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Sample records for cell specific variable

  1. Hydrogen powered fuel cell systems : different designs for variable specifications

    Energy Technology Data Exchange (ETDEWEB)

    Beckhaus, P.; Goessling, S.; Notthoff, T.; Souzani, S.; Schoemaker, M.; Heinzel, A. [ZBT GmbH, Duisburg (Germany). Fuel Cells and Systems

    2009-07-01

    This paper discussed system designs for hydrogen-powered fuel cell systems in the sub-kilowatt range. Designs were presented for the following 3 different types of applications: (1) supplying electrical power for high efficiency drives of 60 per cent, (2) supplying power for small uninterruptible power supply (UPS) devices with high power densities and minimum start-up times in a variety of environmental conditions, and (3) providing the supply of oxygen-reduced breathing air for the high altitude training of athletes. Different control strategies and approaches to system design were discussed. The results of experimental studies conducted to test the fuel cell stack systems were also provided.

  2. Eternity Variables to Simulate Specifications

    NARCIS (Netherlands)

    Hesselink, WH; Boiten, EA; Moller, B

    2002-01-01

    Simulation of specifications is introduced as a unification and generalization of refinement mappings, history variables, forward simulations, prophecy variables, and backward simulations. Eternity variables are introduced as a more powerful alternative for prophecy variables and backward

  3. Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help.

    Science.gov (United States)

    Compeer, Ewoud B; Janssen, Willemijn; van Royen-Kerkhof, Annet; van Gijn, Marielle; van Montfrans, Joris M; Boes, Marianne

    2015-05-10

    Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4(+) T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4(+) T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4(+) T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.

  4. Eternity Variables to Prove Simulation of Specifications

    NARCIS (Netherlands)

    Hesselink, Wim H.

    2005-01-01

    Simulations of specifications are introduced as a unification and generalization of refinement mappings, history variables, forward simulations, prophecy variables, and backward simulations. A specification implements another specification if and only if there is a simulation from the first one to

  5. Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

    Science.gov (United States)

    Martyushev, Alexey P; Petravic, Janka; Grimm, Andrew J; Alinejad-Rokny, Hamid; Gooneratne, Shayarana L; Reece, Jeanette C; Cromer, Deborah; Kent, Stephen J; Davenport, Miles P

    2015-05-01

    CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. Specific discharge variability in a boreal landscape

    Science.gov (United States)

    Lyon, Steve W.; Nathanson, Marcus; Spans, André; Grabs, Thomas; Laudon, Hjalmar; Temnerud, Johan; Bishop, Kevin H.; Seibert, Jan

    2012-08-01

    Specific discharge variations within a mesoscale catchment were studied on the basis of three synoptic sampling campaigns. These were conducted during stable flow conditions within the Krycklan catchment study area in northern Sweden. During each campaign, about 80 individual locations were measured for discharge draining from catchment areas ranging between 0.12 and 67 km2. These discharge samplings allowed for the comparison between years within a given season (September 2005 versus September 2008) and between seasons within a given year (May 2008 versus September 2008) of specific discharge across this boreal landscape. There was considerable variability in specific discharge across this landscape. The ratio of the interquartile range (IQR) defined as the difference between the 75th and 25th percentiles of the specific discharges to the median of the specific discharges ranged from 37% to 43%. Factor analysis was used to explore potential relations between landscape characteristics and the specific discharge observed for 55 of the individual locations that were measured in all three synoptic sampling campaigns. Percentage wet area (i.e., wetlands, mires, and lakes) and elevation were found to be directly related to the specific discharge during the drier September 2008 sampling while potential annual evaporation was found to be inversely related. There was less of a relationship determined during the wetter post spring flood May 2008 sampling and the late summer rewetted September 2005 sampling. These results indicate the ability of forests to "dry out" parts of the catchment over the summer months while wetlands "keep wet" other parts. To demonstrate the biogeochemical implications of such spatiotemporal variations in specific discharge, we estimate dissolved organic carbon (DOC) exports with available data for the May 2008 and September 2008 samplings using both the spatially variable observed specific discharges and the spatially constant catchment average

  7. The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

    Science.gov (United States)

    van den Heuvel, Heleen; Heutinck, Kirstin M; van der Meer-Prins, Ellen M W; Franke-van Dijk, Marry E I; van Miert, Paula P M C; Zhang, Xiaoqian; Ten Berge, Ineke J M; Claas, Frans H J

    2018-01-01

    Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8+ T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8+ T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  8. CCL22-specific T Cells

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Munir Ahmad, Shamaila; Hansen, Morten

    2016-01-01

    Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We...... analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from...... the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using...

  9. Cell-specific precursor processing

    DEFF Research Database (Denmark)

    Rehfeld, Jens F; Bundgaard, Jens R

    2010-01-01

    in different cells and tissues therefore requires control of biogenesis and secretion in order to avoid interference with the function of a specific hormonal peptide from a particular endocrine cell. Several mechanisms are involved in such control, one of them being cell-specific processing of prohormones....... The following pages present four examples of such cell-specific processing and the implications of the phenomenon for the use of peptide hormones as markers of diseases. Notably, sick cells - not least the neoplastic cells - often process prohormones in a manner different from that of the normal endocrine cells....

  10. Relationship between Functional Profile of HIV-1 Specific CD8 T Cells and Epitope Variability with the Selection of Escape Mutants in Acute HIV-1 Infection

    Science.gov (United States)

    Goonetilleke, Nilu; Liu, Michael K. P.; Turnbull, Emma L.; Salazar-Gonzalez, Jesus F.; Hawkins, Natalie; Self, Steve; Watson, Sydeaka; Betts, Michael R.; Gay, Cynthia; McGhee, Kara; Pellegrino, Pierre; Williams, Ian; Tomaras, Georgia D.; Haynes, Barton F.; Gray, Clive M.; Borrow, Persephone; Roederer, Mario; McMichael, Andrew J.; Weinhold, Kent J.

    2011-01-01

    In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants. PMID:21347345

  11. Monitoring of seasonal variability in bronchial hyper-responsiveness and sputum cell counts in non-asthmatic subjects with rhinitis and effect of specific immunotherapy.

    Science.gov (United States)

    Polosa, R; Li Gotti, F; Mangano, G; Mastruzzo, C; Pistorio, M P; Crimi, N

    2003-07-01

    Bronchial hyper-responsiveness (BHR) is documented in a proportion of non-asthmatic individuals with allergic rhinitis (NAAR) and reflects inflammatory events in the lower airways. Natural exposure to allergens is known to modulate BHR and the level of airway inflammation in asthma, but less consistently in NAAR. Specific immunotherapy (SIT) attenuates symptoms possibly by reducing BHR and airway inflammation. The influence of natural exposure to Parietaria pollen on BHR and sputum cell counts of NAAR was investigated and the effect of Parietaria SIT examined. Thirty NAAR, monosensitized to Parietaria judaica, participated in a randomized, double-blind, placebo-controlled, parallel group study of the effects of a Parietaria pollen vaccine on symptoms/medication score, BHR to inhaled methacholine and adenosine 5'-monophosphate (AMP), and cell counts in the sputum collected out of and during the pollen seasons for 36 months. Seasonal variation in BHR to inhaled methacholine and AMP and changes in sputum cell counts were documented. Changes were consistent for AMP, but not methacholine, and invariably associated with modifications in sputum eosinophils and epithelial cells. The clinical efficacy of Parietaria SIT was associated with a decline in the seasonal deterioration of BHR to AMP, whereas no significant effect was observed on BHR to methacholine or sputum cell differentials. Between-groups comparison of the seasonal changes in PC15 methacholine values and sputum cell differentials calculated as the AUC were not statistically significant, whereas a significant difference in PC15 AMP was demonstrated throughout the study (P=0.029), the median (inter-quartile range) AUC values being 2478.5 (1153.3-3600.0) and 1545.5 (755.3-1797.9) for the SIT- and placebo-treated group, respectively. Bronchial airways of NAAR exhibit features of active inflammation that deteriorate during natural allergen exposure, particularly with regard to BHR to AMP. The clinical efficacy of

  12. Intrafamilial phenotypic variability of Specific Language Impairment.

    Science.gov (United States)

    Bartha-Doering, Lisa; Regele, Sabrina; Deuster, Dirk; Seidl, Rainer; Bogdanova, Nadja; Röpke, Albrecht; Wieacker, Peter; Am Zehnhoff-Dinnesen, Antoinette

    2016-08-01

    We investigated language functions in 32 members of a four generation family with several members affected by Specific Language Impairment with an extensive language test battery in order to determine the prevalence, overlap, and homogeneity of linguistic deficits within one pedigree. In sum, one fourth of all family members tested fulfilled the criteria of Specific Language Impairment. Despite of some similarities in language abilities, different combinations of language deficits were observed, and individual language profiles varied substantially. Thus, though there is a high prevalence of language deficits in this family which raises the likelihood of a genetic origin of these deficits, and though all affected study participants displayed selective linguistic deficits with normal non-verbal functioning, language testing showed considerable variance in overlap and homogeneity of linguistic deficits. Thus, even in one genetic population, an underlying linguistic disorder manifests itself in different language abilities to a variant degree. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. 20mN, Variable Specific Impulse Colloid Thruster Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Colloid thrusters have long been known for their exceptional thrust efficiency and ability to operate over a range of specific impulse due to easily variable...

  14. A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

    Directory of Open Access Journals (Sweden)

    Leni Moldovan

    Full Text Available Peripheral blood mononuclear cells (PBMCs, including rare circulating stem and progenitor cells (CSPCs, have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene, defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all p<0.03 with age (R2 = -0.23, vascular stiffness (R2 = -0.24, and central aortic pressure (R2 = -0.19 and positively correlated with body mass index (R2 = 0.72, in women. The co-expression of three neovascular markers was validated at the single-cell level using mRNA in situ hybridization and immunocytochemistry. The overall gene expression in this cardiovascular module was reduced by 72±22% in the patients compared with controls. However, the compactness of both modules was increased in the patients' samples, which was reflected in reduced dispersion of their nodes' degrees of connectivity, suggesting a more primitive character of the patients' CSPCs. In conclusion, our results show that the relationship between CSPCs and vascular function is encoded in modules of the PBMCs transcriptional

  15. PD-L1-specific T cells

    DEFF Research Database (Denmark)

    Ahmad, Shamaila Munir; Borch, Troels Holz; Hansen, Morten

    2016-01-01

    -specific T cells that recognize both PD-L1-expressing immune cells and malignant cells. Thus, PD-L1-specific T cells have the ability to modulate adaptive immune reactions by reacting to regulatory cells. Thus, utilization of PD-L1-derived T cell epitopes may represent an attractive vaccination strategy...... for targeting the tumor microenvironment and for boosting the clinical effects of additional anticancer immunotherapy. This review summarizes present information about PD-L1 as a T cell antigen, depicts the initial findings about the function of PD-L1-specific T cells in the adjustment of immune responses...

  16. Lactobacilli-expressed single-chain variable fragment (scFv) specific for intercellular adhesion molecule 1 (ICAM-1) blocks cell-associated HIV-1 transmission across a cervical epithelial monolayer.

    Science.gov (United States)

    Chancey, Caren J; Khanna, Kristen V; Seegers, Jos F M L; Zhang, Guang Wen; Hildreth, James; Langan, Abigail; Markham, Richard B

    2006-05-01

    The vaginal and cervical epithelia provide an initial barrier to sexually acquired HIV-1 infection in women. To study the interactions between HIV-1-infected cells or cell-free HIV-1 and the reproductive epithelium, the transmission of HIV-1 by infected cells or cell-free virus across human cervical epithelial cells was examined using a Transwell culture system. Cell-associated HIV-1 was transmitted more efficiently than cell-free virus, and monocyte-associated virus was transmitted most efficiently. Abs to ICAM-1 added to the apical side of the epithelium blocked cell-mediated transepithelial HIV-1 transmission in vitro. When used in a previously described model of vaginal HIV-1 transmission in human PBL-SCID mice, anti-murine ICAM-1 Abs (0.4 microg/10 microl) also blocked vaginal transmission of cell-associated HIV-1 in vivo. To evaluate a candidate delivery system for the use of this Ab as an anti-HIV-1 microbicide, anti-ICAM single-chain variable fragment Abs secreted by transformed lactobacilli were evaluated for their protective efficacy in the Transwell model. Like the intact Ab and Fab derived from it, the single-chain variable fragment at a concentration of 6.7 microg/100 microl was able to reduce HIV-1 transmission by 70 +/- 5%. These data support the potential efficacy of an anti-ICAM Ab delivered by lactobacilli for use as an anti-HIV-1 microbicide.

  17. Country-Specific Effects of Climate Variability on Human Migration.

    Science.gov (United States)

    Gray, Clark; Wise, Erika

    2016-04-01

    Involuntary human migration is among the social outcomes of greatest concern in the current era of global climate change. Responding to this concern, a growing number of studies have investigated the consequences of short to medium-term climate variability for human migration using demographic and econometric approaches. These studies have provided important insights, but at the same time have been significantly limited by lack of expertise in the use of climate data, access to cross-national data on migration, and attention to model specification. To address these limitations, we link data on internal and international migration over a 6-year period from 9,812 origin households in Kenya, Uganda, Nigeria, Burkina Faso and Senegal to high-resolution gridded climate data from both station and satellite sources. Analyses of these data using several plausible specifications reveal that climate variability has country-specific effects on migration: Migration tends to increase with temperature anomalies in Uganda, tends to decrease with temperature anomalies in Kenya and Burkina Faso, and shows no consistent relationship with temperature in Nigeria and Senegal. Consistent with previous studies, precipitation shows weak and inconsistent relationships with migration across countries. These results challenge generalizing narratives that foresee a consistent migratory response to climate change across the globe.

