Ngo, Long; Steinkellner, Herta; Whaley, Kevin J.; Olinger, Gene G.

    2011-01-01

    No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED50 = 33 μg). A version with typical heterogenous mammalian glycoforms (ED50 = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED50 = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics. PMID:22143789

  3. Role of Bacillus thuringiensis Cry1 δ Endotoxin Binding in Determining Potency during Lepidopteran Larval Development

    PubMed Central

    Gilliland, Androulla; Chambers, Catherine E.; Bone, Eileen J.; Ellar, David J.

    2002-01-01

    Five economically important crop pests, Manduca sexta, Pieris brassicae, Mamestra brassicae, Spodoptera exigua, and Agrotis ipsilon, were tested at two stages of larval development for susceptibility to Bacillus thuringiensis toxins Cry1Ac, Cry1Ca, Cry1J, and Cry1Ba. Bioassay results for M. sexta showed that resistance to all four Cry toxins increased from the neonate stage to the third-instar stage; the increase in resistance was most dramatic for Cry1Ac, the potency of which decreased 37-fold. More subtle increases in resistance during larval development were seen in M. brassicae for Cry1Ca and in P. brassicae for Cry1Ac and Cry1J. By contrast, the sensitivity of S. exigua did not change during development. At both larval stages, A. ipsilon was resistant to all four toxins. Because aminopeptidase N (APN) is a putative Cry1 toxin binding protein, APN activity was measured in neonate and third-instar brush border membrane vesicles (BBMV). With the exception of S. exigua, APN activity was found to be significantly lower in neonates than in third-instar larvae and thus inversely correlated with increased resistance during larval development. The binding characteristics of iodinated Cry1 toxins were determined for neonate and third-instar BBMV. In M. sexta, the increased resistance to Cry1Ac and Cry1Ba during larval development was positively correlated with fewer binding sites in third-instar BBMV than in neonate BBMV. The other species-instar-toxin combinations did not reveal positive correlations between potency and binding characteristics. The correlation between binding and potency was inconsistent for the species-instar-toxin combinations used in this study, reaffirming the complex mode of action of Cry1 toxins. PMID:11916662

  4. Characterization of a naturally occurring breast cancer subset enriched in EMT and stem cell characteristics

    SciTech Connect

    Hennessy, Bryan T.; Gonzalez-Angulo, Ana-Maria; Stemke-Hale, Katherine; Gilcrease, Michael Z.; Krishnamurthy, Savitri; Lee, Ju-Seog; Fridlyand, Jane; Sahin, Aysegul; Agarwal, Roshan; Joy, Corwin; Liu, Wenbin; Stivers, David; Baggerly, Keith; Carey, Mark; Lluch, Ana; Monteagudo, Carlos; He, Xiaping; Weigman, Victor; Fan, Cheng; Palazzo, Juan; Hortobagyi, Gabriel N.; Nolden, Laura K.; Wang, Nicholas J.; Valero, Vicente; Gray, Joe W.; Perou, Charles M.; Mills, Gordon B.

    2009-05-19

    Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a 'tumorigenic' signature defined using CD44{sup +}/CD24{sup -} breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.

  5. [Dynamic changes of circulating monocyte subsets in high-NaCl diet fed mice].

    PubMed

    Feng, Xiaotong; Luo, Yanwei; Ma, Yongqiang; Zhao, Ying; Zhao, Qian; Ji, Wenjie; Li, Yuming; Zhou, Xin

    2016-06-01

    Objective To observe the dynamic changes of the circulating monocyte subsets in C57BL/6 mice fed with high-NaCl diet. Methods Male C57BL/6 mice were randomly divided into three groups: 9, 40 and 80 g/L NaCl groups. Before the treatment and 4, 8 and 12 weeks after the treatment, the cardiac function was dynamically determined by echocardiography and the blood pressure was measured by tail-cuff plethysmography. Flow cytometry analysis of circulating monocyte subsets was performed. HE staining was used to observe cardiac pathological changes at the time of sacrifice. Results Systolic blood pressure significantly increased with the progression of the high-salt diet. Compared with 9 g/L NaCl group, the ejection fraction of the other two groups slightly increased at week 4, followed by a significant decreasing trend up to week 12, in addition, the percentage of Ly6C(high) monocyte subset showed a progressive increase during high-salt feeding and reached a plateau at week 4, and then abruptly went down up to week 12. On the contrary, Ly6C(low) monocyte subset had an opposite trend, whereas Ly6C(int) monocyte subset remained constant. HE staining showed that cardiomyocyte size, as determined by the myocyte cross-sectional area, became enlarged obviously in the latter two groups. Conclusion Circulating monocyte subsets dynamically changed in the mice fed with high-salt diet. PMID:27371845

  6. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

    PubMed Central

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-01-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c+, CD141+ and CD16+ myeloid DCs and CD123+ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141+ DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  7. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

    PubMed

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-06-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  8. Monocyte Subsets and Related Chemokines in Carotid Artery Stenosis and Ischemic Stroke

    PubMed Central

    Grosse, Gerrit M.; Schulz-Schaeffer, Walter J.; Teebken, Omke E.; Schuppner, Ramona; Dirks, Meike; Worthmann, Hans; Lichtinghagen, Ralf; Maye, Gerrit; Limbourg, Florian P.; Weissenborn, Karin

    2016-01-01

    Carotid stenosis (CS) is an important cause of ischemic stroke. However, reliable markers for the purpose of identification of high-risk, so-called vulnerable carotid plaques, are still lacking. Monocyte subsets are crucial players in atherosclerosis and might also contribute to plaque rupture. In this study we, therefore, aimed to investigate the potential role of monocyte subsets and associated chemokines as clinical biomarkers for vulnerability of CS. Patients with symptomatic and asymptomatic CS (n = 21), patients with cardioembolic ischemic strokes (n = 11), and controls without any cardiovascular disorder (n = 11) were examined. Cardiovascular risk was quantified using the Essen Stroke Risk Score (ESRS). Monocyte subsets in peripheral blood were measured by quantitative flow cytometry. Plaque specimens were histologically analyzed. Furthermore, plasma levels of monocyte chemotactic protein 1 (MCP-1) and fractalkine were measured. Intermediate monocytes (Mon2) were significantly elevated in symptomatic and asymptomatic CS-patients compared to controls. Mon2 counts positively correlated with the ESRS. Moreover, stroke patients showed an elevation of Mon2 compared to controls, independent of the ESRS. MCP-1 levels were significantly higher in patients with symptomatic than in those with asymptomatic CS. Several histological criteria significantly differed between symptomatic and asymptomatic plaques. However, there was no association of monocyte subsets or chemokines with histological features of plaque vulnerability. Due to the multifactorial influence on monocyte subsets, the usability as clinical markers for plaque vulnerability seems to be limited. However, monocyte subsets may be critically involved in the pathology of CS. PMID:27023515

  9. An evaluation of exact methods for the multiple subset maximum cardinality selection problem.

    PubMed

    Brusco, Michael J; Köhn, Hans-Friedrich; Steinley, Douglas

    2016-05-01

    The maximum cardinality subset selection problem requires finding the largest possible subset from a set of objects, such that one or more conditions are satisfied. An important extension of this problem is to extract multiple subsets, where the addition of one more object to a larger subset would always be preferred to increases in the size of one or more smaller subsets. We refer to this as the multiple subset maximum cardinality selection problem (MSMCSP). A recently published branch-and-bound algorithm solves the MSMCSP as a partitioning problem. Unfortunately, the computational requirement associated with the algorithm is often enormous, thus rendering the method infeasible from a practical standpoint. In this paper, we present an alternative approach that successively solves a series of binary integer linear programs to obtain a globally optimal solution to the MSMCSP. Computational comparisons of the methods using published similarity data for 45 food items reveal that the proposed sequential method is computationally far more efficient than the branch-and-bound approach. PMID:27027582

  10. Monocyte Subsets and Related Chemokines in Carotid Artery Stenosis and Ischemic Stroke.

    PubMed

    Grosse, Gerrit M; Schulz-Schaeffer, Walter J; Teebken, Omke E; Schuppner, Ramona; Dirks, Meike; Worthmann, Hans; Lichtinghagen, Ralf; Maye, Gerrit; Limbourg, Florian P; Weissenborn, Karin

    2016-01-01

    Carotid stenosis (CS) is an important cause of ischemic stroke. However, reliable markers for the purpose of identification of high-risk, so-called vulnerable carotid plaques, are still lacking. Monocyte subsets are crucial players in atherosclerosis and might also contribute to plaque rupture. In this study we, therefore, aimed to investigate the potential role of monocyte subsets and associated chemokines as clinical biomarkers for vulnerability of CS. Patients with symptomatic and asymptomatic CS (n = 21), patients with cardioembolic ischemic strokes (n = 11), and controls without any cardiovascular disorder (n = 11) were examined. Cardiovascular risk was quantified using the Essen Stroke Risk Score (ESRS). Monocyte subsets in peripheral blood were measured by quantitative flow cytometry. Plaque specimens were histologically analyzed. Furthermore, plasma levels of monocyte chemotactic protein 1 (MCP-1) and fractalkine were measured. Intermediate monocytes (Mon2) were significantly elevated in symptomatic and asymptomatic CS-patients compared to controls. Mon2 counts positively correlated with the ESRS. Moreover, stroke patients showed an elevation of Mon2 compared to controls, independent of the ESRS. MCP-1 levels were significantly higher in patients with symptomatic than in those with asymptomatic CS. Several histological criteria significantly differed between symptomatic and asymptomatic plaques. However, there was no association of monocyte subsets or chemokines with histological features of plaque vulnerability. Due to the multifactorial influence on monocyte subsets, the usability as clinical markers for plaque vulnerability seems to be limited. However, monocyte subsets may be critically involved in the pathology of CS. PMID:27023515

  11. Anti-Citrullinated Protein Antibodies Induce Macrophage Subset Disequilibrium in RA Patients.

    PubMed

    Zhu, Wei; Li, Xiu; Fang, Shaohong; Zhang, Xiaoli; Wang, Ying; Zhang, Tongshuai; Li, Zhaoying; Xu, Yanwen; Qu, Siying; Liu, Chuanliang; Gao, Fei; Pan, Haile; Wang, Guangyou; Li, Hulun; Sun, Bo

    2015-12-01

    We used samples from rheumatoid arthritis (RA) patients to examine whether Anti-citrullinated protein antibodies (ACPAs) alter macrophage subset distribution and promote RA development. Macrophage subset distributions and interferon regulatory factor 4 (IRF4) and IRF5 expressions were analyzed. ACPAs were purified by affinity column. After RA and osteoarthritis (OA) patients' macrophages were cocultured with ACPAs, macrophage subsets and IRF4 and IRF5 expressions were measured. Small interfering RNAs (siRNAs) were transfected into ACPA-activated cells to suppress IRF4 or IRF5. Fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry were performed. Macrophage subset disequilibrium occurred in RA patient synovial fluids. IRF4 and IRF5 were all expressed in the synovial fluid and synovium. ACPAs (40 IU/ml) could induce macrophages to polarize to M1 subsets, and the percentage of increased M1/M2 ratio of RA patients was higher than that of the OA patients. ACPAs also induce IRF4 and IRF5 protein expressions. IRF5 siRNA transfection impaired ACPA activity significantly. We demonstrated that macrophage subset disequilibrium occurred in RA patients. ACPAs induced IRF5 activity and led to M1 macrophage polarization. PMID:26063186

  12. Comparison of gene expression profiles between human and mouse monocyte subsets

    PubMed Central

    Ingersoll, Molly A.; Spanbroek, Rainer; Lottaz, Claudio; Gautier, Emmanuel L.; Frankenberger, Marion; Hoffmann, Reinhard; Lang, Roland; Haniffa, Muzlifah; Collin, Matthew; Tacke, Frank; Habenicht, Andreas J. R.

    2010-01-01

    Blood of both humans and mice contains 2 main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the 2 species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor γ (PPARγ) signature in mouse monocytes, which is absent in humans, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in mice. PMID:19965649

  13. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases.

    PubMed

    Raphael, Itay; Nalawade, Saisha; Eagar, Todd N; Forsthuber, Thomas G

    2015-07-01

    CD4(+) T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful. PMID:25458968

  14. Isolation and Characterization of New Cannabis Constituents from a High Potency Variety

    PubMed Central

    Radwan, Mohamed M.; Ross, Samir A.; Slade, Desmond; Ahmed, Safwat A.; Zulfiqar, Fazila; ElSohly, Mahmoud A.

    2016-01-01

    Phytochemical investigation of a high potency variety of Cannabis sativa L. resulted in the isolation of six new metabolites, (±)-6,7-trans-epoxycannabigerolic acid (2), (±)-6,7-cis-epoxycannabigerolic acid (3), (±)-6,7-cis-epoxycannabigerol (4), (±)-6,7-trans-epoxycannabigerol (5), 5′-methyl-4-pentylbiphenyl-2,2′,6-triol (7), and 7-methoxycannabispirone (8), along with seven known compounds namely, cannabigerolic acid (1), 5′-methoxycannabigerolic acid (6), cannabispirone (9), β-cannabispiranol (10), dehydrocannabifuran (11), cannflavin B (12) and cannabigerol (13). The antimicrobial as well as the antileishmanial activities were investigated. PMID:18283614

  15. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    SciTech Connect

    Gibbs, Susan; Corsini, Emanuela; Spiekstra, Sander W.; Galbiati, Valentina; Fuchs, Horst W.; DeGeorge, George; Troese, Matthew; Hayden, Patrick; Deng, Wei; Roggen, Erwin

    2013-10-15

    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24 h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC{sub 50}) with human and animal data showed a superior correlation with human DSA{sub 05} (μg/cm{sup 2}) data (Spearman r = 0.8500; P value (two-tailed) = 0.0061) compared to LLNA data (Spearman r = 0.5968; P value (two-tailed) = 0.0542). DSA{sub 05} = induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA{sub 05} data (Spearman r = 0.8333; P value (two-tailed) = 0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model. - Highlights: • A potential epidermal equivalent assay to label and classify sensitizers • Il-18 release distinguishes sensitizers from non sensitizers • IL-18 release can rank sensitizer potency

  16. Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b.

    PubMed

    Rossi, Riccardo L; Rossetti, Grazisa; Wenandy, Lynn; Curti, Serena; Ripamonti, Anna; Bonnal, Raoul J P; Birolo, Roberto Sciarretta; Moro, Monica; Crosti, Maria C; Gruarin, Paola; Maglie, Stefano; Marabita, Francesco; Mascheroni, Debora; Parente, Valeria; Comelli, Mario; Trabucchi, Emilio; De Francesco, Raffaele; Geginat, Jens; Abrignani, Sergio; Pagani, Massimiliano

    2011-08-01

    MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA. PMID:21706005

  17. From the regulatory functions of B cells to the identification of cytokine-producing plasma cell subsets.

    PubMed

    Dang, Van Duc; Hilgenberg, Ellen; Ries, Stefanie; Shen, Ping; Fillatreau, Simon

    2014-06-01

    B lymphocytes have a unique role as antibody-producing cells. Antibodies are key mediators of humoral immunity against infections, and are thought to account for the protection afforded by successful vaccines. B cells can also secrete cytokines and subsequently regulate immune responses mediated by T and innate cells. Remarkably, recent studies identified plasma blasts/plasma cells as the main types of activated B cells producing the cytokines interleukin (IL)-10, IL-35, tumor necrosis factor (TNF)-α, IL-17, and GM-CSF in various contexts in mice. Here, we discuss these observations, which suggest the existence of various subsets of plasma blast/plasma cells distinguishable through their cytokine expression pattern. PMID:24637161

  18. Re-use of SNOMED CT subset in development of the Danish national standard for home care nursing problems.

    PubMed

    Højen, Anne Randorff; Gøeg, Kirstine Rosenbeck; Elberg, Pia Britt

    2015-01-01

    SNOMED CT was chosen as reference terminology for standardisation of homecare nursing documentation to make reporting comparable across the 98 Danish municipalities. The method outlined in this paper for developing a Danish national homecare nursing SNOMED CT subsets is a pragmatic approach to build new SNOMED CT subsets drawing on existing and available SNOMED CT subsets. Combining this approach with awareness of hierarchical coherency in SNOMED CT subsets makes effective retrieval of data possible. PMID:25991118

  19. INPP4B is upregulated and functions as an oncogenic driver through SGK3 in a subset of melanomas

    PubMed Central

    Wilmott, James S.; Guo, Xiang Yun; Yan, Xu Guang; Wang, Chun Yan; Liu, Xiao Ying; Jin, Lei; Tseng, Hsin-Yi; Liu, Tao; Croft, Amanda; Hondermarck, Hubert; Scolyer, Richard A.; Jiang, Chen Chen; Zhang, Xu Dong

    2015-01-01

    Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates PI3K/Akt signalling and has a tumour suppressive role in some types of cancers. However, we have found that it is upregulated in a subset of melanomas. Here we report that INPP4B can function as an oncogenic driver through activation of serum- and glucocorticoid-regulated kinase 3 (SGK3) in melanoma. While INPP4B knockdown inhibited melanoma cell proliferation and retarded melanoma xenograft growth, overexpression of INPP4B enhanced melanoma cell and melanocyte proliferation and triggered anchorage-independent growth of melanocytes. Noticeably, INPP4B-mediated melanoma cell proliferation was not related to activation of Akt, but was mediated by SGK3. Upregulation of INPP4B in melanoma cells was associated with loss of miRNA (miR)-494 and/or miR-599 due to gene copy number reduction. Indeed, overexpression of miR-494 or miR-599 downregulated INPP4B, reduced SGK3 activation, and inhibited melanoma cell proliferation, whereas introduction of anti-miR-494 or anti-miR-599 upregulated INPP4B, enhanced SGK3 activation, and promoted melanoma cell proliferation. Collectively, these results identify upregulation of INPP4B as an oncogenic mechanism through activation of SGK3 in a subset of melanomas, with implications for targeting INPP4B and restoring miR-494 and miR-599 as novel approaches in the treatment of melanomas with high INPP4B expression. PMID:26573229

  20. Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability

    PubMed Central

    Gresser, Amy L.; Gutzwiller, Lisa M.; Gauck, Mackenzie K.; Hartenstein, Volker; Cook, Tiffany A.; Gebelein, Brian

    2015-01-01

    Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability. PMID

  1. Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability.

    PubMed

    Gresser, Amy L; Gutzwiller, Lisa M; Gauck, Mackenzie K; Hartenstein, Volker; Cook, Tiffany A; Gebelein, Brian

    2015-01-01

    Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability. PMID

  2. Inhibitors of the Cysteine Synthase CysM with Antibacterial Potency against Dormant Mycobacterium tuberculosis.

    PubMed

    Brunner, Katharina; Maric, Selma; Reshma, Rudraraju Srilakshmi; Almqvist, Helena; Seashore-Ludlow, Brinton; Gustavsson, Anna-Lena; Poyraz, Ömer; Yogeeswari, Perumal; Lundbäck, Thomas; Vallin, Michaela; Sriram, Dharmarajan; Schnell, Robert; Schneider, Gunter

    2016-07-28

    Cysteine is an important amino acid in the redox defense of Mycobacterium tuberculosis, primarily as a building block of mycothiol. Genetic studies have implicated de novo cysteine biosynthesis in pathogen survival in infected macrophages, in particular for persistent M. tuberculosis. Here, we report on the identification and characterization of potent inhibitors of CysM, a critical enzyme in cysteine biosynthesis during dormancy. A screening campaign of 17 312 compounds identified ligands that bind to the active site with micromolar affinity. These were characterized in terms of their inhibitory potencies and structure-activity relationships through hit expansion guided by three-dimensional structures of enzyme-inhibitor complexes. The top compound binds to CysM with 300 nM affinity and displays selectivity over the mycobacterial homologues CysK1 and CysK2. Notably, two inhibitors show significant potency in a nutrient-starvation model of dormancy of Mycobacterium tuberculosis, with little or no cytotoxicity toward mammalian cells. PMID:27379713

  3. Relative potency as a means of evaluating ELF (Extremely Low Frequency) health risks

    SciTech Connect

    Easterly, C.E.; Glass, L.R.

    1990-09-01

    In the 1970's, a variety of developments took place to heightened public and scientific interest in electromagnetic fields. During this time, biological studies of nonionizing electromagnetic fields were taking place, but no clear evidence of risks to public health was identified. Then came the surprising epidemiological finding suggesting that 60 Hz magnetic fields may be related to some childhood leukemias. Our particular interest at ORNL was how to interpret the available data with respect to human exposures to the nearly ubiquitous fields. A review of the available data showed that consistent biological effects were difficult to identify. Classical toxicological tests used in chemical risk assessment had not been performed with Extremely Low Frequency (ELF) fields but rather a wide range of mechanistic studies had been pursued. To evaluate the level of anticipated hazard or risk there was neither a mechanistic understanding nor a consistent phenomenological outcome. A risk evaluation normally requires one or the other of these two types of information. Two quite different approaches were pursued: meta-analysis and relative potency. The first of these is a method to combine data from similar experiments to enhance the relative statistical power of a collection of small sample size studies, and will not be discussed further. The second, relative potency, will be the focus of this paper. 20 refs., 2 figs.

  4. In vitro potency of doripenem tested against an international collection of rarely isolated bacterial pathogens.

    PubMed

    Jones, Ronald N; Bell, Jan M; Sader, Helio S; Turnidge, John D; Stilwell, Matthew G

    2009-04-01

    Doripenem, a new 1beta-methyl parenteral carbapenem, has very broad-spectrum activity against Gram-positive and Gram-negative aerobic bacteria. As noted here, the spectrum and potency extended to many rarely isolated species sampled by the Doripenem Global Surveillance Program. Among the species or species groups with 0.25 microg/mL for all tested species except Lactococcus garvieae, Listeria monocytogenes, and Micrococcus spp. In conclusion, doripenem exhibited a very wide spectrum but variable potencies against uncommonly cultured aerobic bacterial pathogens isolated in 2003 to 2007. These results confirm the potential use of this new carbapenem for broad-spectrum empiric or directed antimicrobial therapy. PMID:19302927

  5. Bispecific Anti-HIV-1 Antibodies with Enhanced Breadth and Potency.

    PubMed

    Bournazos, Stylianos; Gazumyan, Anna; Seaman, Michael S; Nussenzweig, Michel C; Ravetch, Jeffrey V

    2016-06-16

    Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) suppress viremia in animal models of HIV-1 and humans. To achieve potent activity without the emergence of viral escape mutants, co-administration of different bNAbs is necessary to target distinct epitopes essential for viral fitness. Here, we report the development of bispecific anti-Env neutralizing antibodies (biNAbs) with potent activity. Synergistic activity of biNAbs was achieved by combining an engineered hinge domain of IgG3 to increase Fab domain flexibility necessary for hetero-bivalent binding to the Env trimer while retaining the functional properties of the IgG1-Fc. Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1-infected humanized mice. These findings suggest innovative strategies for generating biNAbs with enhanced neutralization breadth and potency, representing ideal candidate molecules for the control of HIV-1 infection. PMID:27315478

  6. A collaborative study of an alternative in vitro potency assay for the Japanese encephalitis vaccine.

    PubMed

    Kim, Byung-Chul; Kim, Do-Keun; Kim, Hyung-Jin; Hong, Seung-Hwa; Kim, Yeonhee; Lim, Jong-Mi; Hong, JiYoung; Kim, Cheol-Hee; Park, Yong-Keun; Kim, Jaeok

    2016-09-01

    The use of inactivated Japanese encephalitis (JE) vaccines has been ongoing in East Asia for 40 years. A mouse immunogenicity assay followed by a Plaque Reduction Neutralization (PRN) Test (PRNTest) is currently recommended for each lot release of the vaccine by many national authorities. We developed an alternative in vitro ELISA to determine the E antigen content of the Japanese encephalitis virus to observe the 3Rs strategy. A collaborative study for replacing the in vivo potency assay for the Japanese encephalitis vaccine with the in vitro ELISA assay was confirmed comparability between these two methods. The study demonstrated that an in vitro assay could perform faster and was more convenient than the established in vivo PRNTest. Moreover, this assay had better precision and reproducibility compared with the conventional in vivo assay. Additionally, the content of antigen determined using the in vitro ELISA correlated well with the potency of the in vivo assay. Furthermore, this method allowed discrimination between individual lots. Thus, we propose a progressive switch from the in vivo assay to the in vitro ELISA for JE vaccine quality control. PMID:27497622