  18. Near-global freshwater-specific environmental variables for biodiversity analyses in 1 km resolution.

    Science.gov (United States)

    Domisch, Sami; Amatulli, Giuseppe; Jetz, Walter

    2015-12-08

    The lack of freshwater-specific environmental information at sufficiently fine spatial grain hampers broad-scale analyses in aquatic biology, biogeography, conservation, and ecology. Here we present a near-global, spatially continuous, and freshwater-specific set of environmental variables in a standardized 1 km grid. We delineate the sub-catchment for each grid cell along the HydroSHEDS river network and summarize the upstream climate, topography, land cover, surface geology and soil to each grid cell using various metrics (average, minimum, maximum, range, sum, inverse distance-weighted average and sum). All variables were subsequently averaged across single lakes and reservoirs of the Global lakes and Wetlands Database that are connected to the river network. Monthly climate variables were summarized into 19 long-term climatic variables following the 'bioclim' framework. This new set of variables provides a basis for spatial ecological and biodiversity analyses in freshwater ecosystems at near global extent, yet fine spatial grain. To facilitate the generation of freshwater variables for custom study areas and spatial grains, we provide the 'r.stream.watersheds' and 'r.stream.variables' add-ons for the GRASS GIS software.

  19. Primordial Germ Cell Specification and Migration.

    Science.gov (United States)

    Marlow, Florence

    2015-01-01

    Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration.

  20. Cdc42-mediated tubulogenesis controls cell specification

    DEFF Research Database (Denmark)

    Kesavan, Gokul; Sand, Fredrik Wolfhagen; Greiner, Thomas Uwe

    2009-01-01

    Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled......, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later...... for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors. For a video summary of this article, see the PaperFlick file with the Supplemental Data...

  1. Cell-specific cardiac electrophysiology models.

    Directory of Open Access Journals (Sweden)

    Willemijn Groenendaal

    2015-04-01

    Full Text Available The traditional cardiac model-building paradigm involves constructing a composite model using data collected from many cells. Equations are derived for each relevant cellular component (e.g., ion channel, exchanger independently. After the equations for all components are combined to form the composite model, a subset of parameters is tuned, often arbitrarily and by hand, until the model output matches a target objective, such as an action potential. Unfortunately, such models often fail to accurately simulate behavior that is dynamically dissimilar (e.g., arrhythmia to the simple target objective to which the model was fit. In this study, we develop a new approach in which data are collected via a series of complex electrophysiology protocols from single cardiac myocytes and then used to tune model parameters via a parallel fitting method known as a genetic algorithm (GA. The dynamical complexity of the electrophysiological data, which can only be fit by an automated method such as a GA, leads to more accurately parameterized models that can simulate rich cardiac dynamics. The feasibility of the method is first validated computationally, after which it is used to develop models of isolated guinea pig ventricular myocytes that simulate the electrophysiological dynamics significantly better than does a standard guinea pig model. In addition to improving model fidelity generally, this approach can be used to generate a cell-specific model. By so doing, the approach may be useful in applications ranging from studying the implications of cell-to-cell variability to the prediction of intersubject differences in response to pharmacological treatment.

  2. T cells specific for lipid antigens.

    Science.gov (United States)

    Mori, Lucia; De Libero, Gennaro

    2012-09-01

    Lipid-specific T cells are important participants in human immune responses. Recognition of lipid antigens contributes to host defense against pathogens that can cause debilitating diseases, including mycobacterial, viral, and parasitic infections. Lipid-specific T cells also play important roles in various autoimmune diseases, atherosclerosis, and in tumor surveillance. A better understanding of the mechanisms that regulate lipid-reactive T-cell functions will enable the development of novel therapies across a wide range of diseases. In recent years, our laboratory has investigated lipid antigen specificities, mechanisms of lipid antigen presentation, molecular interaction of lipid antigens with CD1 antigen-presenting molecules, and the pathogenic and regulatory functions of lipid-specific T cells in a variety of disease settings. In this review, we present recent data that illustrate the critical role played by lipid-specific immune responses in host protection, with a particular focus on human studies.

  3. Eurythmy therapy increases specific oscillations of heart rate variability.

    Science.gov (United States)

    Edelhäuser, Friedrich; Minnerop, Antje; Trapp, Barbara; Büssing, Arndt; Cysarz, Dirk

    2015-06-06

    Mind-body therapies are beneficial for several diseases (e.g. chronic pain, arterial hypertension, mood disorders). Eurythmy therapy (EYT) is a mind-body therapy from Anthroposophic Medicine. In each EYT exercise a short sequence of body movements and simultaneous guided imagery is repeated several times. In this study, the simultaneous effects of two different EYT exercises on cardiac autonomic regulation as assessed by spectral analysis of heart rate variability (HRV) were investigated. Twenty healthy subjects (age: 29.1 ± 9.3 years, 13 female) performed two different EYT exercises (EYT-A and EYT-B) for 8 min. Each EYT exercise was compared against two matched control exercises: control exercise 1 (CE1-A and CE1-B) consisted of a repetition of the body movements of the EYT exercise but without guided imagery, control exercise 2 consisted of walking on the spot (CE2-A and CE2-B). Spectral power of HRV during each exercise was quantified on the basis of Holter ECG recordings. During EYT-A the frequency of the peak oscillation in the very low frequency (VLF) band matched the repetition rate of the sequence of body movements (0.02 Hz). Low frequency (LF) oscillations were augmented when compared to the control exercises (EYT-A: 7.31 ± 0.84, CE1-A: 6.98 ± 0.90, CE2-A: 6.52 ± 0.87 ln ms(2), p exercises (EYT-B: 9.32 ± 0.82, CE1-B: 6.31 ± 0.75, CE2-B: 6.04 ± 0.80 ln ms(2), p exercises clearly affected cardiac autonomic regulation in a rhythmic manner according to the stimulus of the specific body movements of each EYT exercise. These results offer a physiological basis to develop a rationale for specific clinical indications of these EYT exercises such as stress reduction or prevention of hypertension. DRKS00006760 (registered on 10/10/2014, i.e. retrospective registration); view details at http://www.drks.de/DRKS00006760.

  4. Photoreceptor cell fate specification in vertebrates

    Science.gov (United States)

    Brzezinski, Joseph A.; Reh, Thomas A.

    2015-01-01

    Photoreceptors – the light-sensitive cells in the vertebrate retina – have been extremely well-characterized with regards to their biochemistry, cell biology and physiology. They therefore provide an excellent model for exploring the factors and mechanisms that drive neural progenitors into a differentiated cell fate in the nervous system. As a result, great progress in understanding the transcriptional network that controls photoreceptor specification and differentiation has been made over the last 20 years. This progress has also enabled the production of photoreceptors from pluripotent stem cells, thereby aiding the development of regenerative medical approaches to eye disease. In this Review, we outline the signaling and transcription factors that drive vertebrate photoreceptor development and discuss how these function together in gene regulatory networks to control photoreceptor cell fate specification. PMID:26443631

  5. Learning LM Specificity for Ganglion Cells

    Science.gov (United States)

    Ahumada, Albert J.

    2015-01-01

    Unsupervised learning models have been proposed based on experience (Ahumada and Mulligan, 1990;Wachtler, Doi, Lee and Sejnowski, 2007) that allow the cortex to develop units with LM specific color opponent receptive fields like the blob cells reported by Hubel and Wiesel on the basis of visual experience. These models used ganglion cells with LM indiscriminate wiring as inputs to the learning mechanism, which was presumed to occur at the cortical level.

  6. Cell-Specific Aptamers as Emerging Therapeutics

    Directory of Open Access Journals (Sweden)

    Cindy Meyer

    2011-01-01

    Full Text Available Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by an in vitro process named SELEX (systematic evolution of ligands by exponential enrichment. Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.

  7. Unsteady Flow in a Supersonic Turbine with Variable Specific Heats

    Science.gov (United States)

    Dorney, Daniel J.; Griffin, Lisa W.; Huber, Frank; Sondak, Douglas L.; Turner, James (Technical Monitor)

    2001-01-01

    Modern high-work turbines can be compact, transonic, supersonic, counter-rotating, or use a dense drive gas. The vast majority of modern rocket turbine designs fall into these Categories. These turbines usually have large temperature variations across a given stage, and are characterized by large amounts of flow unsteadiness. The flow unsteadiness can have a major impact on the turbine performance and durability. For example, the Space Transportation Main Engine (STME) fuel turbine, a high work, transonic design, was found to have an unsteady inter-row shock which reduced efficiency by 2 points and increased dynamic loading by 24 percent. The Revolutionary Reusable Technology Turbopump (RRTT), which uses full flow oxygen for its drive gas, was found to shed vortices with such energy as to raise serious blade durability concerns. In both cases, the sources of the problems were uncovered (before turbopump testing) with the application of validated, unsteady computational fluid dynamics (CFD) to the designs. In the case of the RRTT and the Alternate Turbopump Development (ATD) turbines, the unsteady CFD codes have been used not just to identify problems, but to guide designs which mitigate problems due to unsteadiness. Using unsteady flow analyses as a part of the design process has led to turbine designs with higher performance (which affects temperature and mass flow rate) and fewer dynamics problems. One of the many assumptions made during the design and analysis of supersonic turbine stages is that the values of the specific heats are constant. In some analyses the value is based on an average of the expected upstream and downstream temperatures. In stages where the temperature can vary by 300 to 500 K, however, the assumption of constant fluid properties may lead to erroneous performance and durability predictions. In this study the suitability of assuming constant specific heats has been investigated by performing three-dimensional unsteady Navier

  8. Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition.

    Directory of Open Access Journals (Sweden)

    Gaëtan Gruel

    Full Text Available Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from 60Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This

  9. Artesunate exerts specific cytotoxicity in retinoblastoma cells via CD71.

    Science.gov (United States)

    Zhao, Fei; Wang, Han; Kunda, Patricilia; Chen, Xuemei; Liu, Qiu-Ling; Liu, Tao

    2013-09-01

    Retinoblastoma (RB) is an intraocular cancer that affects young children. There is an ongoing effort to find new agents for RB management that are effective, specific and with few side-effects. In the present study, we tested artesunate (ART), a synthetic derivative from the herbal drug artemisinin, used in the clinic for the treatment of malaria. We analyzed ART cytotoxicity in an RB cell line (RB-Y79) and in a retinal epithelial cell line (hTERT-RPE1) by flow cytometric analysis (FCM). We related the effect of ART to the expression of transferrin receptor 1 (TfR-1, also known as CD71) by knocking down CD71 with RNAi and analyzing cell cycle variables by FCM. We found that the cytotoxic action of ART is specific for RB cells in a dose-dependent manner, with low toxicity in normal retina cells. ART is more effective in RB than carboplatin with a markedly strong cytotoxic effect on carboplatin-resistant RB cells. RB had higher CD71 levels at the membrane compared to normal retinal cells. We showed that ART internalization in RB cells is dependent upon the expression of the CD71. In addition, ART blocked the cell cycle progression at the G1 phase, even at low doses, and decreased the proportion of RB cells in the S phase. In conclusion, we showed that ART is a promising drug exhibiting high selective cytotoxicity even against multidrug-resistant RB cells. Thus, we suggest that ART could be used in the treatment of RB.

  10. Primordial germ cell specification from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Wei Wei

    Full Text Available BACKGROUND: Primordial germ cell (PGC specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation. CONCLUSIONS AND SIGNIFICANCE: The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

  11. Specification for dispersed fuel-cell generator

    Science.gov (United States)

    Handley, L. M.; Cohen, R.

    1981-11-01

    A general description and performance definition for a standard 11-mw fuel cell power plant designed for electric utility dispersed-generation applications are provided. Additional features available at the option of the purchaser are also described. The power plant can operate singly or grouped with other power plants to produce larger mutli-megawatt power stations. A 33-mw station is discussed as representative of multiple power plant installations. The power plant specification defines power rating, heat rate, fuels, operating modes, siting characteristics, and available options. A general description included in the attachments covers equipment, typical site arrangement, auxiliary subsystems, maintenance, fuel flexibility, and general fluid and electrical schematics.

  12. SPECIFICITIES PRESENTED IN SOME BASIC AND SPECIFIC MOTOR SKILLS OF VARIABLES TO YOUNG BASKETBALL PLAYERS

    OpenAIRE

    Hazir Salihu; Dardan Dehari

    2016-01-01

    On the basis of the number of 62 entities aged 15-16 years, male gender, address space is composed of the locomotive and the space of ten tests. Factorial procedures are addressed in all basic and specific locomotives, together as results have gained 3 dimensions latency: - Factor of specific speed and accuracy; - Factor complex locomotive specific typical for game and basketball; - Factor of speed and explosive force to the upper extremity. Such a mix of factors, may be justi...

  13. High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

    Science.gov (United States)

    Cerenius, Lage; Liu, Haipeng; Zhang, Yanjiao; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee; Gunnar Andersson, M; Söderhäll, Kenneth; Söderhäll, Irene

    2010-01-01

    Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

  14. Variability and Order in Cytoskeletal Dynamics of Motile Amoeboid Cells

    Science.gov (United States)

    Hsu, Hsin-Fang; Bodenschatz, Eberhard; Westendorf, Christian; Gholami, Azam; Pumir, Alain; Tarantola, Marco; Beta, Carsten

    2017-10-01

    The chemotactic motion of eukaryotic cells such as leukocytes or metastatic cancer cells relies on membrane protrusions driven by the polymerization and depolymerization of actin. Here we show that the response of the actin system to a receptor stimulus is subject to a threshold value that varies strongly from cell to cell. Above the threshold, we observe pronounced cell-to-cell variability in the response amplitude. The polymerization time, however, is almost constant over the entire range of response amplitudes, while the depolymerization time increases with increasing amplitude. We show that cell-to-cell variability in the response amplitude correlates with the amount of Arp2 /3 , a protein that enhances actin polymerization. A time-delayed feedback model for the cortical actin concentration is consistent with all our observations and confirms the role of Arp2 /3 in the observed cell-to-cell variability. Taken together, our observations highlight robust regulation of the actin response that enables a reliable timing of cell movement.

  15. Biomek Cell Workstation: A Variable System for Automated Cell Cultivation.

    Science.gov (United States)

    Lehmann, R; Severitt, J C; Roddelkopf, T; Junginger, S; Thurow, K

    2016-06-01

    Automated cell cultivation is an important tool for simplifying routine laboratory work. Automated methods are independent of skill levels and daily constitution of laboratory staff in combination with a constant quality and performance of the methods. The Biomek Cell Workstation was configured as a flexible and compatible system. The modified Biomek Cell Workstation enables the cultivation of adherent and suspension cells. Until now, no commercially available systems enabled the automated handling of both types of cells in one system. In particular, the automated cultivation of suspension cells in this form has not been published. The cell counts and viabilities were nonsignificantly decreased for cells cultivated in AutoFlasks in automated handling. The proliferation of manual and automated bioscreening by the WST-1 assay showed a nonsignificant lower proliferation of automatically disseminated cells associated with a mostly lower standard error. The disseminated suspension cell lines showed different pronounced proliferations in descending order, starting with Jurkat cells followed by SEM, Molt4, and RS4 cells having the lowest proliferation. In this respect, we successfully disseminated and screened suspension cells in an automated way. The automated cultivation and dissemination of a variety of suspension cells can replace the manual method. © 2015 Society for Laboratory Automation and Screening.

  16. From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects.

    Science.gov (United States)

    Ben-Ari, Meital; Schick, Revital; Barad, Lili; Novak, Atara; Ben-Ari, Erez; Lorber, Avraham; Itskovitz-Eldor, Joseph; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2014-10-01

    We previously reported that induced pluripotent stem cell-derived cardiomyocytes manifest beat rate variability (BRV) resembling heart rate variability (HRV) in the human sinoatrial node. We now hypothesized the BRV-HRV continuum originates in pacemaker cells. To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized 3 levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells, and (3) the in situ sinoatrial node. We measured BRV/HRV properties in single pacemaker cells, induced pluripotent stem cell-derived contracting embryoid bodies (EBs), and electrocardiograms from the same individual. Pronounced BRV/HRV was present at all 3 levels. The coefficient of variance of interbeat intervals and Poincaré plot indices SD1 and SD2 for single cells were 20 times greater than those for EBs (P heart (the latter two were similar; P > .05). We also compared BRV magnitude among single cells, small EBs (~5-10 cells), and larger EBs (>10 cells): BRV indices progressively increased with the decrease in the cell number (P heart rhythm. The decreased BRV magnitude in transitioning from the single cell to the EB suggests that the HRV of in situ hearts originates from the summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca(2+) handling determine HRV in humans and cardiomyocyte networks. Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  17. Abnormality of regulatory T cells in common variable immunodeficiency.

    Science.gov (United States)

    Azizi, Gholamreza; Hafezi, Nasim; Mohammadi, Hamed; Yazdani, Reza; Alinia, Tina; Tavakol, Marzieh; Aghamohammadi, Asghar; Mirshafiey, Abbas

    2017-05-01

    Common variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies (PAD) which is defined by recurrent infections, hypogammaglobulinemia and defects in B-cell differentiation into plasma cells and memory B cells. T cell abnormalities have also been described in CVID patients. Several studies reported that Treg frequencies and their functional characteristics are disturbed and might account for the aberrant immune responses observed in CVID patients. The aim of this review is to describe phenotypic and functional characteristics of Treg cells, and to review the literature with respect to the reported Treg defects and its association with the clinical manifestation in CVID. Copyright © 2016. Published by Elsevier Inc.

  18. Conceptualization and Operationalisation of Specific Variables in Exploratory Researches – An Example for Business Negotiation

    Directory of Open Access Journals (Sweden)

    Zait Adriana

    2016-03-01

    Full Text Available In exploratory researches we are looking for clues, trying to get a better picture of the research variables and relationships. Besides variables already studied, identified in the literature review, we might deal with specific variables - for the investigated subject and socio-cultural region - the ones that might lead us to new explanations and theories. These new variables can be depicted from in-depth interviews and informal discussions with intervening actors, through a process of identification, conceptualization (obtaining theoretical definitions, and operationalisation (suggesting ways for the measurement of the identified variables. The present paper offers an example of such a process for a particular subject - identifying potential specific influences on the business negotiation behaviour. The focus is on the methodological aspects of such a research endeavour.

  19. Specific cell cycle synchronization with butyrate and cell cycle analysis

    Science.gov (United States)

    Synchronized cells have been invaluable for many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. To explore the possibility of using butyrate-blocked cells to obtain synchronized cells, we investigated the property of the cell cyc...

  20. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo.

    Science.gov (United States)

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V S

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner.

  1. Modulating cell-to-cell variability and sensitivity to death ligands by co-drugging

    Science.gov (United States)

    Flusberg, Deborah A.; Sorger, Peter K.

    2013-06-01

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) holds promise as an anti-cancer therapeutic but efficiently induces apoptosis in only a subset of tumor cell lines. Moreover, even in clonal populations of responsive lines, only a fraction of cells dies in response to TRAIL and individual cells exhibit cell-to-cell variability in the timing of cell death. Fractional killing in these cell populations appears to arise not from genetic differences among cells but rather from differences in gene expression states, fluctuations in protein levels and the extent to which TRAIL-induced death or survival pathways become activated. In this study, we ask how cell-to-cell variability manifests in cell types with different sensitivities to TRAIL, as well as how it changes when cells are exposed to combinations of drugs. We show that individual cells that survive treatment with TRAIL can regenerate the sensitivity and death-time distribution of the parental population, demonstrating that fractional killing is a stable property of cell populations. We also show that cell-to-cell variability in the timing and probability of apoptosis in response to treatment can be tuned using combinations of drugs that together increase apoptotic sensitivity compared to treatment with one drug alone. In the case of TRAIL, modulation of cell-to-cell variability by co-drugging appears to involve a reduction in the threshold for mitochondrial outer membrane permeabilization.

  2. Homotopy method for optimization of variable-specific-impulse low-thrust trajectories

    Science.gov (United States)

    Chi, Zhemin; Yang, Hongwei; Chen, Shiyu; Li, Junfeng

    2017-11-01

    The homotopy method has been used as a useful tool in solving fuel-optimal trajectories with constant-specific-impulse low thrust. However, the specific impulse is often variable for many practical solar electric power-limited thrusters. This paper investigates the application of the homotopy method for optimization of variable-specific-impulse low-thrust trajectories. Difficulties arise when the two commonly-used homotopy functions are employed for trajectory optimization. The optimal power throttle level and the optimal specific impulse are coupled with the commonly-used quadratic and logarithmic homotopy functions. To overcome these difficulties, a modified logarithmic homotopy function is proposed to serve as a gateway for trajectory optimization, leading to decoupled expressions of both the optimal power throttle level and the optimal specific impulse. The homotopy method based on this homotopy function is proposed. Numerical simulations validate the feasibility and high efficiency of the proposed method.

  3. Stimulation of adult oligodendrogenesis by myelin-specific T cells

    DEFF Research Database (Denmark)

    Hvilsted Nielsen, Helle; Toft-Hansen, Henrik; Lambertsen, Kate Lykke

    2011-01-01

    In multiple sclerosis (MS), myelin-specific T cells are normally associated with destruction of myelin and axonal damage. However, in acute MS plaque, remyelination occurs concurrent with T-cell infiltration, which raises the question of whether T cells might stimulate myelin repair. We...... investigated the effect of myelin-specific T cells on oligodendrocyte formation at sites of axonal damage in the mouse hippocampal dentate gyrus. Infiltrating T cells specific for myelin proteolipid protein stimulated proliferation of chondroitin sulfate NG2-expressing oligodendrocyte precursor cells early...... after induction via axonal transection, resulting in a 25% increase in the numbers of oligodendrocytes. In contrast, T cells specific for ovalbumin did not stimulate the formation of new oligodendrocytes. In addition, infiltration of myelin-specific T cells enhanced the sprouting response...

  4. Intrinsic Variability Present in Wharton’s Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy

    Directory of Open Access Journals (Sweden)

    Fernanda Vieira Paladino

    2017-01-01

    Full Text Available Wharton’s jelly mesenchymal stem cells (WJ-MSC exhibit immunomodulatory effects on T cell response. WJ-MSC are easy to collect, process, and proliferate rapidly in culture, but information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. We propose to evaluate the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence in order to analyze if expected responses are affected. Our results show that the gene expression of immunomodulatory molecules varied among samples with no specific pattern present. In coculture, all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different degrees, and each PBMC responded with a different level of inhibition. Thus, we suggest that each WJ-MSC displays unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. We believe that variability between samples may play a role in the effectiveness of WJ-MSC employed therapeutically.

  5. Saporin-conjugated tetramers identify efficacious anti-HIV CD8+ T-cell specificities

    DEFF Research Database (Denmark)

    Leitman, Ellen M.; Palmer, Christine D.; Buus, Søren

    2017-01-01

    Antigen-specific T-cells are highly variable, spanning potent antiviral efficacy and damaging auto-reactivity. In virus infections, identifying the most efficacious responses is critical to vaccine design. However, current methods depend on indirect measures or on ex vivo expanded CTL clones. We...... of these reagents is demonstrated here in identifying the CD8+ T-cell specificity most effective in preventing HIV progression in HIV-infected HLA-B*27-positive immune controllers....

  6. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  7. Virus-specific peptide dependent NK cell cytotoxicity.

    Science.gov (United States)

    Tong, Lan; Assenmacher, Mario; Zänker, Kurt S; Jähn, Peter

    2014-01-01

    NK cells do not express recombination-dependent antigen-specific receptors and are traditionally defined as cells of the innate immune response. The activation of NK cells was believed to be controlled by the net balance of signals from a multitude of activating and inhibitory receptors irrespectively of antigen specificity. However, murine antigen-specific memory NK cells in liver have been described to mediate hapten or viral specific recall response and are capable of infiltrating to the site of infection. The mechanisms by which NK cells recognize target cells in an antigen-specific manner are largely unclear. Using a novel multiplex killing assay, we screened the NK cell (human) cytotoxic activity of 35 different donors against different virus peptide pools loaded autologous B cells. We have found that human NK cells from some CMV and EBV positive donors can recognize peptide loaded autologous B cells as targets and perform antigen-specific cytotoxic killing. This may provide evidence that NK cells are able to scan the peptide repertoire on the target cell surface and virus-derived peptides may influence the NK cell activation-inhibition balance.

  8. Integration of aerial imaging and variable-rate technology for site-specific aerial herbicide application

    Science.gov (United States)

    As remote sensing and variable rate technology are becoming more available for aerial applicators, practical methodologies on effective integration of these technologies are needed for site-specific aerial applications of crop production and protection materials. The objectives of this study were to...

  9. Culture-Specific Variables That May Affect Employment Outcomes for Mexican-American Youth with Disabilities.

    Science.gov (United States)

    Meier-Kronick, Nancy

    This paper reviews variables specific to the Mexican-American culture that might influence work-related behavior and outcomes for youths with disabilities from this population. Areas covered include: parental/family network; cultural view of disability; religious influences; acculturation levels; language issues; education and employment…

  10. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  11. SCALE: modeling allele-specific gene expression by single-cell RNA sequencing.

    Science.gov (United States)

    Jiang, Yuchao; Zhang, Nancy R; Li, Mingyao

    2017-04-26

    Allele-specific expression is traditionally studied by bulk RNA sequencing, which measures average expression across cells. Single-cell RNA sequencing allows the comparison of expression distribution between the two alleles of a diploid organism and the characterization of allele-specific bursting. Here, we propose SCALE to analyze genome-wide allele-specific bursting, with adjustment of technical variability. SCALE detects genes exhibiting allelic differences in bursting parameters and genes whose alleles burst non-independently. We apply SCALE to mouse blastocyst and human fibroblast cells and find that cis control in gene expression overwhelmingly manifests as differences in burst frequency.

  12. Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection

    Science.gov (United States)

    Ertelt, James M; Johanns, Tanner M; Mysz, Margaret A; Nanton, Minelva R; Rowe, Jared H; Aguilera, Marijo N; Way, Sing Sing

    2011-01-01

    Typhoid fever is a persistent infection caused by host-adapted Salmonella strains adept at circumventing immune-mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen-specific CD4+ T cells after virulent Salmonella infection requires SPI-2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella-expressed antigens, but extends to CD4+ T cells primed to expand by co-infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen-specific CD4+ T cells identified with MHC class II tetramer. Analysis of T-cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T-cell subsets after Salmonella co-infection that is associated with the purging of antigen-specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection. PMID:22044420

  13. Measles virus-specific murine T cell clones: characterization of fine specificity function.

    NARCIS (Netherlands)

    P. de Vries (Petra); J.P.M. Versteeg-van Oosten (José); I.K.G. Visser (Ilona); R.S. van Binnendijk (Rob); S.A. Langeveld (Sacha); A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

    1989-01-01

    textabstractMeasles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable

  14. Landscape controls on spatiotemporal variability of specific discharge in a boreal region

    Science.gov (United States)

    Karlsen, R.; Grabs, T.; Bishop, K. H.; Laudon, H.; Seibert, J.