  7. Antibacterial and antioxidant potency of floral honeys from different botanical and geographical origins.

    PubMed

    Alzahrani, Hasan A; Alsabehi, Rashid; Boukraâ, Laïd; Abdellah, Fatiha; Bellik, Yuva; Bakhotmah, Balkees A

    2012-01-01

    In order to assess their physicochemical and antioxidant properties as well as their antimicrobial potency, four varieties of honey from different botanical and geographical origins were used. The agar incorporation method was used to determine the antimicrobial potency of honeys. The total phenol content was determined by a modified Folin-Ciocalteu method and the free radical scavenging activity by the Fe(3+)reducing power (FRAP) assay. Manuka honey was the most effective against Staphylococcus aureus Oxa R and S. aureus Oxa S with a Minimum Inhibitory Concentration (MIC) of 6% and 7%, respectively, whereas wild carrot honey was the most effective against Pseudomonas aeruginosa, with a MIC of 12%. Lavender honey was the least effective against all tested strains, even though was found to have the lowest pH and water content. Manuka honey had the highest content of polyphenols, with 899.09 ± 11.75 mg gallic acid/kg, whereas lavender honey had the lowest, with 111.42 ± 3.54 mg gallic acid/kg. A very significant correlation (r value was 0.9079 at P < 0.05) was observed between the total polyphenolic content and the Fe(2+) content formed in the presence of the honey antioxidants. The differences between honey samples in terms of antibacterial and antioxidant activity could be attributed to the natural variations in floral sources of nectar and the different locations. PMID:22948516

  8. Specific increase in potency via structure-based design of a T cell receptor

    PubMed Central

    Malecek, Karolina; Grigoryan, Arsen; Zhong, Shi; Gu, Wei Jun; Johnson, Laura A.; Rosenberg, Steven A.; Cardozo, Timothy; Krogsgaard, Michelle

    2014-01-01

    Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are non-reactive “self” proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable anti-tumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related antigens. Our results showed that the mutated TCRs had improved T cell activation potency, while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. And even though of limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects. PMID:25070852

  9. Concise review: culture mediated changes in fate and/or potency of stem cells.

    PubMed

    Roobrouck, Valerie D; Vanuytsel, Kim; Verfaillie, Catherine M

    2011-04-01

    Although Gurdon demonstrated already in 1958 that the nucleus of intestinal epithelial cells could be reprogrammed to give rise to adult frogs, the field of cellular reprogramming has only recently come of age with the description by Takahashi and Yamanaka in 2006, which defined transcription factors can reprogram fibroblasts to an embryonic stem cell-like fate. With the mounting interest in the use of human pluripotent stem cells and culture-expanded somatic stem/progenitor cells, such as mesenchymal stem cells, increasing attention has been given to the effect of changes in the in vitro microenvironment on the fate of stem cells. These studies have demonstrated that changes in culture conditions may change the potency of pluripotent stem cells or reprogram adult stem/progenitor cells to endow them with a broader differentiation potential. The mechanisms underlying these fate and potency changes by ex vivo culture should be further investigated and considered when designing clinical therapies with stem/progenitor cells. PMID:21305670

  10. Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model

    PubMed Central

    Lagaye, Sylvie; Brun, Sonia; Gaston, Jesintha; Shen, Hong; Stranska, Ruzena; Camus, Claire; Dubray, Clarisse; Rousseau, Géraldine; Massault, Pierre-Philippe; Courcambeck, Jerôme; Bassisi, Firas; Halfon, Philippe; Pol, Stanislas

    2016-01-01

    AIM: To evaluate the antiviral potency of a new anti-hepatitis C virus (HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices (2.7 × 106 cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant (multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription - polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS: The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dose-dependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect (compared to hydroxychloroquine EC50 = 1.17 μmol/L). CONCLUSION: Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dose-dependent manner without cytotoxic effect. PMID:27478540

  11. Potential for Improving Potency and Specificity of Reovirus Oncolysis with Next-Generation Reovirus Variants

    PubMed Central

    Mohamed, Adil; Johnston, Randal N.; Shmulevitz, Maya

    2015-01-01

    Viruses that specifically replicate in tumor over normal cells offer promising cancer therapies. Oncolytic viruses (OV) not only kill the tumor cells directly; they also promote anti-tumor immunotherapeutic responses. Other major advantages of OVs are that they dose-escalate in tumors and can be genetically engineered to enhance potency and specificity. Unmodified wild type reovirus is a propitious OV currently in phase I–III clinical trials. This review summarizes modifications to reovirus that may improve potency and/or specificity during oncolysis. Classical genetics approaches have revealed reovirus variants with improved adaptation towards tumors or with enhanced ability to establish specific steps of virus replication and cell killing among transformed cells. The recent emergence of a reverse genetics system for reovirus has provided novel strategies to fine-tune reovirus proteins or introduce exogenous genes that could promote oncolytic activity. Over the next decade, these findings are likely to generate better-optimized second-generation reovirus vectors and improve the efficacy of oncolytic reotherapy. PMID:26633466

  12. Metrics other than potency reveal systematic variation in responses to cancer drugs

    PubMed Central

    Fallahi-Sichani, Mohammad; Honarnejad, Saman; Heiser, Laura M.; Gray, Joe W.; Sorger, Peter K.

    2014-01-01

    Large-scale analysis of cellular response to anti-cancer drugs typically focuses on variation in potency (IC50) assuming that it is the most important difference between effective/ineffective drugs or sensitive/resistant cells. We took a multi-parametric approach involving analysis of the slope of the dose-response curve (HS), the area under the curve (AUC) and the maximum effect (Emax). We found that some of these parameters vary systematically with cell line and others with drug class. For cell-cycle inhibitors, Emax often but not always correlated with cell proliferation rate. For drugs targeting the Akt/PI3K/mTOR pathway dose-response curves were unusually shallow. Classical pharmacology has no ready explanation for this phenomenon but single-cell analysis showed that it correlated with significant and heritable cell-to-cell variability in the extent of target inhibition. We conclude that parameters other than potency should be considered in the comparative analysis of drug response, particularly at clinically relevant concentrations near and above IC50. PMID:24013279

  13. Metrics other than potency reveal systematic variation in responses to cancer drugs.

    PubMed

    Fallahi-Sichani, Mohammad; Honarnejad, Saman; Heiser, Laura M; Gray, Joe W; Sorger, Peter K

    2013-11-01

    Large-scale analysis of cellular response to anticancer drugs typically focuses on variation in potency (half-maximum inhibitory concentration, (IC50)), assuming that it is the most important difference between effective and ineffective drugs or sensitive and resistant cells. We took a multiparametric approach involving analysis of the slope of the dose-response curve, the area under the curve and the maximum effect (Emax). We found that some of these parameters vary systematically with cell line and others with drug class. For cell-cycle inhibitors, Emax often but not always correlated with cell proliferation rate. For drugs targeting the Akt/PI3K/mTOR pathway, dose-response curves were unusually shallow. Classical pharmacology has no ready explanation for this phenomenon, but single-cell analysis showed that it correlated with significant and heritable cell-to-cell variability in the extent of target inhibition. We conclude that parameters other than potency should be considered in the comparative analysis of drug response, particularly at clinically relevant concentrations near and above the IC50. PMID:24013279

  14. Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

    PubMed

    Lobato, Francisco C F; Lima, Catarina G R D; Assis, Ronnie A; Pires, Prhiscylla S; Silva, Rodrigo O S; Salvarani, Felipe M; Carmo, Anderson O; Contigli, Christiane; Kalapothakis, Evanguedes

    2010-08-31

    Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia. PMID:20670910

  15. Resveratrol derivatives as promising chemopreventive agents with improved potency and selectivity

    PubMed Central

    Kondratyuk, Tamara P.; Park, Eun-Jung; Marler, Laura E.; Ahn, Soyoun; Yuan, Yang; Choi, Yongsoo; Yu, Rui; van Breemen, Richard B.; Sun, Bin; Hoshino, Juma; Cushman, Mark; Jermihov, Katherine C.; Mesecar, Andrew D.; Grubbs, Clinton J.; Pezzuto, John M.

    2014-01-01

    Despite scores of investigations, the actual impact of resveratrol (3,5,4′-trihydroxy-trans-stilbene) on human health, as a dietary component or supplement, remains moot. This is due to many factors, such as relatively low potency, pleiotropic mechanisms, and rapid metabolism. Nonetheless, as a promiscuous molecule that interacts with numerous targets, resveratrol can be viewed as a scaffold for designing structural relatives potentially capable of mediating more intense responses with greater mechanistic stringency. We currently report the synthesis and biological evaluation of 92 stilbene analogs. The compounds were tested with in vitro assays for activation of quinone reductase 1, inhibition of QR2, nitric oxide production, aromatase, NFκB, TPA-induced ornithine decarboxylase, or cyclooxygenase-1 and -2, quenching of 2,2-diphenyl-1-picrylhydrazyl free radical, interaction with estrogen receptors, and as antiproliferative agents. Several compounds were found to mediate responses with much greater potency than resveratrol; some mediated pleiotropic responses, as is the case with the parent molecule, but others were highly specific or totally inactive. When administered to rats, higher serum concentrations and greater stability was demonstrated with prototype lead molecules. Due to structural simplicity, facile syntheses are available for large-scale production. These data support the promise of more advanced development of novel resveratrol derivatives as drug entities. PMID:21714126

  16. European regulatory framework and practices for veterinary Leptospira vaccine potency testing.

    PubMed

    Bruckner, Lukas

    2013-09-01

    In Europe, the legal basis for requirements for medicinal products is described in the European Pharmacopoeia (Ph. Eur.) In the European Union, the Ph. Eur. is supplemented by several guidelines issued by the European Medicines Agency. Immunological veterinary products must comply with the Ph. Eur. monograph on veterinary vaccines and the accompanying texts, as well as specific monographs. The Ph. Eur. includes monographs on canine leptospirosis and bovine leptospirosis vaccines (inactivated). Both monographs require that an immunogenicity test be performed once in the target species during the life of a vaccine. The hamster challenge test is applied for batch potency testing of canine vaccines. Alternatively, serological tests or suitable validated in vitro tests to determine the content of one or more antigenic components indicative of protection may be performed. Vaccines for use in cattle are tested in a serological test in guinea pigs. The acceptance criteria in alternative tests are set with reference to a batch of vaccine that has given satisfactory results in the immunogenicity test. At a January 2012 European workshop, the suitability of the hamster potency test was questioned and unanimous agreement was reached that moving toward complete in vitro testing is possible and should be promoted. PMID:23886926

  17. Agmatine enhances antidepressant potency of MK-801 and conventional antidepressants in mice.

    PubMed

    Neis, Vivian Binder; Moretti, Morgana; Manosso, Luana Meller; Lopes, Mark W; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2015-03-01

    Agmatine, an endogenous guanidine amine, has been shown to produce antidepressant-like effects in animal studies. This study investigated the effects of the combined administration of agmatine with either conventional monoaminergic antidepressants or the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 in the tail suspension test (TST) in mice. The aim was to evaluate the extent of the antidepressant synergism by examining the ability of a fixed dose of agmatine to shift the antidepressant potency of fluoxetine, imipramine, bupropion and MK-801. A sub-effective dose of agmatine (0.0001 mg/kg, p.o.) significantly increased the potency by which fluoxetine, imipramine, bupropion and MK-801 decreased immobility time in the TST by 2-fold (fluoxetine), 10-fold (imipramine and bupropion) and 100-fold (MK-801). Combined with previous evidence indicating a role of monoaminergic systems in the effect of agmatine, the current data suggest that agmatine may modulate monoaminergic neurotransmission and augment the activity of conventional antidepressants. Moreover, this study found that agmatine substantially augmented the antidepressant-like effect of MK-801, reinforcing the notion that this compound modulates NMDA receptor activation. These preclinical data may stimulate future clinical studies testing the effects of augmentation therapy with agmatine for the management of depressive disorders. PMID:25553821

  18. Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic α-Helical Peptides*

    PubMed Central

    Vermeer, Louic S.; Lan, Yun; Abbate, Vincenzo; Ruh, Emrah; Bui, Tam T.; Wilkinson, Louise J.; Kanno, Tokuwa; Jumagulova, Elmira; Kozlowska, Justyna; Patel, Jayneil; McIntyre, Caitlin A.; Yam, W. C.; Siu, Gilman; Atkinson, R. Andrew; Lam, Jenny K. W.; Bansal, Sukhvinder S.; Drake, Alex F.; Mitchell, Graham H.; Mason, A. James

    2012-01-01

    We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens. PMID:22869378

  19. Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment.