    2014-12-01

    Spatial and temporal variability of specific discharge is rarely measured at the small-catchment-scale and is commonly ignored by most studies which instead assume spatially uniform specific discharge. This assumption is convenient but can lead to fundamentally wrong results, e.g., when calculating solute fluxes at the catchment scale. Pioneering work on 14, partly nested, sub-catchments in a boreal meso-scale (67 km2) catchment in Northern Sweden revealed substantial spatial and temporal variations in both the magnitude and timing of specific discharge. We explore the structure of this variability and its connection to the landscape characteristics using a 5-year gap filled time series of continuous flow records.For the long term (5 years) flow magnitudes for the various sub-catchments varied between 73 % and 132 % relative to the flow at the main outlet, with higher flows from wetland dominated catchments (Spearman rank correlation R=0.81). Looking at seasonal and short term flows, both more pronounced variability and stronger links to different landscape properties are seen. Spring flood magnitudes were correlated (R=0.61 to 0.80) with wet areas, while summer flows were negatively correlated to catchment tree volumes (R=-0.61 to -0.75) and potential evaporation (R=-0.48 to -0.78).On shorter timescales, from daily to monthly, also other catchment properties explained the observed spatial discharge variability. During dry periods of low summer baseflows, sub-catchments with deep fluvial deposits maintained a higher discharge than catchments with shallow soils. This pattern was reversed during summer stormflow events.The results show that there is a spatial structure in the specific discharge and that this structure is temporally variable. Different landscape characteristics influence the flows at different time scales, and the spatiotemporal discharge variability depends on seasonal climatic variability. The observed structure does not only influence mass balance

  15. Temporal variability and stability in infant-directed sung speech: evidence for language-specific patterns.

    Science.gov (United States)

    Falk, Simone

    2011-06-01

    In this paper, sung speech is used as a methodological tool to explore temporal variability in the timing of word-internal consonants and vowels. It is hypothesized that temporal variability/stability becomes clearer under the varying rhythmical conditions induced by song.This is explored crosslinguistically in German - a language that exhibits a potential vocalic quantity distinction - and the non-quantity languages French and Russian. Songs by non-professional singers, i.e. parents that sang to their infants aged 2 to 13 months in a non-laboratory setting, were recorded and analyzed.Vowel and consonant durations at syllable contacts of trochaic word types with CVCV or CV:CV structure were measured under varying rhythmical conditions. Evidence is provided that in German non-professional singing, the two syllable structures can be differentiated by two distinct temporal variability patterns: vocalic variability (and consonantal stability) was found to be dominant in CV:CV structures whereas consonantal variability (and vocalic stability) was characteristic for CVCV structures. In French and Russian, however, only vocalic variability seemed to apply.Additionally, findings suggest that the different temporal patterns found in German were also supported by the stability pattern at the tonal level. These results point to subtle (supra) segmental timing mechanisms in sung speech that affect temporal targets according to the specific prosodic nature of the language in question.

  16. Examining the sources of variability in cell culture media used for biopharmaceutical production.

    Science.gov (United States)

    McGillicuddy, Nicola; Floris, Patrick; Albrecht, Simone; Bones, Jonathan

    2018-01-01

    Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

  17. Cell type-specific bipolar cell input to ganglion cells in the mouse retina.

    Science.gov (United States)

    Neumann, S; Hüser, L; Ondreka, K; Auler, N; Haverkamp, S

    2016-03-01

    Many distinct ganglion cell types, which are the output elements of the retina, were found to encode for specific features of a visual scene such as contrast, color information or movement. The detailed composition of retinal circuits leading to this tuning of retinal ganglion cells, however, is apart from some prominent examples, largely unknown. Here we aimed to investigate if ganglion cell types in the mouse retina receive selective input from specific bipolar cell types or if they sample their synaptic input non-selectively from all bipolar cell types stratifying within their dendritic tree. To address this question we took an anatomical approach and immunolabeled retinae of two transgenic mouse lines (GFP-O and JAM-B) with markers for ribbon synapses and type 2 bipolar cells. We morphologically identified all green fluorescent protein (GFP)-expressing ganglion cell types, which co-stratified with type 2 bipolar cells and assessed the total number of bipolar input synapses and the proportion of synapses deriving from type 2 bipolar cells. Only JAM-B ganglion cells received synaptic input preferentially from bipolar cell types other than type 2 bipolar cells whereas the other analyzed ganglion cell types sampled their bipolar input most likely from all bipolar cell terminals within their dendritic arbor. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Target-specific delivery of doxorubicin to human glioblastoma cell ...

    Indian Academy of Sciences (India)

    Abdullah Tahir Bayraç

    2018-01-29

    Jan 29, 2018 ... nano-sized targeted drug delivery approach adorned with A-172 glioblastoma cell-line-specific single stranded DNA. (ssDNA) ... GMT-3 aptamer-mediated therapeutic drug transportation in the treatment of gliomas specifically. ..... Nanomedicine to overcome radioresistance in glioblastoma stem-like cells ...

  19. Sex Specification and Heterogeneity of Primordial Germ Cells in Mice.

    Science.gov (United States)

    Sakashita, Akihiko; Kawabata, Yukiko; Jincho, Yuko; Tajima, Shiun; Kumamoto, Soichiro; Kobayashi, Hisato; Matsui, Yasuhisa; Kono, Tomohiro

    2015-01-01

    In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells.

  20. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  1. On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium.

    Science.gov (United States)

    Long, Yan; Huang, He

    2015-12-01

    Hematopoietic stem cells (HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells (PSCs-embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.

  2. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam

    2009-01-01

    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  3. Organ-Specific and Memory Treg Cells: Specificity, Development, Function, and Maintenance

    Science.gov (United States)

    Gratz, Iris K.; Campbell, Daniel J.

    2014-01-01

    Foxp3+ regulatory T cells (Treg cells) are essential for establishing and maintaining self-tolerance, and also inhibit immune responses to innocuous environmental antigens. Imbalances and dysfunction in Treg cells lead to a variety of immune-mediated diseases, as deficits in Treg cell function contribute to the development autoimmune disease and pathological tissue damage, whereas overabundance of Treg cells can promote chronic infection and tumorigenesis. Recent studies have highlighted the fact that Treg cells themselves are a diverse collection of phenotypically and functionally specialized populations, with distinct developmental origins, antigen-specificities, tissue-tropisms, and homeostatic requirements. The signals directing the differentiation of these populations, their specificities and the mechanisms by which they combine to promote organ-specific and systemic tolerance, and how they embody the emerging property of regulatory memory are the focus of this review. PMID:25076948

  4. Same same but different - quantifying the importance of intra-specific variability to plankton biodiversity

    Science.gov (United States)

    Menden-Deuer, S.; Rowlett, J. M.

    2016-02-01

    Planktonic microbes are vastly more diverse than theoretically explicable (Hutchinson 1961). We recently suggested that intra-specific variability was the key characteristic that allowed co-existence of dozens of planktonic species because the outcome of competitions between individuals with variable competitive abilities was unpredictable (Menden-Deuer and Rowlett 2014). Building on this game-theoretic model, here we examine the quantitative consequences of different degrees of intra-specific variability on species survival and co-existence probability. Frequency distributions of species competitive abilities (i.e. species behavior distributions SBD) vary, including invariant distributions with each individual's competitive ability identical to the mean to entirely bimodal distributions, consisting only of individuals with the highest and lowest competitive ability. In total, we explore the effect of 14 different SBDs on species survival probability in individual-based competition model simulations across varying durations and population sizes. We find that particularly at small population sizes intra-specific diversity enhances survival probability and for some SBDs extinctions are not observed. These results have implications for anticipating species ability to withstand changing environmental conditions and understand diverse planktonic communities in a seemingly uniform ocean.

  5. Tissue specific heterogeneity in effector immune cell response

    Directory of Open Access Journals (Sweden)

    Saba eTufail

    2013-08-01

    Full Text Available Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct ‘homing codes’ (adhesion molecules and chemokine receptors during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A and sunlight (vitamin D3 prime dendritic cells, imprinting them to play centrestage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues alongwith giving an overview of tissue tropism in B cells.

  6. Variability in expression of antigens responsible for serotype specificity in Candida albicans.

    Science.gov (United States)

    Barturen, B; Bikandi, J; San Millán, R; Moragues, M D; Regulez, P; Quindós, G; Pontón, J

    1995-07-01

    The monoclonal antibody (mAb) B9E, which reacts with a cell wall surface determinant of Candida albicans serotype A, and a polyclonal monospecific antiserum against the antigen 6 (IF6) were used to investigate the expression of the antigens responsible for the serotype specificity in C. albicans under different growth conditions. By indirect immunofluorescence, both antibodies reacted with the cell wall surface of serotype A yeast cells and germ tubes grown in vitro but no reactivity was observed with serotype B yeast cells. In some cases, only a weak reactivity restricted to a zone close to the parent yeast cell was observed in serotype B germ tubes stained with mAb B9E. Both antibodies reacted strongly with yeast cells and germ tubes present in kidney abscesses from rabbits infected with both serotypes, but only serotype A yeast cells and germ tubes present in smears from patients with vulvovaginal candidiasis reacted with B9E and IF6 antibodies. The expression of antigens reactive with both antibodies was modulated by the pH of the environment in which the fungus was grown. Both antibodies showed a similar pattern of reactivity when studied with a spectrofluorometer. Serotype A yeast cells showed maximum reactivity when cells were grown on Sabouraud dextrose broth supplemented with yeast extract at pH 4.6. The lowest reactivity was observed in cells grown at pH 2.0. Conversely, the reactivity of serotype B yeast cells increased at alkaline pH values, the highest being in cells grown at pH values of 7.2 and 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution.

    Science.gov (United States)

    Parham, Peter; Moffett, Ashley

    2013-02-01

    Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, in which they have progressively co-evolved with MHC class I molecules. The emergence of the MHC-C gene in hominids drove the evolution of a system of NK cell receptors for MHC-C molecules that is most elaborate in chimpanzees. By contrast, the human system of MHC-C receptors seems to have been subject to different selection pressures that have acted in competition on the immunological and reproductive functions of MHC class I molecules. We suggest that this compromise facilitated the development of the bigger brains that enabled archaic and modern humans to migrate out of Africa and populate other continents.

  8. Countermovement jump height: gender and sport-specific differences in the force-time variables.

    Science.gov (United States)

    Laffaye, Guillaume; Wagner, Phillip P; Tombleson, Tom I L

    2014-04-01

    The goal of this study was to assess (a) the eccentric rate of force development, the concentric force, and selected time variables on vertical performance during countermovement jump, (b) the existence of gender differences in these variables, and (c) the sport-specific differences. The sample was composed of 189 males and 84 females, all elite athletes involved in college and professional sports (primarily football, basketball, baseball, and volleyball). The subjects performed a series of 6 countermovement jumps on a force plate (500 Hz). Average eccentric rate of force development (ECC-RFD), total time (TIME), eccentric time (ECC-T), Ratio between eccentric and total time (ECC-T:T) and average force (CON-F) were extracted from force-time curves and the vertical jumping performance, measured by impulse momentum. Results show that CON-F (r = 0.57; p height (JH), whereas the time variables are slightly and negatively correlated (r = -0.21-0.23, p sport-specific signatures: volleyball players revealed a temporal-prevailing profile, a weak-force with large ECC-T:T for basketball players and explosive and powerful profiles for football and baseball players.

  9. Early specification of dopaminergic phenotype during ES cell differentiation

    Directory of Open Access Journals (Sweden)

    Li Meng

    2007-07-01

    Full Text Available Abstract Background Understanding how lineage choices are made during embryonic stem (ES cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA. However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. Results Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. Conclusion Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.

  10. β-CELL SPECIFIC CYTOPROTECTION BY PROLACTIN ON HUMAN ISLETS

    Science.gov (United States)

    Yamamoto, Toshiyuki; Ricordi, Camillo; Mita, Atsuyoshi; Miki, Atsushi; Sakuma, Yasunaru; Damaris Molano, R.; Fornoni, Alessia; Pileggi, Antonello; Inverardi, Luca; Ichii, Hirohito

    2008-01-01

    Many cytoprotective agents have been reported to improve islet isolation and transplantation outcomes. However, several cytoprotective agents may improve all cell subsets within an islet preparation, and selected non- β-cells components may have a negative effect on β-cell function and survival (e.g., acinar cells). In this study, we examined the effect of prolactin (PRL) supplementation to the culture medium, to determine whether it could exert β-cell-selective cytoprotection (islet viability and function) towards a possible use of PRL during pre transplant islet culture. Materials and Methods Human islets pre-cultured or not with recombinant human PRL (500 μg/L) for 48 hours. Non-β-cells and β-cell-specific fractional viability and cellular composition were assessed by FACS and Laser Scanning Cytometry (LSC). Islet potency was assessed in vivo by transplantation into chemically-induced diabetic immunodeficient mice. Results The relative viable β-cell mass and the relative islet β-cell content in PRL group were 28% higher (p=0.018) and 19% higher (p=0.029) than control group, respectively. All transplanted mice achieved normoglycemia in both groups, indicating that PRL treatment did not alter islet function. Conclusions PRL treatment improves β-cell-specific viability and survival in human islets in vitro. The development of novel β-cell-specific cytoprotective strategies will be of assistance in improving islet transplantation. PMID:18374075

  11. Contributions of weather variables for specific adaptation of rubber tree (Hevea brasiliensis Muell.- Arg clones

    Directory of Open Access Journals (Sweden)

    Priyadarshan P.M.

    2003-01-01

    Full Text Available The specific adaptation of 15 rubber tree (Hevea brasiliensis clones was assessed by analyzing yield during a normal year (1997-98 and a year (1998-99 in which the yield was exceptional. Differences in yield in response to changes in weather conditions over the years were evident with clones RRII 203, RRIM 703, PB 5/51 and PB 235 which all exhibited a negative trend with increasing wind velocity during 1997-98, these clones also exhibited a negative correlation with minimum temperature during 1998-99. The prominent yield differences across the years made selection based on both yield and stability inevitable through computing weather variables and environmental index as covariant. To determine the contribution of variable(s to genotype-environment (GE interactions, the GE interaction was partitioned into heterogeneity and residual GE interaction. Heterogeneity only for environmental index was highly significant (p = 0.01, meaning that stability or instability of clones was due to a linear effect of the environmental index. The non-significant values of heterogeneity for the weather variables revealed that none of these factors individually was sufficient to explain heterogeneity. A QBASIC computer program called STABLE was used to select simultaneously for yield and stability. Clones PB 235, RRII 118, RRII 203, RRIM 703 and RRIM 600 were stable over the years investigated.

  12. Measles virus–specific plasma cells are prominent in subacute sclerosing panencephalitis CSF

    Science.gov (United States)

    Owens, G.P.; Ritchie, A.M.; Gilden, D.H.; Burgoon, M.P.; Becker, D.; Bennett, J.L.

    2012-01-01

    Objective To demonstrate the specificity of expanded CD138+ plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). Methods IgG variable region sequences of single-antibody-secreting CD138+ cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138+ clones and assayed for immunoreactivity against MV proteins. Results Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. Conclusions Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders. PMID:17515543

  13. Osteocalcin and bone-specific alkaline phosphatase in Sickle cell ...

    African Journals Online (AJOL)

    specific alkaline phosphatase (b-AP) total protein levels were evaluated as indicators of bone turnover in twenty patients with sickle cell haemoglobinopathies and in twenty normal healthy individuals. The serum bonespecific alkaline phosphatase ...

  14. Specific cell surface labeling of GPCRs using split GFP

    National Research Council Canada - National Science Library

    Jiang, Wen-Xue; Dong, Xu; Jiang, Jing; Yang, Yu-Hong; Yang, Ju; Lu, Yun-Bi; Fang, San-Hua; Wei, Er-Qing; Tang, Chun; Zhang, Wei-Ping

    2016-01-01

    ...) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP...

  15. Biological Variation of Hemostasis Variables in Thrombosis and Bleeding: Consequences for Performance Specifications.

    Science.gov (United States)

    de Maat, Moniek P M; van Schie, Marianne; Kluft, Cornelis; Leebeek, Frank W G; Meijer, Piet

    2016-12-01

    Levels of hemostasis factors vary between and within individuals as a result of genetic and environmental factors and analytical variation of the assays. The current state of the art for defining analytical precision requirements for analytical testing is based on this between- and within-individual (biological) variation. However, information on biological variation in hemostasis variables is still limited.The aim of this study was to determine the biological variation of coagulation variables involved in thrombosis and bleeding to provide a recommendation for performance specifications and to assess whether hemostasis assays fulfill the recommendation. We performed a longitudinal study by repeated blood sampling (in total 13 times over a 1-year period) in 40 healthy individuals and measured prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, antithrombin, factor VIII, factor IX, von Willebrand factor (VWF), protein C, and protein S. We evaluated the effect of the biological variation on parameters of analytical variation and propose required performance specifications. Biological variation was highly different for various hemostasis variables: the within-subject variation ranged from 2.6% (PT) to 25.6% [VWF collagen binding (CB) activity], the between-subject variation varied from 4.1% (PT) to 31.2% (VWF:ristocetin cofactor acitivity), and the assay variation from 1.3% (PT) to 12.9% (VWF:CB). With the reagents and analyzers used in this study, most of the hemostasis tests variables fulfill the current quality criteria for diagnosis and monitoring of routine hemostasis assays. © 2016 American Association for Clinical Chemistry.

  16. Divergence of zebrafish and mouse lymphatic cell fate specification pathways

    NARCIS (Netherlands)

    van Impel, Andreas; Zhao, Zhonghua; Hermkens, Dorien M A; Roukens, M Guy; Fischer, Johanna C; Peterson-Maduro, Josi; Duckers, Henricus; Ober, Elke A; Ingham, Philip W; Schulte-Merker, Stefan

    In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis.

  17. Target-specific delivery of doxorubicin to human glioblastoma cell ...

    Indian Academy of Sciences (India)

    Celltype-specific toxicity of GMT-3:DOX complex was showed by XTT assay and terminated cytotoxic effects were screenedfor both target cell and a control breast cancer cell line. The result of this contribution demonstrated the potential utility ofGMT-3 aptamer-mediated therapeutic drug transportation in the treatment of ...

  18. Determinants of cell-to-cell variability in protein kinase signaling.

    Directory of Open Access Journals (Sweden)

    Matthias Jeschke

    Full Text Available Cells reliably sense environmental changes despite internal and external fluctuations, but the mechanisms underlying robustness remain unclear. We analyzed how fluctuations in signaling protein concentrations give rise to cell-to-cell variability in protein kinase signaling using analytical theory and numerical simulations. We characterized the dose-response behavior of signaling cascades by calculating the stimulus level at which a pathway responds ('pathway sensitivity' and the maximal activation level upon strong stimulation. Minimal kinase cascades with gradual dose-response behavior show strong variability, because the pathway sensitivity and the maximal activation level cannot be simultaneously invariant. Negative feedback regulation resolves this trade-off and coordinately reduces fluctuations in the pathway sensitivity and maximal activation. Feedbacks acting at different levels in the cascade control different aspects of the dose-response curve, thereby synergistically reducing the variability. We also investigated more complex, ultrasensitive signaling cascades capable of switch-like decision making, and found that these can be inherently robust to protein concentration fluctuations. We describe how the cell-to-cell variability of ultrasensitive signaling systems can be actively regulated, e.g., by altering the expression of phosphatase(s or by feedback/feedforward loops. Our calculations reveal that slow transcriptional negative feedback loops allow for variability suppression while maintaining switch-like decision making. Taken together, we describe design principles of signaling cascades that promote robustness. Our results may explain why certain signaling cascades like the yeast pheromone pathway show switch-like decision making with little cell-to-cell variability.

  19. Dysregulation of Innate Lymphoid Cells in Common Variable Immunodeficiency.

    Science.gov (United States)

    Maglione, Paul J; Cols, Montserrat; Cunningham-Rundles, Charlotte

    2017-10-05

    Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immune deficiency. With widespread use of immunoglobulin replacement therapy, non-infectious complications, such as autoimmunity, chronic intestinal inflammation, and lung disease, have replaced infections as the major cause of morbidity and mortality in this immune deficiency. The pathogenic mechanisms that underlie the development of these complications in CVID are not known; however, there have been numerous associated laboratory findings. Among the most intriguing of these associations is elevation of interferon signature genes in CVID patients with inflammatory/autoimmune complications, as a similar gene expression profile is found in systemic lupus erythematosus and other chronic inflammatory diseases. Linked with this heightened interferon signature in CVID is an expansion of circulating IFN-γ-producing innate lymphoid cells. Innate lymphoid cells are key regulators of both protective and pathogenic immune responses that have been extensively studied in recent years. Further exploration of innate lymphoid cell biology in CVID may uncover key mechanisms underlying the development of inflammatory complications in these patients and may inspire much needed novel therapeutic approaches.

  20. Sensitivity, Specificity and Predictive Value of Heart Rate Variability Indices in Type 1 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Anne Kastelianne França da Silva

    Full Text Available Abstract Background: Heart rate variability (HRV indices may detect autonomic changes with good diagnostic accuracy. Type diabetes mellitus (DM individuals may have changes in autonomic modulation; however, studies of this nature in this population are still scarce. Objective: To compare HRV indices between and assess their prognostic value by measurements of sensitivity, specificity and predictive values in young individuals with type 1 DM and healthy volunteers. Methods: In this cross-sectional study, physical and clinical assessment was performed in 39 young patients with type 1 DM and 43 young healthy controls. For HRV analysis, beat-to-beat heart rate variability was measured in dorsal decubitus, using a Polar S810i heart rate monitor, for 30 minutes. The following indices were calculated: SDNN, RMSSD, PNN50, TINN, RRTri, LF ms2, HF ms2, LF un, HF un, LF/HF, SD1, SD2, SD1/SD2, and ApEn. Results: Type 1 DM subjects showed a decrease in sympathetic and parasympathetic activities, and overall variability of autonomic nervous system. The RMSSD, SDNN, PNN50, LF ms2, HF ms2, RRTri, SD1 and SD2 indices showed greater diagnostic accuracy in discriminating diabetic from healthy individuals. Conclusion: Type 1 DM individuals have changes in autonomic modulation. The SDNN, RMSSD, PNN50, RRtri, LF ms2, HF ms2, SD1 and SD2 indices may be alternative tools to discriminate individuals with type 1 DM.

  1. Sensitivity, Specificity and Predictive Value of Heart Rate Variability Indices in Type 1 Diabetes Mellitus

    Science.gov (United States)

    da Silva, Anne Kastelianne França; Christofaro, Diego Giuliano Destro; Bernardo, Aline Fernanda Barbosa; Vanderlei, Franciele Marques; Vanderlei, Luiz Carlos Marques

    2017-01-01

    Background Heart rate variability (HRV) indices may detect autonomic changes with good diagnostic accuracy. Type diabetes mellitus (DM) individuals may have changes in autonomic modulation; however, studies of this nature in this population are still scarce. Objective To compare HRV indices between and assess their prognostic value by measurements of sensitivity, specificity and predictive values in young individuals with type 1 DM and healthy volunteers. Methods In this cross-sectional study, physical and clinical assessment was performed in 39 young patients with type 1 DM and 43 young healthy controls. For HRV analysis, beat-to-beat heart rate variability was measured in dorsal decubitus, using a Polar S810i heart rate monitor, for 30 minutes. The following indices were calculated: SDNN, RMSSD, PNN50, TINN, RRTri, LF ms2, HF ms2, LF un, HF un, LF/HF, SD1, SD2, SD1/SD2, and ApEn. Results Type 1 DM subjects showed a decrease in sympathetic and parasympathetic activities, and overall variability of autonomic nervous system. The RMSSD, SDNN, PNN50, LF ms2, HF ms2, RRTri, SD1 and SD2 indices showed greater diagnostic accuracy in discriminating diabetic from healthy individuals. Conclusion Type 1 DM individuals have changes in autonomic modulation. The SDNN, RMSSD, PNN50, RRtri, LF ms2, HF ms2, SD1 and SD2 indices may be alternative tools to discriminate individuals with type 1 DM. PMID:28443958

  2. Connecting social environment variables to the onset of major specific health outcomes.

    Science.gov (United States)

    Hill, Patrick L; Weston, Sara J; Jackson, Joshua J

    2014-01-01

    The present research examined the effects of the social environment on the onset of specific health ailments. Using data from the Health and Retirement Study, we examined participants' responses to social environment questions in 2006 as predictors of onset of different health conditions over the next four years. Healthy participants (n = 7514) reported on their number of social partners, interaction frequency, positive social support and negative social support with respect to both their family and friends. These variables were used to predict onset of seven conditions in 2010: high blood pressure, heart condition, lung disease, cancer, stroke, diabetes and arthritis. Logistic regressions indicated that the social environment provided some predictive value for onset of most health outcomes, with more positive and less negative social support appearing to buffer against onset. Social environmental variables related to friendships appeared to play a greater role than the family indicators. However, no variable proved universally adaptive, and social indicators had little value in predicting onset of chronic conditions. The current findings point to the potential for the social environment to influence later health, while demonstrating the nuanced role that our social lives play with respect to health.

  3. Intraindividual Variability in Domain-Specific Cognition and Risk of Mild Cognitive Impairment and Dementia

    Directory of Open Access Journals (Sweden)

    Leslie Vaughan

    2013-01-01

    Full Text Available Intraindividual variability among cognitive domains may predict dementia independently of interindividual differences in cognition. A multidomain cognitive battery was administered to 2305 older adult women (mean age 74 years enrolled in an ancillary study of the Women’s Health Initiative. Women were evaluated annually for probable dementia and mild cognitive impairment (MCI for an average of 5.3 years using a standardized protocol. Proportional hazards regression showed that lower baseline domain-specific cognitive scores significantly predicted MCI (N=74, probable dementia (N=45, and MCI or probable dementia combined (N=101 and that verbal and figural memory predicted each outcome independently of all other cognitive domains. The baseline intraindividual standard deviation across test scores (IAV Cognitive Domains significantly predicted probable dementia and this effect was attenuated by interindividual differences in verbal episodic memory. Slope increases in IAV Cognitive Domains across measurement occasions (IAV Time explained additional risk for MCI and MCI or probable dementia, beyond that accounted for by interindividual differences in multiple cognitive measures, but risk for probable dementia was attenuated by mean decreases in verbal episodic memory slope. These findings demonstrate that within-person variability across cognitive domains both at baseline and longitudinally independently accounts for risk of cognitive impairment and dementia in support of the predictive utility of within-person variability.

  4. Patient-Specific Classification of ICU Sedation Levels From Heart Rate Variability.

    Science.gov (United States)

    Nagaraj, Sunil B; Biswal, Siddharth; Boyle, Emily J; Zhou, David W; McClain, Lauren M; Bajwa, Ednan K; Quraishi, Sadeq A; Akeju, Oluwaseun; Barbieri, Riccardo; Purdon, Patrick L; Westover, M Brandon

    2017-07-01

    To develop a personalizable algorithm to discriminate between sedation levels in ICU patients based on heart rate variability. Multicenter, pilot study. Several ICUs at Massachusetts General Hospital, Boston, MA. We gathered 21,912 hours of routine electrocardiogram recordings from a heterogenous group of 70 adult ICU patients. All patients included in the study were mechanically ventilated and were receiving sedatives. As "ground truth" for developing our method, we used Richmond Agitation Sedation Scale scores grouped into four levels denoted "comatose" (-5), "deeply sedated" (-4 to -3), "lightly sedated" (-2 to 0), and "agitated" (+1 to +4). We trained a support vector machine learning algorithm to calculate the probability of each sedation level from heart rate variability measures derived from the electrocardiogram. To estimate algorithm performance, we calculated leave-one-subject out cross-validated accuracy. The patient-independent version of the proposed system discriminated between the four sedation levels with an overall accuracy of 59%. Upon personalizing the system supplementing the training data with patient-specific calibration data, consisting of an individual's labeled heart rate variability epochs from the preceding 24 hours, accuracy improved to 67%. The personalized system discriminated between light- and deep-sedation states with an average accuracy of 75%. With further refinement, the methodology reported herein could lead to a fully automated system for depth of sedation monitoring. By enabling monitoring to be continuous, such technology may help clinical staff to monitor sedation levels more effectively and to reduce complications related to over- and under sedation.