    PubMed

    Hauger, Richard L; Olivares-Reyes, J Alberto; Braun, Sandra; Hernandez-Aranda, Judith; Hudson, Christine C; Gutknecht, Eric; Dautzenberg, Frank M; Oakley, Robert H

    2013-09-10

    The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response. PMID:23820308

  20. Non-suppressive regulatory T cell subset expansion in pulmonary arterial hypertension.

    PubMed

    Sada, Yoshiharu; Dohi, Yoshihiro; Uga, Sayuri; Higashi, Akifumi; Kinoshita, Hiroki; Kihara, Yasuki

    2016-08-01

    Regulatory T cells (Tregs) have been reported to play a pivotal role in the vascular remodeling of pulmonary arterial hypertension (PAH). Recent studies have revealed that Tregs are heterogeneous and can be characterized by three phenotypically and functionally different subsets. In this study, we investigated the roles of Treg subsets in the pathogenesis of PAH in eight patients with PAH and 14 healthy controls. Tregs and their subsets in peripheral blood samples were analyzed by flow cytometry. Treg subsets were defined as CD4(+)CD45RA(+)FoxP3(low) resting Tregs (rTregs), CD4(+)CD45RA(-)FoxP3(high) activated Tregs (aTregs), and CD4(+)CD45RA(-)FoxP3(low) non-suppressive Tregs (non-Tregs). The proportion of Tregs among CD4(+) T cells was significantly higher in PAH patients than in controls (6.54 ± 1.10 vs. 3.81 ± 0.28 %, p < 0.05). Of the three subsets, the proportion of non-Tregs was significantly elevated in PAH patients compared with controls (4.06 ± 0.40 vs. 2.79 ± 0.14 %, p < 0.01), whereas those of rTregs and aTregs were not different between the two groups. Moreover, the expression levels of cytotoxic T lymphocyte antigen 4, a functional cell surface molecule, in aTregs (p < 0.05) and non-Tregs (p < 0.05) were significantly higher in PAH patients compared with controls. These results suggested the non-Treg subset was expanded and functionally activated in peripheral lymphocytes obtained from IPAH patients. We hypothesize that immunoreactions involving the specific activation of the non-Treg subset might play a role in the vascular remodeling of PAH. PMID:26319442

  1. Caffeine's antioxidant potency optically sensed with double-stranded DNA-encased single-walled carbon nanotubes.

    PubMed

    Zhao, Edward H; Ergul, Busra; Zhao, Wei

    2015-03-12

    There is great interest in developing a sensitive method being able to quantitatively measure and compare antioxidant potencies of samples of interest against multiple reactive oxygen species (ROS) whose imbalance could cause oxidative stress. Here, a sensitive nanoprobe, double-stranded DNA encased single-walled carbon nanotubes (SWNTs) has been developed to determine antioxidant potencies of selected samples (caffeine, regular coffee, and decaffeinated coffee) against ROS, hydrogen peroxide, and hydroxyl radicals. Antioxidant vitamin C and uric acid are used as standards. The method focuses on unique dual optical sensing capability of SWNTs, the rate of spectral suppression when exposed to ROS, and the magnitude of spectral recovery of the ROS-suppressed SWNTs when an antioxidant is added. It is found that the dual sensing capability of SWNTs is still sustained when reacting with the reactive hydroxyl radicals. The results show that caffeine's antioxidant potency is weak, about one millionth of those of vitamin C and uric acid. It is a better scavenger of hydrogen peroxide and a little less effective for hydroxyl radicals. In comparison, coffee, regardless of regular or decaffeinated, is a more efficient antioxidant than caffeine, having an antioxidant potency about ten thousand times stronger. This work provides a versatile detection method for evaluating the antioxidant potencies of samples of interest against various ROS for chemical, biological, and medical applications. PMID:25714428

  2. A multi-dose serological assay suitable to quantify the potency of inactivated rabies vaccines for veterinary use.

    PubMed

    Krämer, Beate; Kamphuis, Elisabeth; Hanschmann, Kay-Martin; Milne, Catherine; Daas, Arnold; Duchow, Karin

    2013-11-01

    The mouse vaccination-challenge test, which is the most widely used method for determining the potency of inactivated rabies vaccines, is imprecise, time-consuming, and causes severe distress to the test animals. An alternative single-dose serological method has been implemented in the European Pharmacopoeia Monograph 0451 to replace the mouse challenge test for batch release. This single-dose limit method provides semi-quantitative results, but is not suitable for quantifying potency. We have now extended this serological method to a multi-dose format which allows a quantification of vaccine potency. In studies including all rabies vaccine strains relevant for Europe, we found dose-dependency for all vaccines and standard preparations. We have demonstrated that the multi-dose serological approach provides reliable quantitative potency results and is more precise than the mouse vaccination-challenge test. We have shown that adjuvanted vaccines can be calibrated against non-adjuvanted material, and that reference material can be calibrated against the International Standard. The method is therefore capable of assigning potency with the additional advantage of requiring fewer animals and reducing distress. Once the applicability of the method has been further verified in a collaborative study, it can complement the single-dose assay and eventually eliminate the need for the mouse challenge test. PMID:24144483

  3. B Cell Anergy Modulated by TLR1/2 and the miR-17∼92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4.

    PubMed

    Ntoufa, Stavroula; Papakonstantinou, Nikos; Apollonio, Benedetta; Gounari, Maria; Galigalidou, Chrysi; Fonte, Eleonora; Anagnostopoulos, Achilles; Belessi, Chrysoula; Muzio, Marta; Ghia, Paolo; Stamatopoulos, Kostas

    2016-05-15

    Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca(2+) or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR-17∼92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17∼92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease. PMID:27059597

  4. Allergen-Specific Immunotherapy Alters the Frequency, as well as the FcR and CLR Expression Profiles of Human Dendritic Cell Subsets

    PubMed Central

    Lundberg, Kristina; Rydnert, Frida; Broos, Sissela; Andersson, Morgan; Greiff, Lennart; Lindstedt, Malin

    2016-01-01

    Allergen-specific immunotherapy (AIT) induces tolerance and shifts the Th2 response towards a regulatory T-cell profile. The underlying mechanisms are not fully understood, but dendritic cells (DC) play a vital role as key regulators of T-cell responses. DCs interact with allergens via Fc receptors (FcRs) and via certain C-type lectin receptors (CLRs), including CD209/DC-SIGN, CD206/MR and Dectin-2/CLEC6A. In this study, the effect of AIT on the frequencies as well as the FcR and CLR expression profiles of human DC subsets was assessed. PBMC was isolated from peripheral blood from seven allergic donors before and after 8 weeks and 1 year of subcutaneous AIT, as well as from six non-allergic individuals. Cells were stained with antibodies against DC subset-specific markers and a panel of FcRs and CLRs and analyzed by flow cytometry. After 1 year of AIT, the frequency of CD123+ DCs was increased and a larger proportion expressed FcεRI. Furthermore, the expression of CD206 and Dectin-2 was reduced on CD141+ DCs after 1 year of treatment and CD206 as well as Dectin-1 was additionally down regulated in CD1c+ DCs. Interestingly, levels of DNGR1/CLEC9A on CD141+ DCs were increased by AIT, reaching levels similar to cells isolated from non-allergic controls. The modifications in phenotype and occurrence of specific DC subsets observed during AIT suggest an altered capacity of DC subsets to interact with allergens, which can be part of the mechanisms by which AIT induces allergen tolerance. PMID:26863539

  5. T cell subset profile in healthy Zambian adults at the University Teaching Hospital

    PubMed Central

    Chisenga, Caroline Cleopatra; Kelly, Paul

    2016-01-01

    Introduction Symptom-free human immunodeficiency virus antibody-negative Zambian adults (51 subjects, aged 20 to 62 years, 33.3% women and 66.7% men) were studied to establish T cell subset reference ranges. Methods We carried out across sectional study at the University Teaching Hospital, Lusaka. Blood samples were collected from healthy donor volunteers from hospital health care staff, between February and March 2015. Immunopheno typing was undertaken to characterize Tcell subsets using the markers CD3, CD4, CD8, α4β7, Ki67, CD25, CCR7, CD54RA, CD57, CD28, CD27 and HLA-DR. Results Among 51 volunteers, Women had significantly higher absolute CD4 count (median 1042; IQR 864, 1270) than in men (671; 545, 899) (p=0.003). Women also had more CD4 cells expressing homing, naïve, effector and effector memory T cell subsets compared to men. However, in the CD8 population, only the effector cells were significantly different with women expressing more than the males. Conclusion We provide early reference range for T cell subsets in Zambian adults and conclude that among the African women some T cell subsets are higher than men. PMID:27231509

  6. Identifying informative subsets of the Gene Ontology with information bottleneck methods

    PubMed Central

    Jin, Bo; Lu, Xinghua

    2010-01-01

    Motivation: The Gene Ontology (GO) is a controlled vocabulary designed to represent the biological concepts pertaining to gene products. This study investigates the methods for identifying informative subsets of GO terms in an automatic and objective fashion. This task in turn requires addressing the following issues: how to represent the semantic context of GO terms, what metrics are suitable for measuring the semantic differences between terms, how to identify an informative subset that retains as much as possible of the original semantic information of GO. Results: We represented the semantic context of a GO term using the word-usage-profile associated with the term, which enables one to measure the semantic differences between terms based on the differences in their semantic contexts. We further employed the information bottleneck methods to automatically identify subsets of GO terms that retain as much as possible of the semantic information in an annotation database. The automatically retrieved informative subsets align well with an expert-picked GO slim subset, cover important concepts and proteins, and enhance literature-based GO annotation. Availability: http://carcweb.musc.edu/TextminingProjects/ Contact: xinghua@pitt.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20702400

  7. Subsemble: an ensemble method for combining subset-specific algorithm fits

    PubMed Central

    Sapp, Stephanie; van der Laan, Mark J.; Canny, John

    2013-01-01

    Ensemble methods using the same underlying algorithm trained on different subsets of observations have recently received increased attention as practical prediction tools for massive datasets. We propose Subsemble: a general subset ensemble prediction method, which can be used for small, moderate, or large datasets. Subsemble partitions the full dataset into subsets of observations, fits a specified underlying algorithm on each subset, and uses a clever form of V-fold cross-validation to output a prediction function that combines the subset-specific fits. We give an oracle result that provides a theoretical performance guarantee for Subsemble. Through simulations, we demonstrate that Subsemble can be a beneficial tool for small to moderate sized datasets, and often has better prediction performance than the underlying algorithm fit just once on the full dataset. We also describe how to include Subsemble as a candidate in a SuperLearner library, providing a practical way to evaluate the performance of Subsemlbe relative to the underlying algorithm fit just once on the full dataset. PMID:24778462

  8. Impact of Toxoplasma gondii on Dendritic Cell Subset Function in the Intestinal Mucosa.

    PubMed

    Cohen, Sara B; Denkers, Eric Y

    2015-09-15

    The function of mucosal dendritic cell (DC) subsets in immunity and inflammation is not well understood. In this study, we define four DC subsets present within the lamina propria and mesenteric lymph node compartments based on expression of CD103 and CD11b. Using IL-12p40 YFP (Yet40) reporter mice, we show that CD103(+)CD11b(-) mucosal DCs are primary in vivo sources of IL-12p40; we also identified CD103(-)CD11b(-) mucosal DCs as a novel population producing this cytokine. Infection was preferentially found in CD11b(+) DCs that were negative for CD103. Lamina propria DCs containing parasites were negative for IL-12p40. Instead, production of the cytokine was strictly a property of noninfected cells. We also show that vitamin A metabolism, as measured by ALDH activity, was preferentially found in CD103(+)CD11b(+) DC and was strongly downregulated in all mucosal DC subsets during infection. Finally, overall apoptosis of lamina propria DC subsets was increased during infection. Combined, these results highlight the ability of intestinal Toxoplasma infection to alter mucosal DC activity at both the whole population level and at the level of individual subsets. PMID:26283477

  9. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    NASA Astrophysics Data System (ADS)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  10. Sources of variation in the mutagenic potency of complex chemical mixtures based on the salmonella/microsome assay

    SciTech Connect

    Krewski, D.; Leroux, B.G.; Creason, J.; Claxton, L.