  5. Dissection of T-cell antigen specificity in human melanoma.

    Science.gov (United States)

    Andersen, Rikke Sick; Thrue, Charlotte Albæk; Junker, Niels; Lyngaa, Rikke; Donia, Marco; Ellebæk, Eva; Svane, Inge Marie; Schumacher, Ton N; Thor Straten, Per; Hadrup, Sine Reker

    2012-04-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes predominantly from differentiation and cancer-testis antigens. Notably, the majority of these responses were of low frequency and tumor-specific T-cell frequencies decreased during rapid expansion. A further notable observation was a large variation in the T-cell specificities detected in cultures established from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined.

  6. Electrical detection of specific versus non-specific binding events in breast cancer cells

    Science.gov (United States)

    King, Benjamin C.; Clark, Michael; Burkhead, Thomas; Sethu, Palaniappan; Rai, Shesh; Kloecker, Goetz; Panchapakesan, Balaji

    2012-10-01

    Detection of circulating tumor cells (CTCs) from patient blood samples offers a desirable alternative to invasive tissue biopsies for screening of malignant carcinomas. A rigorous CTC detection method must identify CTCs from millions of other formed elements in blood and distinguish them from healthy tissue cells also present in the blood. CTCs are known to overexpress surface receptors, many of which aid them in invading other tissue, and these provide an avenue for their detection. We have developed carbon nanotube (CNT) thin film devices to specifically detect these receptors in intact cells. The CNT sidewalls are functionalized with antibodies specific to Epithelial Cell Adhesion Molecule (EpCAM), a marker overexpressed by breast and other carcinomas. Specific binding of EpCAM to anti-EpCAM antibodies causes a change in the local charge environment of the CNT surface which produces a characteristic electrical signal. Two cell lines were tested in the device: MCF7, a mammary adenocarcinoma line which overexpresses EpCAM, and MCF10A, a non-tumorigenic mammary epithelial line which does not. Introduction of MCF7s caused significant changes in the electrical conductance of the devices due to specific binding and associated charge environment change near the CNT sidewalls. Introduction of MCF10A displays a different profile due to purely nonspecific interactions. The profile of specific vs. nonspecific interaction signatures using carbon based devices will guide development of this diagnostic tool towards clinical sample volumes with wide variety of markers.

  7. Specific eradication of HIV-1 from infected cultured cells

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    Levin Aviad

    2010-08-01

    Full Text Available Abstract A correlation between increase in the integration of Human Immunodeficiency virus-1 (HIV-1 cDNA and cell death was previously established. Here we show that combination of peptides that stimulate integration together with the protease inhibitor Ro 31-8959 caused apoptotic cell death of HIV infected cells with total extermination of the virus. This combination did not have any effect on non-infected cells. Thus it appears that cell death is promoted only in the infected cells. It is our view that the results described in this work suggest a novel approach to specifically promote death of HIV-1 infected cells and thus may eventually be developed into a new and general anti-viral therapy.

  8. Targeted DNA vaccines for enhanced induction of Idiotype (Id-specific B and T cells

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    Agnete Brunsvik Fredriksen

    2012-10-01

    Full Text Available Background: Idiotopes (Id are antigenic determinants localized in variable (V regions of Ig. Id-specific T and B cells (antibodies play a role in immunotherapy of Id+ tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id+ single chain Fragment variable (scFv from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen presenting cells (APC. APC-specific targeting units were either scFv from APC-specific mAb (anti-MHCII, anti-CD40 or chemokines (MIP-1α, RANTES. Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i Transfected cells secreted plasmid-encoded Id+ fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii Targeted vaccine molecules increased Id-specific B and T cell responses. (iii Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv Targeted Id DNA vaccines induced tumor resistance against challenges with Id+ tumors. (v Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi Human Id+ scFv from 4 multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii Human MIP-1α vaccine proteins enhanced human T cell responses in vitro (viii A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusions: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

  9. Dissection of T-cell antigen specificity in human melanoma

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

    2012-01-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma......-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes...

  10. Cell-specific monitoring of protein synthesis in vivo.

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    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  11. Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons

    Science.gov (United States)

    Chowdhury, Sayan Mullick; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Yun; Neville, Kayla; Sitharaman, Balaji

    2012-01-01

    types. Additional analysis indicates that this increased uptake is the dominant cause of the significantly higher toxicity exhibited by HeLa cells. The results suggest that water-solubilized O-GNR-PEG-DSPEs have a heterogeneous cell-specific cytotoxicity, and have significantly different cytotoxicity profile compared to graphene nanoparticles prepared by the modified Hummer’s method (graphene nanoparticles prepared by oxidation of graphite, and its mechanical exfoliation) or its variations. PMID:23072942

  12. Epibacterial community patterns on marine macroalgae are host-specific but temporally variable.

    Science.gov (United States)

    Lachnit, Tim; Meske, Diana; Wahl, Martin; Harder, Tilmann; Schmitz, Ruth

    2011-03-01

    Marine macroalgae are constantly exposed to epibacterial colonizers. The epiphytic bacterial patterns and their temporal and spatial variability on host algae are poorly understood. To investigate the interaction between marine macroalgae and epiphytic bacteria, this study tested if the composition of epibacterial communities on different macroalgae was specific and persisted under varying biotic and abiotic environmental conditions over a 2-year observation time frame. Epibacterial communities on the co-occurring macroalgae Fucus vesiculosus, Gracilaria vermiculophylla and Ulva intestinalis were repeatedly sampled in summer and winter of 2007 and 2008. The epibacterial community composition was analysed by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene libraries. Epibacterial community profiles did not only differ significantly at each sampling interval among algal species, but also showed consistent seasonal differences on each algal species at a bacterial phylum level. These compositional patterns re-occurred at the same season of two consecutive years. Within replicates of the same algal species, the composition of bacterial phyla was subject to shifts at the bacterial species level, both within the same season but at different years and between different seasons. However, 7-16% of sequences were identified as species specific to the host alga. These findings demonstrate that marine macroalgae harbour species-specific and temporally adapted epiphytic bacterial biofilms on their surfaces. Since several algal host-specific bacteria were highly similar to other bacteria known to either avoid subsequent colonization by eukaryotic larvae or to exhibit potent antibacterial activities, algal host-specific bacterial associations are expected to play an important role for marine macroalgae. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  13. Cell-specific DNA methylation patterns of retina-specific genes.

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    Shannath L Merbs

    Full Text Available Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO, retinal binding protein 3 (RBP3, IRBP cone opsin, short-wave-sensitive (OPN1SW, cone opsin, middle-wave-sensitive (OPN1MW, and cone opsin, long-wave-sensitive (OPN1LW was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods. These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA

  14. Regulation of cell-to-cell variability in divergent gene expression.

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-24

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically 'leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  15. Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

    Science.gov (United States)

    Chesnokova, Vera; Zonis, Svetlana; Zhou, Cuiqi; Ben-Shlomo, Anat; Wawrowsky, Kolja; Toledano, Yoel; Tong, Yunguang; Kovacs, Kalman; Scheithauer, Bernd; Melmed, Shlomo

    2011-01-01

    Although pituitary adenomas are usually benign, unique trophic mechanisms restraining cell proliferation are unclear. As GH-secreting adenomas are associated with p53/p21-dependent senescence, we tested mechanisms constraining non-functioning pituitary adenoma growth. Thirty six gonadotroph-derived non-functioning pituitary adenomas all exhibited DNA damage, but undetectable p21 expression. However, these adenomas all expressed p16, and >90% abundantly expressed cytoplasmic clusterin associated with induction of the Cdk inhibitor p15 in 70% of gonadotroph and in 26% of somatotroph lineage adenomas (p = 0.006). Murine LβT2 and αT3 gonadotroph pituitary cells, and αGSU.PTTG transgenic mice with targeted gonadotroph cell adenomas also abundantly expressed clusterin and exhibited features of oncogene-induced senescence as evidenced by C/EBPβ and C/EBPδ induction. In turn, C/EBPs activated the clusterin promoter ∼5 fold, and elevated clusterin subsequently elicited p15 and p16 expression, acting to arrest murine gonadotroph cell proliferation. In contrast, specific clusterin suppression by RNAis enhanced gonadotroph proliferation. FOXL2, a tissue-specific gonadotroph lineage factor, also induced the clusterin promoter ∼3 fold in αT3 pituitary cells. As nine of 12 pituitary carcinomas were devoid of clusterin expression, this protein may limit proliferation of benign adenomatous pituitary cells. These results point to lineage-specific pathways restricting uncontrolled murine and human pituitary gonadotroph adenoma cell growth. PMID:21464964

  16. Specific Movement of Cell Membranes Fused with HVJ (Sendai Virus)

    Science.gov (United States)

    Okada, Yoshio; Kim, Jeman; Maeda, Yumiko; Koseki, Ignez

    1974-01-01

    When Ehrlich ascites tumor cells fused with UV-inactivated HVJ (Sendai virus) were cultured, the viral envelope antigens, which had been integrated at random into the cell membranes, accumulated at one site on the fused cell. The site showed a specific structure, namely, a projection of the cytoplasm from the spherical fused cell surface with clusters of many long microvillilike protrusions from it. When the fused cells were conjugated with antibody against Ehrlich ascites tumor cells and cultured, the antigen-antibody complex also accumulated on the projection. Associated with the development of the projection and accumulation of viral antigens, vacuoles developed at the base of the projection in the cytoplasm of the fused cells in culture. The development of the projection and vacuoles and the accumulation of viral antigens were all inhibited by glucose, mannose, galactose, sodium azide, and cycloheximide, and these inhibitions were reversible. All these structures were observed in multinucleated cells, but few in mononucleated cells after reaction with HVJ (Sendai virus). We consider that these three phenomena correlate with each other and may result from changes in cell-membrane systems caused by fusion of cells. Images PMID:4365583

  17. Cell-type specific four-component hydrogel.

    Directory of Open Access Journals (Sweden)

    Timo Aberle

    Full Text Available In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin to generate a blend (technical term: quattroGel, an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

  18. Specification of Epidermal Cell Fate in Plant Shoots

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    Shinobu eTakada

    2014-02-01

    Full Text Available Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots.

  19. Type-specific cell line models for type-specific ovarian cancer research.

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    Michael S Anglesio

    Full Text Available BACKGROUND: OVARIAN CARCINOMAS CONSIST OF AT LEAST FIVE DISTINCT DISEASES: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies. METHODS: We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 "ovarian cancer" cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis. RESULTS: Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements. CONCLUSIONS: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic "ovarian carcinoma" cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of

  20. Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells.

    Science.gov (United States)

    Maul, Robert W; Cao, Zheng; Venkataraman, Lakshmi; Giorgetti, Carol A; Press, Joan L; Denizot, Yves; Du, Hansen; Sen, Ranjan; Gearhart, Patricia J

    2014-10-20

    Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

  1. Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo–activated cells

    Science.gov (United States)

    Maul, Robert W.; Cao, Zheng; Venkataraman, Lakshmi; Giorgetti, Carol A.; Press, Joan L.; Denizot, Yves; Du, Hansen; Sen, Ranjan

    2014-01-01

    Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo–activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment. PMID:25288395

  2. High specific energy, high capacity nickel-hydrogen cell design

    Science.gov (United States)

    Wheeler, James R.

    1993-01-01

    A 3.5 inch rabbit-ear-terminal nickel-hydrogen cell was designed and tested to deliver high capacity at steady discharge rates up to and including a C rate. Its specific energy yield of 60.6 wh/kg is believed to be the highest yet achieved in a slurry-process nickel-hydrogen cell, and its 10 C capacity of 113.9 AH the highest capacity yet of any type in a 3.5 inch diameter size. The cell also demonstrated a pulse capability of 180 amps for 20 seconds. Specific cell parameters and performance are described. Also covered is an episode of capacity fading due to electrode swelling and its successful recovery by means of additional activation procedures.

  3. Fungus-Specific CD4 T Cells as Specific Sensors for Identification of Pulmonary Fungal Infections.

    Science.gov (United States)

    Scheffold, Alexander; Schwarz, Carsten; Bacher, Petra

    2018-02-01

    Patients with cystic fibrosis (CF) suffer from chronic lung infections, caused by bacterial, viral or fungal pathogens, which determine morbidity and mortality. The contribution of individual pathogens to chronic disease and acute lung exacerbations is often difficult to determine due to the complex composition of the lung microbiome in CF. In particular, the relevance of fungal pathogens in CF airways remains poorly understood due to limitations of current diagnostics to identify the presence of fungal pathogens and to resolve the individual host-pathogen interaction status. T-lymphocytes play an essential role in host defense against pathogens, but also in inappropriate immune reactions such as allergies. They have the capacity to specifically recognize and discriminate the different pathogens and orchestrate a diverse array of effector functions. Thus, the analysis of the fungus-specific T cell status of an individual can in principle provide detailed information about the identity of the fungal pathogen(s) encountered and the actual fungus-host interaction status. This may allow to classify patients, according to appropriate (protective) or inappropriate (pathology-associated) immune reactions against individual fungal pathogens. However, T cell-based diagnostics are currently not part of the clinical routine. The identification and characterization of fungus-specific T cells in health and disease for diagnostic purposes are associated with significant challenges. Recent technological developments in the field of fungus-specific T helper cell detection provide new insights in the host T cell-fungus interaction. In this review, we will discuss basic principles and the potential of T cell-based diagnostics, as well as the perspectives and further needs for use of T cells for improved clinical diagnostics of fungal diseases.

  4. Site-Specific Variability in the Chemical Diversity of the Antarctic Red Alga Plocamium cartilagineum

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    Ryan M. Young

    2013-06-01

    Full Text Available Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like “P. cartilagineum” from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km2 area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

  5. Genomic imprinting is variably lost during reprogramming of mouse iPS cells.

    Science.gov (United States)

    Takikawa, Sachiko; Ray, Chelsea; Wang, Xin; Shamis, Yulia; Wu, Tien-Yuan; Li, Xiajun

    2013-09-01

    Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals. Copyright © 2013. Published by Elsevier B.V.

  6. Progress towards human primordial germ cell specification in vitro.

    Science.gov (United States)

    Canovas, S; Campos, R; Aguilar, E; Cibelli, J B

    2017-01-01

    Primordial germ cells (PGCs) have long been considered the link between one generation and the next. PGC specification begins in the early embryo as a result of a highly orchestrated combination of transcriptional and epigenetic mechanisms. Understanding the molecular events that lead to proper PGC development will facilitate the development of new treatments for human infertility as well as species conservation. This article describes the latest, most relevant findings about the mechanisms of PGC formation, emphasizing human PGC. It also discusses our own laboratory's progress in using transdifferentiation protocols to derive human PGCs (hPGCs). Our preliminary results arose from our pursuit of a sequential hPGC induction strategy that starts with the repression of lineage-specific factors in the somatic cell, followed by the reactivation of germ cell-related genes using specific master regulators, which can indeed reactivate germ cell-specific genes in somatic cells. While it is still premature to assume that fully functional human gametes can be obtained in a dish, our results, together with those recently published by others, provide strong evidence that generating their precursors, PGCs, is within reach. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Engineering Specificity and Function of Therapeutic Regulatory T Cells

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    Jenny L. McGovern

    2017-11-01

    Full Text Available Adoptive therapy with polyclonal regulatory T cells (Tregs has shown efficacy in suppressing detrimental immune responses in experimental models of autoimmunity and transplantation. The lack of specificity is a potential limitation of Treg therapy, as studies in mice have demonstrated that specificity can enhance the therapeutic potency of Treg. We will discuss that vectors encoding T cell receptors or chimeric antigen receptors provide an efficient gene-transfer platform to reliably produce Tregs of defined antigen specificity, thus overcoming the considerable difficulties of isolating low-frequency, antigen-specific cells that may be present in the natural Treg repertoire. The recent observations that Tregs can polarize into distinct lineages similar to the Th1, Th2, and Th17 subsets described for conventional T helper cells raise the possibility that Th1-, Th2-, and Th17-driven pathology may require matching Treg subsets for optimal therapeutic efficacy. In the future, genetic engineering may serve not only to enforce FoxP3 expression and a stable Treg phenotype but it may also enable the expression of particular transcription factors that drive differentiation into defined Treg subsets. Together, established and recently developed gene transfer and editing tools provide exciting opportunities to produce tailor-made antigen-specific Treg products with defined functional activities.

  8. Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

    Science.gov (United States)

    Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

    2011-06-01

    Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

  9. Automated size-specific CT dose monitoring program: assessing variability in CT dose.

    Science.gov (United States)

    Christianson, Olav; Li, Xiang; Frush, Donald; Samei, Ehsan

    2012-11-01

    The potential health risks associated with low levels of ionizing radiation have created a movement in the radiology community to optimize computed tomography (CT) imaging protocols to use the lowest radiation dose possible without compromising the diagnostic usefulness of the images. Despite efforts to use appropriate and consistent radiation doses, studies suggest that a great deal of variability in radiation dose exists both within and between institutions for CT imaging. In this context, the authors have developed an automated size-specific radiation dose monitoring program for CT and used this program to assess variability in size-adjusted effective dose from CT imaging. The authors radiation dose monitoring program operates on an independent health insurance portability and accountability act compliant dosimetry server. Digital imaging and communication in medicine routing software is used to isolate dose report screen captures and scout images for all incoming CT studies. Effective dose conversion factors (k-factors) are determined based on the protocol and optical character recognition is used to extract the CT dose index and dose-length product. The patient's thickness is obtained by applying an adaptive thresholding algorithm to the scout images and is used to calculate the size-adjusted effective dose (ED(adj)). The radiation dose monitoring program was used to collect data on 6351 CT studies from three scanner models (GE Lightspeed Pro 16, GE Lightspeed VCT, and GE Definition CT750 HD) and two institutions over a one-month period and to analyze the variability in ED(adj) between scanner models and across institutions. No significant difference was found between computer measurements of patient thickness and observer measurements (p = 0.17), and the average difference between the two methods was less than 4%. Applying the size correction resulted in ED(adj) that differed by up to 44% from effective dose estimates that were not adjusted by patient size

  10. Effect of concurrent training on gender-specific biochemical variables and adiposity in obese adolescents.

    Science.gov (United States)

    Antunes, Barbara de Moura Mello; Christofaro, Diego Giuliano Destro; Monteiro, Paula Alves; Silveira, Loreana Sanches; Fernandes, Rômulo Araújo; Mota, Jorge; Freitas Júnior, Ismael Forte

    2015-08-01

    The purpose of the present study was to analyze the effects of a 20-week concurrent training (20 WCT) intervention program on gender-specific body composition and metabolic variables in obese adolescents. Sample was composed of twenty-five obese adolescents, aged between 12 and 15 (13.4 ± 0.96) years. Fat-free mass (FFM), percentage trunk fat mass (TFM%) and percentage fat mass (%FM) were evaluated through dual-energy X-ray absorptiometry (DXA). Measurement of intra-abdominal adiposity (IAAT) was performed using ultrasound. Blood pressure was measured and blood samples analyzed for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), triglycerides (TG) and plasma glucose. All participants performed the concurrent training (combination of weight training and aerobic training) three times per week, one hour per day, for 20 weeks. Descriptive analysis and analysis of variance (ANOVA) for repeated measures were used to compare baseline, 10 week and 20 week moments using the Bonferroni post-hoc test. Statistical significance was set at p concurrent training. A significant increase in height was found in both the male and female groups (p = 0.001 and p = 0.047, respectively), after 20 weeks of concurrent training. In addition, several modifications were observed in body composition and metabolic variables, with a significant decrease in BMI (p = 0.002 and p = 0.017), BMI z-score (p = 0.033 and p = 0.004), FM% (p = 0.002 and p = 0.002), TFM% (p = 0.009 and p = 0.018), TC (p = 0.042 and p = 0.001) and LDL-c (p = 0.006 and p = 0.001) in the male and female groups, respectively, after 20 weeks of intervention when compared with baseline. Our results identified that concurrent training was an effective intervention for treating metabolic variable and body composition disorders, in both genders, by decreasing adiposity with consequent improvement in BMI and BMI z-scores, and enhancement in lipid profile variables.

  11. Micro-magnet arrays for specific single bacterial cell positioning

    Science.gov (United States)

    Pivetal, Jérémy; Royet, David; Ciuta, Georgeta; Frenea-Robin, Marie; Haddour, Naoufel; Dempsey, Nora M.; Dumas-Bouchiat, Frédéric; Simonet, Pascal

    2015-04-01

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications.

  12. Optimization Manufacture of Virus- and Tumor-Specific T Cells

    Science.gov (United States)

    Lapteva, Natalia; Vera, Juan F.

    2011-01-01

    Although ex vivo expanded T cells are currently widely used in pre-clinical and clinical trials, the complexity of manufacture remains a major impediment for broader application. In this review we discuss current protocols for the ex vivo expansion of virus- and tumor-specific T cells and describe our experience in manufacture optimization using a gas-permeable static culture flask (G-Rex). This innovative device has revolutionized the manufacture process by allowing us to increase cell yields while decreasing the frequency of cell manipulation and in vitro culture time. It is now being used in good manufacturing practice (GMP) facilities for clinical cell production in our institution as well as many others in the US and worldwide. PMID:21915183

  13. Optimization Manufacture of Virus- and Tumor-Specific T Cells

    Directory of Open Access Journals (Sweden)

    Natalia Lapteva

    2011-01-01

    Full Text Available Although ex vivo expanded T cells are currently widely used in pre-clinical and clinical trials, the complexity of manufacture remains a major impediment for broader application. In this review we discuss current protocols for the ex vivo expansion of virus- and tumor-specific T cells and describe our experience in manufacture optimization using a gas-permeable static culture flask (G-Rex. This innovative device has revolutionized the manufacture process by allowing us to increase cell yields while decreasing the frequency of cell manipulation and in vitro culture time. It is now being used in good manufacturing practice (GMP facilities for clinical cell production in our institution as well as many others in the US and worldwide.

  14. Sequence-specific and domain-specific DNA repair in xeroderma pigmentosum and Cockayne syndrome cells.

    Science.gov (United States)

    Tu, Y; Bates, S; Pfeifer, G P

    1997-08-15

    Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells have specific DNA repair defects. We had previously analyzed repair rates of cyclobutane pyrimidine dimers at nucleotide resolution along the human JUN gene in normal fibroblasts and found very efficient repair of sequences near the transcription initiation site but slow repair along the promoter. To investigate sequence-specific repair rate patterns in XP and CS cells, we conducted a similar analysis in XPA, XPB, XPC, XPD, and CSB fibroblasts. XPA cells were almost completely repair-deficient at all sequences analyzed. XPC cells repaired only the transcribed DNA strand beginning at position -20 relative to the transcription start site. Both XBP and XPD cells were deficient in repair of nontranscribed DNA and also very inefficiently repaired the transcribed strand including sequences near the transcription start site. CSB cells exhibited rapid repair near the transcription initiation site but were deficient in repair of sequences encountered by RNA polymerase during elongation (beginning at position +20). Since transcription of the JUN gene was UV-induced in all fibroblast strains, including CSB, the defective repair of the transcribed strand in CSB cannot be explained by a lack of transcription; rather, it appears to be a true DNA repair defect.

  15. Association of Hematological Variables with Team-Sport Specific Fitness Performance.

    Science.gov (United States)

    Brocherie, Franck; Millet, Grégoire P; Hauser, Anna; Steiner, Thomas; Wehrlin, Jon P; Rysman, Julien; Girard, Olivier

    2015-01-01

    We investigated association of hematological variables with specific fitness performance in elite team-sport players. Hemoglobin mass (Hbmass) was measured in 25 elite field hockey players using the optimized (2 min) CO-rebreathing method. Hemoglobin concentration ([Hb]), hematocrit and mean corpuscular hemoglobin concentration (MCHC) were analyzed in venous blood. Fitness performance evaluation included a repeated-sprint ability (RSA) test (8 x 20 m sprints, 20 s of rest) and the Yo-Yo intermittent recovery level 2 (YYIR2). Hbmass was largely correlated (r = 0.62, PRSA-derived parameters (r ranging from -0.06 to -0.32; all P>0.05). [Hb] and MCHC displayed moderate correlations with both YYIR2TD (r = 0.44 and 0.41; both PRSA sprint decrement score (r = -0.41 and -0.44; both PRSA best and total sprint times (r = -0.46, PRSA sprint decrement score (r = -0.19, P>0.05). Hbmass is positively correlated with specific aerobic fitness, but not with RSA, in elite team-sport players. Additionally, the negative relationships between YYIR2 and RSA tests performance imply that different hematological mechanisms may be at play. Overall, these results indicate that these two fitness tests should not be used interchangeably as they reflect different hematological mechanisms.

  16. Association of Hematological Variables with Team-Sport Specific Fitness Performance.

    Directory of Open Access Journals (Sweden)

    Franck Brocherie

    Full Text Available We investigated association of hematological variables with specific fitness performance in elite team-sport players.Hemoglobin mass (Hbmass was measured in 25 elite field hockey players using the optimized (2 min CO-rebreathing method. Hemoglobin concentration ([Hb], hematocrit and mean corpuscular hemoglobin concentration (MCHC were analyzed in venous blood. Fitness performance evaluation included a repeated-sprint ability (RSA test (8 x 20 m sprints, 20 s of rest and the Yo-Yo intermittent recovery level 2 (YYIR2.Hbmass was largely correlated (r = 0.62, P0.05. [Hb] and MCHC displayed moderate correlations with both YYIR2TD (r = 0.44 and 0.41; both P0.05.Hbmass is positively correlated with specific aerobic fitness, but not with RSA, in elite team-sport players. Additionally, the negative relationships between YYIR2 and RSA tests performance imply that different hematological mechanisms may be at play. Overall, these results indicate that these two fitness tests should not be used interchangeably as they reflect different hematological mechanisms.

  17. Specific uptake of serotonin by murine lymphoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, J.C.; Walker, R.F.; Brooks, W.H.; Roszman, T.L.