    1992-01-01

    Twenty laboratories worldwide participated in a collaborative trial sponsored by the International Program on Chemical Safety on the mutagenicity of complex mixtures as expressed in the Salmonella/microsome assay. The U.S. National Institute of Standards and Technology provided homogeneous reference samples of urban air and diesel particles and a coal tar solution to each participating laboratory, along with samples of benzo(a)pyrene and 1-nitropyrene which served as positive controls. Mutagenic potency was characterized by the slope of the initial linear component of the dose response curve. Analysis of variance revealed significant interlaboratory variation in mutagenic potency, which accounted for 57-96% of the total variance on a logarithmic scale, depending on the sample, strain and activation conditions. No significant differences were noted in the average potency reported for air and diesel particles between laboratories using soxhlet extracts and those using sonication, although there was larger interlaboratory variation for the soxhlet method.

  11. Effects of alcohols on murine preimplantation development: relationship to relative membrane disordering potency.

    PubMed

    Kowalczyk, C L; Stachecki, J J; Schultz, J F; Leach, R E; Armant, D R

    1996-05-01

    During in vitro culture of murine preimplantation embryos, we have observed that exposure to 0.1% ethanol induces an immediate increase in intracellular calcium levels and subsequently accelerates embryogenesis. If the observed effects of ethanol on developing embryos is mediated by its membrane disordering potency, we hypothesized that the relative membrane disordering potencies of related alcohols would correspondingly effect embryonic intracellular calcium levels and developmental rates. Two-cell embryos were exposed to 0.1% ethanol or 0.05 to 1.0% (w/v) n-butanol, n-propanol, isopropanol, 1,2-propanediol, glycerol, or methanol for 24 hr at 37 degrees C, and development to the blastocyst stage was monitored after 5 days. n-Butanol, n-propanol, isopropanol, and methanol treatment caused a dose-dependent inhibition (p < 0.01) of development to the blastocyst stage, whereas 1,2-propanediol or glycerol neither accelerated nor inhibited development. In a second experiment, 8-cell morulae were treated with 1,2-propanediol or glycerol, and cavitation rates were examined. There was no significant difference from control embryos in the onset of cavitation or the blastocoel expansion rate of 1,2-propanediol- or glycerol-exposed embryos, whereas exposure to 0.1% ethanol accelerate cavitation (p > 0.05). In a third experiment, morulae were exposed to 0.1% or 1.0% of each alcohol and were monitored for changes in intracellular calcium levels using the fluorescent indicator, fluo-3-acetoxymethyl ester. There was an immediate increase in intracellular calcium levels when morulae were treated with 1.0% ethanol or n-butanol, but only ethanol induced an increase (p < 0.05) in the level of intracellular calcium at 0.1%. These data suggest that ethanol is unique in its ability to accelerate embryogenesis and that the membrane disordering potency of ethanol does not directly underlie its effects on intracellular calcium release and the acceleration of preimplantation development

  12. Digital image correlation involves an inverse problem: A regularization scheme based on subset size constraint

    NASA Astrophysics Data System (ADS)

    Zhan, Qin; Yuan, Yuan; Fan, Xiangtao; Huang, Jianyong; Xiong, Chunyang; Yuan, Fan

    2016-06-01

    Digital image correlation (DIC) is essentially implicated in a class of inverse problem. Here, a regularization scheme is developed for the subset-based DIC technique to effectively inhibit potential ill-posedness that likely arises in actual deformation calculations and hence enhance numerical stability, accuracy and precision of correlation measurement. With the aid of a parameterized two-dimensional Butterworth window, a regularized subpixel registration strategy is established, in which the amount of speckle information introduced to correlation calculations may be weighted through equivalent subset size constraint. The optimal regularization parameter associated with each individual sampling point is determined in a self-adaptive way by numerically investigating the curve of 2-norm condition number of coefficient matrix versus the corresponding equivalent subset size, based on which the regularized solution can eventually be obtained. Numerical results deriving from both synthetic speckle images and actual experimental images demonstrate the feasibility and effectiveness of the set of newly-proposed regularized DIC algorithms.

  13. Optimal search-based gene subset selection for gene array cancer classification.

    PubMed

    Li, Jiexun; Su, Hua; Chen, Hsinchun; Futscher, Bernard W

    2007-07-01

    High dimensionality has been a major problem for gene array-based cancer classification. It is critical to identify marker genes for cancer diagnoses. We developed a framework of gene selection methods based on previous studies. This paper focuses on optimal search-based subset selection methods because they evaluate the group performance of genes and help to pinpoint global optimal set of marker genes. Notably, this paper is the first to introduce tabu search (TS) to gene selection from high-dimensional gene array data. Our comparative study of gene selection methods demonstrated the effectiveness of optimal search-based gene subset selection to identify cancer marker genes. TS was shown to be a promising tool for gene subset selection. PMID:17674622

  14. MIClique: An algorithm to identify differentially coexpressed disease gene subset from microarray data.

    PubMed

    Zhang, Huanping; Song, Xiaofeng; Wang, Huinan; Zhang, Xiaobai

    2009-01-01

    Computational analysis of microarray data has provided an effective way to identify disease-related genes. Traditional disease gene selection methods from microarray data such as statistical test always focus on differentially expressed genes in different samples by individual gene prioritization. These traditional methods might miss differentially coexpressed (DCE) gene subsets because they ignore the interaction between genes. In this paper, MIClique algorithm is proposed to identify DEC gene subsets based on mutual information and clique analysis. Mutual information is used to measure the coexpression relationship between each pair of genes in two different kinds of samples. Clique analysis is a commonly used method in biological network, which generally represents biological module of similar function. By applying the MIClique algorithm to real gene expression data, some DEC gene subsets which correlated under one experimental condition but uncorrelated under another condition are detected from the graph of colon dataset and leukemia dataset. PMID:20169000

  15. An algorithm for extracting maximum cardinality subsets with perfect dominance or anti-Robinson structures.

    PubMed

    Brusco, Michael J; Stahl, Stephanie

    2007-11-01

    A common criterion for seriation of asymmetric matrices is the maximization of the dominance index, which sums the elements above the main diagonal of a reordered matrix. Similarly, a popular seriation criterion for symmetric matrices is the maximization of an anti-Robinson gradient index, which is associated with the patterning of elements in the rows and columns of a reordered matrix. Although perfect dominance and perfect anti-Robinson structure are rarely achievable for empirical matrices, we can often identify a sizable subset of objects for which a perfect structure is realized. We present and demonstrate an algorithm for obtaining a maximum cardinality (i.e. the largest number of objects) subset of objects such that the seriation of the proximity matrix corresponding to the subset will have perfect structure. MATLAB implementations of the algorithm are available for dominance, anti-Robinson and strongly anti-Robinson structures. PMID:17971276

  16. Comparison of in vivo genotoxic and carcinogenic potency to augment mode of action analysis: Case study with hexavalent chromium.

    PubMed

    Thompson, Chad M; Bichteler, Anne; Rager, Julia E; Suh, Mina; Proctor, Deborah M; Haws, Laurie C; Harris, Mark A

    2016-04-01

    Recent analyses-highlighted by the International Workshops on Genotoxicity Testing Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment-have identified a correlation between (log) estimates of a carcinogen's in vivo genotoxic potency and in vivo carcinogenic potency in typical laboratory animal models, even when the underlying data have not been matched for tissue, species, or strain. Such a correlation could have important implications for risk assessment, including informing the mode of action (MOA) of specific carcinogens. When in vivo genotoxic potency is weak relative to carcinogenic potency, MOAs other than genotoxicity (e.g., endocrine disruption or regenerative hyperplasia) may be operational. Herein, we review recent in vivo genotoxicity and carcinogenicity data for hexavalent chromium (Cr(VI)), following oral ingestion, in relevant tissues and species in the context of the aforementioned correlation. Potency estimates were generated using benchmark doses, or no-observable-adverse-effect-levels when data were not amenable to dose-response modeling. While the ratio between log values for carcinogenic and genotoxic potency was ≥1 for many compounds, the ratios for several Cr(VI) datasets (including in target tissue) were less than unity. In fact, the ratios for Cr(VI) clustered closely with ratios for chloroform and diethanolamine, two chemicals posited to have non-genotoxic MOAs. These findings suggest that genotoxicity may not play a major role in the cancers observed in rodents following exposure to high concentrations of Cr(VI) in drinking water-a finding consistent with recent MOA and adverse outcome pathway (AOP) analyses concerning Cr(VI). This semi-quantitative analysis, therefore, may be useful to augment traditional MOA and AOP analyses. More case examples will be needed to further explore the general applicability and validity of this approach for human health risk assessment. PMID:27085472

  17. CD4+ T-cell subsets in inflammatory diseases: beyond the Th1/Th2 paradigm.

    PubMed

    Hirahara, Kiyoshi; Nakayama, Toshinori

    2016-04-01

    CD4(+)T cells are crucial for directing appropriate immune responses during host defense and for the pathogenesis of inflammatory diseases. In addition to the classical biphasic model of differentiation of T-helper 1 (Th1) and Th2 cells, unexpected increases in the numbers of CD4(+)T-cell subsets, including Th17, Th9, T follicular-helper (Tfh) and T-regulatory (Treg) cells, have been recognized. In the present review, we focus on how these various T-helper cell subsets contribute to the pathogenesis of immune-mediated inflammatory diseases. In particular, we focus on multiple sclerosis, psoriasis and asthma as typical model diseases in which multiple T-helper cell subsets have recently been suggested to play a role. We will also discuss various unique sub-populations of T-helper cells that have been identified. First, we will introduce the heterogeneous T-helper cell subsets, which are classified by their simultaneous expression of multiple key transcription factors. We will also introduce different kinds of memory-type Th2 cells, which are involved in the pathogenesis of chronic type-2 immune-related diseases. Finally, we will discuss the molecular mechanisms underlying the generation of the plasticity and heterogeneity of T-helper cell subsets. The latest progress in the study of T-helper cell subsets has forced us to reconsider the etiology of immune-mediated inflammatory diseases beyond the model based on the Th1/Th2 balance. To this end, we propose another model--the pathogenic T-helper population disease-induction model--as a possible mechanism for the induction and/or persistence of immune-mediated inflammatory diseases. PMID:26874355

  18. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

    PubMed

    Cron, Andrew; Gouttefangeas, Cécile; Frelinger, Jacob; Lin, Lin; Singh, Satwinder K; Britten, Cedrik M; Welters, Marij J P; van der Burg, Sjoerd H; West, Mike; Chan, Cliburn

    2013-01-01

    Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM) approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM) naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC) samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a consistent labeling

  19. Potency testing of veterinary rabies vaccines: replacement of challenge by in vitro testing: considerations for development of alternative assays.

    PubMed

    Lewis, C E; Fry, A M; Hermann, J R; Siev, D; Dusek, D M; Gatewood, D M

    2012-01-01

    Vaccination of domestic animals against rabies creates a critical barrier between wildlife reservoirs and the human population. Ensuring these vaccines are potent and effective is paramount in preventing human exposure to this deadly and costly disease. The National Institutes of Health (NIH) test is, at present, the most widely used and internationally recommended potency assay for batch testing inactivated rabies vaccines. This test has numerous inherent limitations and disadvantages, including a lack of precision. The NIH test requires a large number of animals and involves unrelieved pain and suffering. A relevant in vitro assay should provide a more accurate, reproducible, rapid, safe, and humane rabies vaccine potency test. PMID:22888592

  20. Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains.

    SciTech Connect

    Zhou, H.; Sheng, S.; Compton, D.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.; Katzenellenbogen, B.; Nettles, K.; Greene, G.; Katzenellenbogen, J.; Biosciences Division; Univ. of Illinois; Univ. of Chicago; The Scripps Research Inst.

    2007-01-01

    2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

  1. Strictly ordered minimal subsets of a class of convex monotone skew-product semiflows

    NASA Astrophysics Data System (ADS)

    Novo, Sylvia; Obaya, Rafael

    We study the topological and ergodic structure of a class of convex and monotone skew-product semiflows. We assume the existence of two strongly ordered minimal subsets K 1, K 2 and we obtain an ergodic representation of their upper Lyapunov exponents. In the case of null upper Lyapunov exponents, we obtain a lamination into minimal subsets of an intermediate region where the restriction of the semiflow is affine. In the hyperbolic case, we deduce the long-time behaviour of every trajectory ordered with K2. Some examples of skew-product semiflows generated by non-autonomous differential equations and satisfying the assumptions of monotonicity and convexity are also presented.