    1986-03-01

    Recently the authors confirmed and extended earlier observations that serotonin (5-hydroxytryptamine, 5HT) can influence immune function. Both 5HT and its precursor, 5-hydroxytryptophan inhibit the primary, in vivo antibody response to sheep red blood cells, in mice. Here, the authors report specific in vitro association of this amine with mouse splenocytes. Spleen cells from 6-8 week old CBA/J mice incorporated /sup 3/H-5HT(10/sup -8/ to 2.5 x 10/sup -6/M) in a saturable manner, at 37/sup 0/C. Specificity of uptake was indicated by competition with excess (10/sup -5/M) unlabelled 5HT and with 10/sup -5/M fluoxetine, a selective inhibitor of active 5HT reuptake in rat brain. The 5HT receptor antagonists, methysergide and cyproheptadine, also blocked 5HT uptake. Cell lysis and displacement studies revealed largely intracellular accumulation of /sup 3/H-5HT with little membrane association, in splenocytes. Hofstee analysis of uptake kinetics yielded an apparent Km of 0.82 +/- 0.22 x 10/sup -7/M and Vmax of 501 +/- 108 pM/3 x 10/sup 6/ cells/10 min. Spleen cells fractionated on Sephadex G10 showed virtually no specific 5HT uptake while peritoneal exudate cells from thioglycollate treated mice displayed 5HT uptake kinetics similar to those of splenocytes. The site of specific /sup 3/H-5HT incorporation within a population of spleen cells and the functional significance of this phenomenon to immunomodulation by 5HT remain to be elucidated.

  18. Specification of primordial germ cells in medaka (Oryzias latipes

    Directory of Open Access Journals (Sweden)

    Raz Erez

    2007-01-01

    Full Text Available Abstract Background Primordial germ cells (PGCs give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression – the vasa homologue in another teleost, medaka (Oryzias latipes. Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species. Results In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm. Conclusion Taken together, these results are consistent with the idea that in medaka the inheritance of

  19. Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation in Vivo

    Science.gov (United States)

    2009-11-01

    as N-CoR and SMRT to the AR transcription complex. Alternatively, ART-27 or its binding partner, URI may interact with components of the chromatin...Prostate Cancer Cells Is Independent of NCoR and SMRT Corepressors. Cancer Res 2007;67: 8388–95. 36. Klokk TI, Kurys P, Elbi C, et al. Ligand-specific

  20. Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

    Directory of Open Access Journals (Sweden)

    Naira F. Z. Schneider

    2018-03-01

    Full Text Available Cardiac glycosides (CGs are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

  1. Specific organization of Golgi apparatus in plant cells.

    Science.gov (United States)

    Vildanova, M S; Wang, W; Smirnova, E A

    2014-09-01

    Microtubules, actin filaments, and Golgi apparatus are connected both directly and indirectly, but it is manifested differently depending on the cell organization and specialization, and these connections are considered in many original studies and reviews. In this review we would like to discuss what underlies differences in the structural organization of the Golgi apparatus in animal and plant cells: specific features of the microtubule cytoskeleton organization, the use of different cytoskeleton components for Golgi apparatus movement and maintenance of its integrity, or specific features of synthetic and secretory processes. We suppose that a dispersed state of the Golgi apparatus in higher plant cells cannot be explained only by specific features of the microtubule system organization and by the absence of centrosome as an active center of their organization because the Golgi apparatus is organized similarly in the cells of other organisms that possess the centrosome and centrosomal microtubules. One of the key factors determining the Golgi apparatus state in plant cells is the functional uniformity or functional specialization of stacks. The functional specialization does not suggest the joining of the stacks to form a ribbon; therefore, the disperse state of the Golgi apparatus needs to be supported, but it also can exist "by default". We believe that the dispersed state of the Golgi apparatus in plants is supported, on one hand, by dynamic connections of the Golgi apparatus stacks with the actin filament system and, on the other hand, with the endoplasmic reticulum exit sites distributed throughout the endoplasmic reticulum.

  2. Red blood cell components: time to revisit the sources of variability.

    Science.gov (United States)

    Sparrow, Rosemary L

    2017-03-01

    Quality and safety of red blood cell (RBC) components is managed by screening of donors and strict regulatory controls of blood collection, processing and storage procedures. Despite these efforts, variations in RBC component quality exist as exemplified by the wide range in storage-induced haemolysis. This article provides a brief overview of the variables that contribute or potentially contribute to the quality of stored RBC components, including blood collection, processing, and donor-related variables. Particular focus is made on donor health and lifestyle factors that are not specifically screened and may impact on the physicobiochemical properties of RBCs and their storability. Inflammatory and oxidative stress states may be especially relevant as RBCs are susceptible to oxidative injury. Few studies have investigated the effect of specific donor-related variables on the quality of stored RBC components. Donor-related variables may be unaccounted confounders in the "age of blood" clinical studies that compared outcomes following transfusion of fresher or longer-stored RBC components. The conclusion is drawn that the blood donor is the greatest source of RBC component variability and the least "regulated" aspect of blood component production. It is proposed that more research is needed to better understand the connection between donor-related variables and quality consistency of stored RBC components. This could be very important given the impact of modern lifestyles that sees escalating rates of non-communicable health conditions that are associated with increased oxidative stress, such as hypertension, obesity and diabetes in children and adults, as well as an ageing population in many countries. The effect of these changes to global health and population demographics will impact on blood donor panels, and without significant new research, the consequences on the quality of stored blood components and transfusion outcomes are unknown.

  3. High efficiency cell-specific targeting of cytokine activity

    Science.gov (United States)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  4. Specification of tapetum and microsporocyte cells within the anther.

    Science.gov (United States)

    Zhang, Dabing; Yang, Li

    2014-02-01

    Flowering plants form male reproductive cells (microsporocytes) during sporophytic generation, which subsequently differentiate into multicellular male gametes in the gametophytic generation. The tapetum is a somatic helper tissue neighboring microsporocytes and supporting gametogenesis. The mechanism controlling the specification of the tapetum and microsporocyte cell fate within the anther has long been a mystery in biology. Recent investigations have revealed molecular switches and signaling pathways underlying the establishment of somatic and reproductive cells in plants. In this review we discuss common and diversified signaling molecules and regulatory pathways including receptor-like protein kinases, redox status, glycoprotein, transcription factors, hormones and microRNA implicated in the specification of tapetum and microsporocytes in plants. Copyright © 2013. Published by Elsevier Ltd.

  5. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  6. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  7. Circadian control of antigen-specific T cell responses

    Directory of Open Access Journals (Sweden)

    Nobis CC

    2016-09-01

    Full Text Available Chloé C Nobis,1–3 Nathalie Labrecque,2–4 Nicolas Cermakian1,5–8 1Douglas Mental Health University Institute, 2Maisonneuve-Rosemont Hospital Research Centre, 3Department of Microbiology, Infectious Diseases and Immunology, 4Department of Medicine, University of Montreal, 5Department of Psychiatry, 6Department of Microbiology and Immunology, 7Department of Neurology and Neurosurgery, 8Department of Physiology, McGill University, Montreal, QC, Canada Abstract: The immune system is composed of two arms, the innate and the adaptive immunity. While the innate response constitutes the first line of defense and is not specific for a particular pathogen, the adaptive response is highly specific and allows for long-term memory of the pathogen encounter. T lymphocytes (or T cells are central players in the adaptive immune response. Various aspects of T cell functions vary according to the time of day. Circadian clocks located in most tissues and cell types generate 24-hour rhythms of various physiological processes. These clocks are based on a set of clock genes, and this timing mechanism controls rhythmically the expression of numerous other genes. Clock genes are expressed in cells of the immune system, including T cells. In this review, we provide an overview of the circadian control of the adaptive immune response, with emphasis on T cells, including their development, trafficking, response to antigen, and effector functions. Keywords: circadian clock, adaptive immune response, T lymphocyte, antigen, cytokine, proliferation

  8. Logical modeling of lymphoid and myeloid cell specification and transdifferentiation.

    Science.gov (United States)

    Collombet, Samuel; van Oevelen, Chris; Sardina Ortega, Jose Luis; Abou-Jaoudé, Wassim; Di Stefano, Bruno; Thomas-Chollier, Morgane; Graf, Thomas; Thieffry, Denis

    2017-06-06

    Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.

  9. Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio).

    Science.gov (United States)

    Yamamoto, K; Mukamoto, M; Watarai, S; Kodama, H; Nakayasu, C; Okamoto, N

    2001-04-01

    To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. 22 carp. Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.

  10. Purging Exhausted Virus-Specific CD8 T Cell Phenotypes by Somatic Cell Reprogramming.

    Science.gov (United States)

    Chan, Joshua; Kim, Patrick Y; Kranz, Emiko; Nagaoka, Yoshiko; Lee, YooJin; Wen, Jing; Elsaesser, Heidi J; Qin, Meng; Brooks, David G; Ringpis, Gene-Errol; Chen, Irvin S Y; Kamata, Masakazu

    2017-11-01

    Cytotoxic T cells are critical in controlling virus infections. However, continuous antigen stimulation and negative regulatory factors cause CD8 T cells to enter a dysfunctional state (T cell exhaustion), resulting in viral persistence. We hypothesized that the exhausted T cell state could be molecularly rejuvenated using a somatic cell reprogramming technology, which is technically able to convert any types of cells to induced pluripotent stem cells (iPSCs), to regenerate functional T cells capable of purging chronic infection. We generated a new mouse line (B6/129OKSM) in which every somatic cell contains four doxycycline-inducible reprogramming genes (Oct4, Klf4, Sox2, and c-Myc: OKSM), and infected them with lymphocytic choriomeningitis virus (LCMV) clone 13 to establish chronic infection. Exhausted LCMV-specific T cells isolated by flow sorting were successfully reprogrammed ex vivo into iPSCs in the presence of doxycycline. Upon injection into blastocysts and subsequent transfer into foster females, the reprogrammed cells differentiated into functional naive T cells that maintained their original antigen specificity. These results provide proof of concept that somatic cell reprogramming of exhausted T cells into iPSCs can erase imprints of their previous exhausted state and in turn regenerate functional virus-specific T cells.

  11. Specification of regional intestinal stem cell identity during Drosophila metamorphosis.

    Science.gov (United States)

    Driver, Ian; Ohlstein, Benjamin

    2014-05-01

    In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. Here, we show that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are only capable of transforming ISC and enterocyte identity during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Our data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance.

  12. [Establishment of hemophilia A patient-specific inducible pluripotent stem cells with urine cells].

    Science.gov (United States)

    Hu, Zhiqing; Hu, Xuyun; Pang, Jialun; Wang, Xiaolin; Lin Peng, Siyuan; Li, Zhuo; Wu, Yong; Wu, Lingqian; Liang, Desheng

    2015-10-01

    OBJECTIVE To generate hemophilia A (HA) patient-specific inducible pluripotent stem cells (iPSCs) and induce endothelial differentiation. METHODS Tubular epithelial cells were isolated and cultured from the urine of HA patients. The iPSCs were generated by forced expression of Yamanaka factors (Oct4, Sox2, c-Myc and Klf4) using retroviruses and characterized by cell morphology, pluripotent marker staining and in vivo differentiation through teratoma formation. Induced endothelial differentiation of the iPSCs was achieved with the OP9 cell co-culture method. RESULTS Patient-specific iPSCs were generated from urine cells of the HA patients, which could be identified by cell morphology, pluripotent stem cell surface marker staining and in vivo differentiation of three germ layers. The teratoma experiment has confirmed that such cells could differentiate into endothelial cells expressing the endothelial-specific markers CD144, CD31 and vWF. CONCLUSION HA patient-specific iPSCs could be generated from urine cells and can differentiate into endothelial cells. This has provided a new HA disease modeling approach and may serve as an applicable autologous cell source for gene correction and cell therapy studies for HA.

  13. On the Interpretation of Instrumental Variables in the Presence of Specification Errors

    Directory of Open Access Journals (Sweden)

    P.A.V.B. Swamy

    2015-01-01

    Full Text Available The method of instrumental variables (IV and the generalized method of moments (GMM, and their applications to the estimation of errors-in-variables and simultaneous equations models in econometrics, require data on a sufficient number of instrumental variables that are both exogenous and relevant. We argue that, in general, such instruments (weak or strong cannot exist.

  14. Sequence variability, recombination analysis, and specific detection of the W strain of Plum pox virus.

    Science.gov (United States)

    Glasa, Miroslav; Malinowski, Tadeusz; Predajňa, Lukáš; Pupola, Neda; Dekena, Dzintra; Michalczuk, Lech; Candresse, Thierry

    2011-08-01

    Plum pox virus (PPV), a member of the genus Potyvirus, is the causal agent of Sharka, the most detrimental disease of stone-fruit trees worldwide. PPV isolates are grouped into seven distinct strains. The minor PPV-W strain was established recently for the divergent W3174 isolate found in Canada. Here, the partial or complete genomic sequences of four PPV-W isolates from Latvia have been determined. The completely sequenced isolates LV-141pl and LV-145bt share 93.1 and 92.1% nucleotide identity, respectively, with isolate W3174, with two regions of higher (>20%) divergence in the P1/HC-Pro and NIa (VPg) regions. Further analyses demonstrated that these two regions correspond to two independent recombination events in the W3174 genome, one involving PPV-M (approximate genome positions 692 to 1424) and the other PPV-D (nucleotides 5672 to 5789). The LV-141pl and LV-145bt isolates appear to be representatives of the "ancestral" PPV-W strain, not affected by recombination. The PPV-W intrastrain variability is substantially higher than that of all other PPV strains, with potential implications for the serological detection of PPV-W isolates. A PPV-W-specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of all five presently available W isolates. The characterization of these new PPV-W isolates sheds light on PPV-W evolutionary history, further supports the hypothesis of its East-European origin, and opens the way for the biological and epidemiological characterization of this poorly known PPV strain.

  15. Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

    Directory of Open Access Journals (Sweden)

    Joana Gomes

    2015-08-01

    Full Text Available Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs and total cell membranes (MBs from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101, CD63, CD9, annexin-I, and MBs contained markers of cellular membrane compartments, including calnexin, GRASP65, GS28, LAMP-1, and L1CAM. The