  2. A unifying framework for seed sensitivity and its application to subset seeds.

    PubMed

    Kucherov, Gregory; Noé, Laurent; Roytberg, Mikhail

    2006-04-01

    We propose a general approach to compute the seed sensitivity, that can be applied to different definitions of seeds. It treats separately three components of the seed sensitivity problem--a set of target alignments, an associated probability distribution, and a seed model--that are specified by distinct finite automata. The approach is then applied to a new concept of subset seeds for which we propose an efficient automaton construction. Experimental results confirm that sensitive subset seeds can be efficiently designed using our approach, and can then be used in similarity search producing better results than ordinary spaced seeds. PMID:16819802

  3. Altered cytokine production by specific human peripheral blood cell subsets immediately following space flight

    NASA Technical Reports Server (NTRS)

    Crucian, B. E.; Cubbage, M. L.; Sams, C. F.

    2000-01-01

    In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the

  4. Adoptive Therapy with Chimeric Antigen Receptor Modified T Cells of Defined Subset Composition

    PubMed Central

    Riddell, Stanley R.; Sommermeyer, Daniel; Berger, Carolina; Liu, Lingfeng (Steven); Balakrishnan, Ashwini; Salter, Alex; Hudecek, Michael; Maloney, David G.; Turtle, Cameron J.

    2014-01-01

    The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in in targeting CD19 on B cell malignancies. The clinical trials of CD19 CAR therapy have thus far not attempted to select defined subsets prior to transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to utilizing adoptive therapy with genetically modified T cells of defined subset and phenotypic composition. PMID:24667960

  5. A unifying framework for seed sensitivity and its application to subset seeds

    PubMed Central

    Kucherov, Gregory; Noé, Laurent; Roytberg, Mihkail

    2006-01-01

    We propose a general approach to compute the seed sensitivity, that can be applied to different definitions of seeds. It treats separately three components of the seed sensitivity problem – a set of target alignments, an associated probability distribution, and a seed model – that are specified by distinct finite automata. The approach is then applied to a new concept of subset seeds for which we propose an efficient automaton construction. Experimental results confirm that sensitive subset seeds can be efficiently designed using our approach, and can then be used in similarity search producing better results than ordinary spaced seeds. PMID:16819802

  6. Discovery and computer aided potency optimization of a novel class of small molecule CXCR4 antagonists.

    PubMed

    Vinader, Victoria; Ahmet, Djevdet S; Ahmed, Mohaned S; Patterson, Laurence H; Afarinkia, Kamyar

    2013-01-01

    Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12 acting on chemokine receptor CXCR4 is particularly significant since it orchestrates migration of cancer cells in a tissue-specific metastatic process. High CXCR4 tumour expression is associated with poor prognosis of lung, brain, CNS, blood and breast cancers. We have identified a new class of small molecule CXCR4 antagonists based on the use of computational modelling studies in concert with experimental determination of in vitro activity against CXCL12-induced intracellular calcium mobilisation, proliferation and chemotaxis. Molecular modelling proved to be a useful tool in rationalising our observed potencies, as well as informing the direction of the synthetic efforts aimed at producing more potent compounds. PMID:24205302

  7. Discovery and Computer Aided Potency Optimization of a Novel Class of Small Molecule CXCR4 Antagonists

    PubMed Central

    Vinader, Victoria; Ahmet, Djevdet S.; Ahmed, Mohaned S.; Patterson, Laurence H.; Afarinkia, Kamyar

    2013-01-01

    Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12 acting on chemokine receptor CXCR4 is particularly significant since it orchestrates migration of cancer cells in a tissue-specific metastatic process. High CXCR4 tumour expression is associated with poor prognosis of lung, brain, CNS, blood and breast cancers. We have identified a new class of small molecule CXCR4 antagonists based on the use of computational modelling studies in concert with experimental determination of in vitro activity against CXCL12-induced intracellular calcium mobilisation, proliferation and chemotaxis. Molecular modelling proved to be a useful tool in rationalising our observed potencies, as well as informing the direction of the synthetic efforts aimed at producing more potent compounds. PMID:24205302

  8. Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle.

    PubMed

    Dulal, Pawan; Wright, Daniel; Ashfield, Rebecca; Hill, Adrian V S; Charleston, Bryan; Warimwe, George M

    2016-04-29

    Development of safe and efficacious vaccines whose potency is unaffected by long-term storage at ambient temperature would obviate major vaccine deployment hurdles and limit wastage associated with breaks in the vaccine cold chain. Here, we evaluated the immunogenicity of a novel chimpanzee adenovirus vectored Rift Valley Fever vaccine (ChAdOx1-GnGc) in cattle, following its thermostabilisation by slow desiccation on glass fiber membranes in the non-reducing sugars trehalose and sucrose. Thermostabilised ChAdOx1-GnGc vaccine stored for 6 months at 25, 37 or 45 ° C elicited comparable Rift Valley Fever virus neutralising antibody titres to those elicited by the 'cold chain' vaccine (stored at -80 ° C throughout) at the same dose, and these were within the range associated with protection against Rift Valley Fever in cattle. The results support the use of sugar-membrane thermostabilised vaccines in target livestock species. PMID:27020712

  9. Non-cannabinoid constituents from a high potency Cannabis sativa variety

    PubMed Central

    Radwan, Mohamed M.; ElSohly, Mahmoud A.; Slade, Desmond; Ahmed, Safwat A.; Wilson, Lisa; El-Alfy, Abir T.; Khan, Ikhlas A.; Ross, Samir A.

    2016-01-01

    Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and β-sitosteryl-3-O-β-D-glucopyranoside-2'-O-palmitate (6). In addition, five known compounds, α-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2–4 were inactive as analgesics. PMID:18774146

  10. Evaluation of models for predicting the phototoxicity potency of polycyclic aromatic hydrocarbons

    SciTech Connect

    Erickson, R.J.; Ankley, G.T.; Sheedy, B.R.; Kosian, P.A.; Mattson, V.R.; Cox, J.S.; Defoe, D.L.

    1995-12-31

    The acute phototoxicities of selected polycyclic aromatic hydrocarbons (PAHs) to the oligochaete Lumbriculus variegatus were investigated in order to evaluate a predictive structure/activity relationship (SAR) for the phototoxic potential of PAHs and to determine the relationship of phototoxicity to PAH accumulation, light intensity, and exposure duration. Test organisms were exposed to multiple concentrations of anthracene, pyrene, fluorene, and fluoranthene in water for 96 h and then to various intensities of ultraviolet light for 96 h in clean water. In agreement with the SAR model, fluorene was not phototoxic while pyrene, fluoranthene, and anthracene were. Based upon measured accumulations of PAHs, anthracene and pyrene had similar potencies, and both were 3--4 fold more toxic than fluoranthene. Time-to-death was found to adhere well to a model based on damage accumulating as a function of the product of chemical accumulation and light intensity. Additivity of PAH phototoxicity was evaluated in exposures using mixtures of these chemicals.

  11. Combination strategies to enhance the potency of monocyte-derived dendritic cell-based cancer vaccines.

    PubMed

    Fecek, Ronald J; Storkus, Walter J

    2016-10-01

    Dendritic cells (DCs) are potent inducers of adaptive immunity and their clinical use in cancer vaccine formulations remains an area of active translational and clinical investigation. Although cancer vaccines applied as monotherapies have had a modest history of clinical success, there is great enthusiasm for novel therapeutic strategies combining DC-based cancer vaccines with agents that 'normalize' immune function in the tumor microenvironment (TME). Broadly, these combination vaccines are designed to antagonize/remove immunosuppressive networks within the TME that serve to limit the antitumor action of vaccine-induced T cells and/or to condition the TME to facilitate the recruitment and optimal function and durability of vaccine-induced T cells. Such combination regimens are expected to dramatically enhance the clinical potency of DC-based cancer vaccine platforms. PMID:27605069

  12. Thioamide Hydroxypyrothiones Supersede Amide Hydroxypyrothiones in Potency Against Anthrax Lethal Factor

    PubMed Central

    Agrawal, Arpita; de Oliveira, César Augusto F.; Cheng, Yuhui; Jacobsen, Jennifer A.; McCammon, J. Andrew; Cohen, Seth M.

    2009-01-01

    Anthrax lethal factor (LF) is a critical virulence factor in the pathogenesis of anthrax. A structure-activity relationship (SAR) of potential lethal factor inhibitors (LFi) is presented in which the zinc-binding group (ZBG), linker, and backbone moieties for a series of hydroxypyrone-based compounds were systematically varied. It was found that hydroxypyrothione ZBGs generate more potent inhibitors than hydroxypyrone ZBGs. Furthermore, coupling the hydroxypyrothione to a backbone group via a thioamide bond improves potency when compared to an amide linker. QM/MM studies show that the thioamide bond in these inhibitors allows for the formation of two additional hydrogen bonds with the protein active site. In both types of hydroxypyrothione compounds, ligand efficiencies of 0.29-0.54 kcal mol-1 per heavy atom were achieved. The results highlight the need for a better understanding to optimize the interplay between the ZBG, linker, and backbone to get improved LFi. PMID:19170530

  13. A comparison of the potencies of several diluted and undiluted corticosteroid preparations using the vasoconstrictor assay.

    PubMed

    Kirsch, J; Gibson, J R; Darley, C R; Burke, C A

    1983-01-01

    10 subjects were assessed using the vasoconstrictor assay technique in a double-blind study in order to evaluate the relative potencies of several diluted and undiluted proprietary corticosteroid preparations. Dermovate ointment achieved a significantly higher score than any other preparation but there was no significant difference between the scores for Betnovate ointment (betamethasone valerate 0.1%), Propaderm Forte cream (beclomethasone dipropionate 0.5%), Propaderm ointment (beclomethasone dipropionate 0.025%), Nerisone Forte ointment (diflucortolone valerate 0.3%), and Nerisone ointment (diflucortolone valerate 0.1%). Furthermore, no significant difference in scores could be demonstrated between Adcortyl ointment (triamcinolone acetonide 0.1%), Ledercort ointment (triamcinolone acetonide 0.1%) and extemporaneous dilutions of these ointments 1 part in 4 in their recommended diluents (triamcinolone acetonide 0.025%). The relevance of these findings to clinical practice is discussed. PMID:6195026

  14. A Cusp Catastrophe Model for Team Learning, Team Potency and Team Culture.

    PubMed

    Rebelo, Teresa; Stamovlasis, Dimitrios; Lourenco, Paulo Renato; Dimas, Isabel; Pinheiro, Margarida

    2016-10-01

    This paper examines team learning behaviors within a nonlinear dynamical system (NDS) perspective. The present research is based on a sample of 36 project workgroups, where data were collected at two moments of their life cycle, with visual analogue scales. Using both the least squares method and maximum likelihood, it proposes a cusp catastrophe model for explaining team learning. The cusp model is superior to its linear alternatives and implements team culture as the asymmetry variable and team potency as bifurcation. The findings of cusp structure in the data support the existence of discontinuous shifts in learning behavior and furthermore a proposition that the punctuated equilibrium model (PEM) might be a reasonable model for describing group functioning, since it encompasses such sudden changes between distinct stages (attractors). A discussion on small group research is also provided by highlighting the nonlinear dynamics of team processes, along with further implications for research and practice. PMID:27550707

  15. Treatment of thromboangiitis obliterans (Buerger's disease) with high-potency vasodilators.

    PubMed

    Jiménez-Gallo, David; Albarrán-Planelles, Cristina; Arjona-Aguilera, Cintia; Blanco-Sánchez, German; Rodríguez-Mateos, María Eugenia; Linares-Barrios, Mario

    2015-01-01

    Thromboangiitis obliterans (TAO) or Buerger's disease is a vascular inflammatory thrombotic occlusive and segmental disease affecting distal small and medium-sized arteries of the limbs. Tobacco is the main trigger factor and avoiding it is the main treatment. Frequently, it is necessary to use high-potency vasodilators, such as iloprost, bosentan, sildenafil, or alprostadil, to relieve symptoms and reduce the risk of amputation. Iloprost is the only one that has been shown to be effective in randomized clinical trials. We report the third case of TAO treated with sildenafil and a new case treated with bosentan. This condition can be diagnosed and treated by a dermatologist. We would like to highlight the excellent clinical response despite the fact that patients continued to smoke. This finding may have important therapeutic implications because early treatment may prevent amputation and can be effective even during the process of giving up smoking. PMID:25649782

  16. Inositol 1,3,4,6-tetrakisphosphate mobilizes calcium in Xenopus oocytes with high potency.

    PubMed Central

    Ivorra, I; Gigg, R; Irvine, R F; Parker, I

    1991-01-01

    Injection of Ins(1,3,4,6)P4 into Xenopus oocytes evoked Ca2(+)-dependent membrane currents with a potency 5-10 times less than Ins(1,4,5)P3, whereas Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 were almost ineffective. Responses to Ins(1,3,4,6)P4 arose through liberation of intracellular Ca2+ and through entry of extracellular Ca2+. These results, together with the observation that Ins(1,3,4,6)P4 facilitated responses to Ins(1,4,5)P3, suggests that both of these compounds may act on the same intracellular receptors. PMID:1991032

  17. Effects of Pomegranate Seed Oil on the Fertilization Potency of Rat’s Sperm

    PubMed Central

    Nikseresht, Mohsen; Fallahzadeh, Ali Reza; Toori, Mehdi Akbartabar

    2015-01-01

    Background Pomegranate has been taken great scientific attention in recent years due to its health benefits. Pomegranate seed oil is a rich source of 9-cis, and 11-trans conjugate linolenic acid. The aim of this study was to evaluate the effect of dietary pomegranate seed oil on the fertilization potency of rat’s sperm. Materials and Methods Twenty-four male Wistar rats were divided into four groups. The first group, which served as the control group, received 1 mL of corn oil for seven weeks. Groups II, III, IV served as the experimental groups received 200, 500 and 1000 mg/kg of pomegranate seed oil, for the same period of time respectively. After seven weeks, all of the rats were sacrificed, and their epididymis sperm was collected and added to IVF medium (T6) containing metaphase II oocytes. Almost 21 oocytes had been removed from every female rat oviduct. In this medium, oocyte fertilization, cleavage rates, and embryo development into blastocysts, were evaluated by inverted microscopy. Results Levels of LD50 in the oral route in male rats were more than 5000 mg/kg body weight. Our data showed that the rates of fertilization, cleavage and embryo development into blastocysts were higher in the groups that had received 500 and 1000 mg/kg body weight of pomegranate seed oil. Conclusion This study demonstrated that pomegranate seed oil had a positive effect on the fertilization potency of male rats. These beneficial effects may be useful in assisted reproductive technology. PMID:26816914

  18. Structural Reevaluation of Streptococcus pneumoniae Lipoteichoic Acid and New Insights into Its Immunostimulatory Potency*

    PubMed Central

    Gisch, Nicolas; Kohler, Thomas; Ulmer, Artur J.; Müthing, Johannes; Pribyl, Thomas; Fischer, Kathleen; Lindner, Buko; Hammerschmidt, Sven; Zähringer, Ulrich

    2013-01-01

    Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity. PMID:23603911

  19. Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives.

    PubMed

    Penov Gaši, Katarina M; Djurendić, Evgenija A; Szécsi, Mihály; Gardi, János; Csanádi, János J; Klisurić, Olivera R; Dojčinović-Vujašković, Sanja V; Nikolić, Andrea R; Savić, Marina P; Ajduković, Jovana J; Oklješa, Aleksandar M; Kojić, Vesna V; Sakač, Marija N; Jovanović-Šanta, Suzana S

    2015-02-01

    A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ(5)-3βHSD, 17βHSD2 and 17βHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17βHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant. PMID:25541058

  20. (/sup 125/I)diiodoinsulins. Binding affinities, biologic potencies, and properties of their decay products

    SciTech Connect

    Perez Maceda, B.; Linde, S.; Sonne, O.; Gliemann, J.

    1982-07-01

    Insulin was iodinated with 0.3-0.4 mol /sup 125/I/mol insulin using the lactoperoxidase method. About one-third of the radioactivity incorporated into insulin was in diiodoinsulins and about 40% of these molecules contained diiodotyrosine in residue 14 of the A chain. Most of the remaining molecules contained one A14-monoiodotyrosine and one monoiodotyrosine in either position A19, B16, or B26. The binding affinity and biologic potency of this heterogeneous diiodoinsulin preparation was not significantly different from that of A14-(/sup 125/I)monoiodoinsulin in rat adipocytes, whereas it was slightly reduced in hepatocytes and IM-9 lymphocytes. From the iodine distribution and previous data on the binding affinity of each of the four monoiodoinsulin isomers it was calculated that A14-diiodotyrosine-insulin possesses full binding affinity and biologic potency in adipocytes. Diiodoinsulins isolated from another iodoinsulin preparation (iodate method) contained 58% A19-diiodotyrosine-insulin, and most remaining molecules contained one A19-monoiodotyrosine. The binding affinity of this mixed diiodoinsulin preparation was approximately one-fourth of that of A14-monoiodoinsulin in adipocytes, IM-9 lymphocytes, and hepatocytes. It was calculated that A19-diiodotyrosine-insulin is nearly devoid of binding affinity. The diiodoinsulins (lactoperoxidase method) decayed to iodide (probably from diiodotyrosine-insulin) or to polymers with little specific but a markedly increased nonspecific binding. In addition, the polymers had a marked tendency to adsorb to cellulose acetate filters. Conclusions: 1. The binding affinities of diiodoinsulins range from very low values to values at least as high as that of insulin depending on the positions of the iodine moieties. 2. The relative binding affinities vary among tissues. 3. Polymeric decay products give high nonspecific binding.

  1. Chronological supplement to the Carcinogenic Potency Database: standardized results of animal bioassays published through December 1982.

    PubMed Central

    Gold, L S; de Veciana, M; Backman, G M; Magaw, R; Lopipero, P; Smith, M; Blumenthal, M; Levinson, R; Bernstein, L; Ames, B N

    1986-01-01

    This paper is a chronological supplement to our earlier publication, "A Carcinogenic Potency Database of the Standardized Results of Animal Bioassays.¿ We report here results of carcinogenesis bioassays published in Technical Reports of the National Cancer Institute/National Toxicology Program between July 1980 and December 1982, and the general literature between July 1981 and December 1982. This supplement includes results of 280 long-term, chronic experiments of 114 test compounds, and reports the same information about each experiment in the same plot format as the earlier paper: e.g., the species and strain of test animal, the route and duration of compound administration, dose level and other aspects of experimental protocol, histopathology and tumor incidence, TD50 and its statistical significance, dose response, author's opinion about carcinogenicity, and literature reference. While a number of appendices are provided to facilitate use of this supplement, we have not duplicated here the material published earlier. Instead, we refer the reader to the earlier publications (Peto et al. and Gold et al.) for a thorough description of the numerical index of carcinogenic potency (TD50), a guide to the plot of the database, and a discussion of the sources of data, the rationale for the inclusion of particular experiments and particular target sites, and the conventions adopted in summarizing the literature. For 44 of the 114 chemicals reported in this second plot, results of earlier experiments are also given in the first plot; since only 1981-1982 results are reported here, the first plot is required for these repeated compounds. In this paper we also give corrections for errors that appeared in the earlier publication. PMID:3530736

  2. Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo

    PubMed Central

    Hassan, Hatem A.F.M.; Smyth, Lesley; Rubio, Noelia; Ratnasothy, Kulachelvy; Wang, Julie T.-W.; Bansal, Sukhvinder S.; Summers, Huw D.; Diebold, Sandra S.; Lombardi, Giovanna; Al-Jamal, Khuloud T.

    2016-01-01

    Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~ 386 nm), bearing net positive charge (5.8 mV), or short MWNTs-OVA (~ 122 nm) of increasing negative charges (− 23.4, − 35.8 or − 39 mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system. PMID:26802552

  3. Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions.

    PubMed

    Aasa, Jenny; Vare, Daniel; Motwani, Hitesh V; Jenssen, Dag; Törnqvist, Margareta

    2016-07-01

    Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration-time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration×cell treatment time. Gly induced mutations in the hprt-gene at a rate of 0.08±0.01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study. PMID:27402481

  4. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

    PubMed

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T; Cui, Jiajia; Cheng, Christopher J; Saltzman, W Mark

    2015-12-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities. PMID:26627251

  5. Reassessment of MTBE cancer potency considering modes of action for MTBE and its metabolites.

    PubMed

    Bogen, Kenneth T; Heilman, Jacqueline M

    2015-01-01

    A 1999 California state agency cancer potency (CP) evaluation of methyl tert-butyl ether (MTBE) assumed linear risk extrapolations from tumor data were plausible because of limited evidence that MTBE or its metabolites could damage DNA, and based such extrapolations on data from rat gavage and rat and mouse inhalation studies indicating elevated tumor rates in male rat kidney, male rat Leydig interstitial cells, and female rat leukemia/lymphomas. More recent data bearing on MTBE cancer potency include a rodent cancer bioassay of MTBE in drinking water; several new studies of MTBE genotoxicity; several similar evaluations of MTBE metabolites, formaldehyde, and tert-butyl alcohol or TBA; and updated evaluations of carcinogenic mode(s) of action (MOAs) of MTBE and MTBE metabolite's. The lymphoma/leukemia data used in the California assessment were recently declared unreliable by the U.S. Environmental Protection Agency (EPA). Updated characterizations of MTBE CP, and its uncertainty, are currently needed to address a variety of decision goals concerning historical and current MTBE contamination. To this end, an extensive review of data sets bearing on MTBE and metabolite genotoxicity, cytotoxicity, and tumorigenicity was applied to reassess MTBE CP and related uncertainty in view of MOA considerations. Adopting the traditional approach that cytotoxicity-driven cancer MOAs are inoperative at very low, non-cytotoxic dose levels, it was determined that MTBE most likely does not increase cancer risk unless chronic exposures induce target-tissue toxicity, including in sensitive individuals. However, the corresponding expected (or plausible upper bound) CP for MTBE conditional on a hypothetical linear (e.g., genotoxic) MOA was estimated to be ∼2 × 10(-5) (or 0.003) per mg MTBE per kg body weight per day for adults exposed chronically over a lifetime. Based on this conservative estimate of CP, if MTBE is carcinogenic to humans, it is among the weakest 10% of chemical

  6. The efflux pump inhibitor timcodar improves the potency of antimycobacterial agents.

    PubMed

    Grossman, Trudy H; Shoen, Carolyn M; Jones, Steven M; Jones, Peter L; Cynamon, Michael H; Locher, Christopher P

    2015-03-01

    Previous studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs against Mycobacterium tuberculosis in in vitro and in vivo combination studies. When used alone, timcodar weakly inhibited M. tuberculosis growth in broth culture (MIC, 19 μg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. When M. tuberculosis was cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 μg/ml) in the growth inhibition of M. tuberculosis and demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log10 reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted. PMID:25534740

  7. Searching for Drug Synergy in Complex Dose–Response Landscapes Using an Interaction Potency Model

    PubMed Central

    Yadav, Bhagwan; Wennerberg, Krister; Aittokallio, Tero; Tang, Jing

    2015-01-01

    Rational design of multi-targeted drug combinations is a promising strategy to tackle the drug resistance problem for many complex disorders. A drug combination is usually classified as synergistic or antagonistic, depending on the deviation of the observed combination response from the expected effect calculated based on a reference model of non-interaction. The existing reference models were proposed originally for low-throughput drug combination experiments, which make the model assumptions often incompatible with the complex drug interaction patterns across various dose pairs that are typically observed in large-scale dose–response matrix experiments. To address these limitations, we proposed a novel reference model, named zero interaction potency (ZIP), which captures the drug interaction relationships by comparing the change in the potency of the dose–response curves between individual drugs and their combinations. We utilized a delta score to quantify the deviation from the expectation of zero interaction, and proved that a delta score value of zero implies both probabilistic independence and dose additivity. Using data from a large-scale anticancer drug combination experiment, we demonstrated empirically how the ZIP scoring approach captures the experimentally confirmed drug synergy while keeping the false positive rate at a low level. Further, rather than relying on a single parameter to assess drug interaction, we proposed the use of an interaction landscape over the full dose–response matrix to identify and quantify synergistic and antagonistic dose regions. The interaction landscape offers an increased power to differentiate between various classes of drug combinations, and may therefore provide an improved means for understanding their mechanisms of action toward clinical translation. PMID:26949479

  8. Studies with bioengineered Nisin peptides highlight the broad-spectrum potency of Nisin V.

    PubMed

    Field, Des; Quigley, Lisa; O'Connor, Paula M; Rea, Mary C; Daly, Karen; Cotter, Paul D; Hill, Colin; Ross, R Paul

    2010-07-01

    Nisin A is the most thoroughly investigated member of the lantibiotic family of antimicrobial peptides. In addition to a long history of safe use as a food antimicrobial, its activity against multi-drug resistant pathogens has resulted in a renewed interest in applying nisin as a chemotherapeutic to treat bacterial infections. The wealth of Nisin-related information that has been generated has also led to the development of the biotechnological capacity to engineer novel Nisin variants with a view to improving the function and physicochemical properties of this already potent peptide. However, the identification of bioengineered Nisin derivatives with enhanced antimicrobial activity against Gram-positive targets is a recent event. In this study, we created stable producers of the most promising derivatives of Nisin A generated to date [M21V (hereafter Nisin V) and K22T (hereafter Nisin T)] and assessed their potency against a range of drug-resistant clinical, veterinary and food pathogens. Nisin T exhibited increased activity against all veterinary isolates, including streptococci and staphylococci, and against a number of multi-drug resistant clinical isolates including MRSA, but not vancomycin-resistant enterococci. In contrast, Nisin V displayed increased potency against all targets tested including hVISA strains and the hyper-virulent Clostridium difficile ribotype 027 and against important food pathogens such as Listeria monocytogenes and Bacillus cereus. Significantly, this enhanced activity was validated in a model food system against L. monocytogenes. We conclude that Nisin V possesses significant potential as a novel preservative or chemotherapeutic compound. PMID:21255345

  9. Studies with bioengineered Nisin peptides highlight the broad‐spectrum potency of Nisin V

    PubMed Central

    Field, Des; Quigley, Lisa; O'Connor, Paula M.; Rea, Mary C.; Daly, Karen; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

    2010-01-01

    Summary Nisin A is the most thoroughly investigated member of the lantibiotic family of antimicrobial peptides. In addition to a long history of safe use as a food antimicrobial, its activity against multi‐drug resistant pathogens has resulted in a renewed interest in applying nisin as a chemotherapeutic to treat bacterial infections. The wealth of Nisin‐related information that has been generated has also led to the development of the biotechnological capacity to engineer novel Nisin variants with a view to improving the function and physicochemical properties of this already potent peptide. However, the identification of bioengineered Nisin derivatives with enhanced antimicrobial activity against Gram‐positive targets is a recent event. In this study, we created stable producers of the most promising derivatives of Nisin A generated to date [M21V (hereafter Nisin V) and K22T (hereafter Nisin T)] and assessed their potency against a range of drug‐resistant clinical, veterinary and food pathogens. Nisin T exhibited increased activity against all veterinary isolates, including streptococci and staphylococci, and against a number of multi‐drug resistant clinical isolates including MRSA, but not vancomycin‐resistant enterococci. In contrast, Nisin V displayed increased potency against all targets tested including hVISA strains and the hyper‐virulent Clostridium difficile ribotype 027 and against important food pathogens such as Listeria monocytogenes and Bacillus cereus. Significantly, this enhanced activity was validated in a model food system against L. monocytogenes. We conclude that Nisin V possesses significant potential as a novel preservative or chemotherapeutic compound. PMID:21255345

  10. In vitro studies with five quinolones: evidence for changes in relative potency as quinolone resistance rises.

    PubMed Central

    Thomson, K S; Sanders, C C; Hayden, M E

    1991-01-01

    A panel of 203 staphylococci, Enterobacteriaceae, Pseudomonas aeruginosa, and miscellaneous nonfermentative gram-negative bacilli were chosen for their various susceptibilities to ciprofloxacin. On the basis of agar dilution susceptibilities, each of the four taxonomic groups was divided into ciprofloxacin-susceptible, moderately resistant, and highly resistant subgroups, and each subgroup was then further analyzed for its susceptibility to the fluoroquinolones CI-960, CI-990, sparfloxacin, and ofloxacin. Although the MICs of each quinolone increased as ciprofloxacin resistance increased, the potency of CI-960 appeared to increase relative to the potencies of the other quinolones. Similarly, the MICs of sparfloxacin and ofloxacin appeared to be less affected by ciprofloxacin resistance than were the MICs of ciprofloxacin or CI-990. Single-step mutants of representative clinical isolates with different levels of ciprofloxacin resistance were selected to determine whether the study quinolones differed in their propensity to select resistant mutants and whether the presence of preexisting ciprofloxacin resistance influenced the subsequent development of resistance. Each of the five fluoroquinolones and nalidixic acid selected mutants that exhibited generally modest decreases in quinolone susceptibility (4- to 16-fold). However, CI-960 inhibited significantly more mutants (80%) than did the other quinolones (39 to 59%) at a concentration of 1 microgram/ml. The presence of preexisting ciprofloxacin resistance appeared to be associated with higher mutational frequencies in coagulase-negative staphylococci exposed to each of the fluoroquinolones and in Serratia marcescens exposed to nalidixic acid. Preexisting ciprofloxacin resistance did not influence the development of resistance in the strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa that were studied. The results of this study suggest that quinolones are not affected

  11. Analysis of cannabis seizures in NSW, Australia: cannabis potency and cannabinoid profile.

    PubMed

    Swift, Wendy; Wong, Alex; Li, Kong M; Arnold, Jonathon C; McGregor, Iain S

    2013-01-01

    Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ(9)-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales "Cannabis Cautioning" scheme. A further 26 "Known Provenance" samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The "Cannabis Cautioning" samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (<0.1%). "Known Provenance" samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed. PMID:23894589

  12. Analysis of Cannabis Seizures in NSW, Australia: Cannabis Potency and Cannabinoid Profile

    PubMed Central

    Li, Kong M.; Arnold, Jonathon C.; McGregor, Iain S.

    2013-01-01

    Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ9-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales “Cannabis Cautioning” scheme. A further 26 “Known Provenance” samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The “Cannabis Cautioning” samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (<0.1%). “Known Provenance” samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed. PMID:23894589

  13. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

    PubMed Central

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T.; Cui, Jiajia; Cheng, Christopher J.; Saltzman, W. Mark

    2015-01-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities. PMID:26627251

  14. Cold chain monitoring of OPV at transit levels in India: correlation of VVM and potency status.

    PubMed

    Jain, R; Sahu, A K; Tewari, S; Malik, N; Singh, S; Khare, S; Bhatia, R

    2003-12-01

    We have conducted a study to analyze monitoring of the cold chain of 674 OPV field samples collected at four different levels of vaccine distribution viz., immunization clinics, district stores, hospitals and Primary Health Centers (PHC) from states of Uttar Pradesh, Madhya Pradesh, and Delhi. The study design included: collection and scoring of vaccine vial monitor (VVM) status of the samples and testing for total oral polio virus concentration (TOPV) by standard WHO protocol. Ten samples each were exposed to 25 degrees C and 37 degrees C, and 10 samples as controls were kept at -20 degrees C. VVM were scored daily till they attained grade 4 and each sample was subsequently subjected to potency testing for individual polio serotypes 1, 2 and 3, and TOPV. Of the 674 samples tested it was observed that: samples from immunization clinics and district stores had an acceptable VVM score of grade 1 and 2; however the probable risk that a sub potent vaccine could have been administered was 2.15%. In 2.5% samples received from district stores vaccine had a VVM score of grade 3 (i.e., discard point), although vaccine when tested was found to be potent (i.e., leading to the vaccine wastage). With exposure to higher temperatures, VVM changed score to grade 2 and 3 when the vaccine was kept at 25 degrees C/37 degrees C, and the titres of individual serotypes 1, 2 and 3 and TOPV were beyond the acceptable limits. Important observations at the different levels of vaccine distribution network and correlation of VVM and potency status of OPV are discussed in the paper which will be of help to the EPI program managers at different transit levels. PMID:14624793

  15. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    SciTech Connect

    Milton, Flora Aparecida; Cvoro, Aleksandra; Amato, Angelica A.; Sieglaff, Douglas H.; Filgueira, Carly S.; Arumanayagam, Anithachristy Sigamani; Caro Alves de Lima, Maria do; Rocha Pitta, Ivan; Assis Rocha Neves, Francisco de; Webb, Paul

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.

  16. Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury

    PubMed Central

    Rantakari, Pia; Patten, Daniel A.; Valtonen, Joona; Karikoski, Marika; Gerke, Heidi; Dawes, Harriet; Laurila, Juha; Ohlmeier, Steffen; Elima, Kati; Hübscher, Stefan G.; Jalkanen, Sirpa; Adams, David H.; Salmi, Marko; Shetty, Shishir

    2016-01-01

    Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1+ macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP+ fibrogenic cells. Stabilin-1−/− macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6Clo monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1+ monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage–specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1+ macrophages shape the tissue microenvironment during liver injury and healing. PMID:27474165

  17. c-kit+AT2R+ Bone Marrow Mononuclear Cell Subset Is a Superior Subset for Cardiac Protection after Myocardial Infarction

    PubMed Central

    Du, Mingjun; Zhang, Wentian; Wang, Chenxi; Lian, Feng; Xue, Song

    2016-01-01

    Although the bone marrow mononuclear cell (BMMNC) is known as an ideal cell type for cell-based therapy for MI treatment, the effective subpopulation still remains unknown. Our study aimed at identifying the optimal subset of BMMNCs suited for cardiac regeneration. In this study, we observed that MI led to (i) a significant increase of the c-kit+AT2R+ BMMNC subpopulation in mice and (ii) a modest increase of AT2R+ BMMNCs in humans. c-kit+AT2R+ and c-kit+AT2R− BMMNC subpopulations were obtained from mice after MI. Then, we cocultured cardiac H9C2 cells with c-kit+AT2R+, c-kit+AT2R−, and unfractionated BMMNCs; finally, we found that the c-kit+AT2R+ subset is superior to the c-kit+AT2R− subset in improving cardiomyocyte protection in vitro. Of note, c-kit+AT2R+ BMMNCs showed a more robust migration capacity than c-kit+AT2R− and unfractionated BMMNCs in vitro and in vivo. Additionally, compared to c-kit+AT2R− and unfractionated BMMNCs, intravenous transplantation of c-kit+AT2R+ BMMNC resulted in smaller infarct size and lower levels of inflammatory reactions in heart tissue, leading to a higher global heart function improvement. In conclusion, our results indicate that the c-kit+AT2R+ BMMNC subpopulation exerts a protective effect against MI and shows promising therapeutic possibilities with regard to the treatment of ischemic heart disease. PMID:27429622

  18. Regulatory B cells are enriched within the IgM memory and transitional subsets in healthy donors but are deficient in chronic GVHD

    PubMed Central

    Khoder, Ahmad; Sarvaria, Anushruti; Alsuliman, Abdullah; Chew, Claude; Sekine, Takuya; Cooper, Nichola; Mielke, Stephan; de Lavallade, Hugues; Muftuoglu, Muharrem; Fernandez Curbelo, Irina; Liu, Enli; Muraro, Paolo A.; Alousi, Amin; Stringaris, Kate; Parmar, Simrit; Shah, Nina; Shaim, Hila; Yvon, Eric; Molldrem, Jeffrey; Rouce, Rayne; Champlin, Richard; McNiece, Ian; Mauri, Claudia; Shpall, Elizabeth J.

    2014-01-01

    A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19+IgM+CD27+ memory and CD19+CD24hiCD38hi transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4+ T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell–based therapy in the treatment of this disease. PMID:25051962

  19. αCP binding to a cytosine-rich subset of polypyrimidine tracts drives a novel pathway of cassette exon splicing in the mammalian transcriptome

    PubMed Central

    Ji, Xinjun; Park, Juw Won; Bahrami-Samani, Emad; Lin, Lan; Duncan-Lewis, Christopher; Pherribo, Gordon; Xing, Yi; Liebhaber, Stephen A.

    2016-01-01

    Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5′ to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5′ to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome. PMID:26896798

  20. Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

    PubMed Central

    Wen, Ke; Li, Guohua; Zhang, Wei; Azevedo, Marli SP; Saif, Linda J; Liu, Fangning; Bui, Tammy; Yousef, Ahmed; Yuan, Lijuan

    2011-01-01

    γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2−CD8−, CD2+CD8− and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3–5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In naive pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8–10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8− subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs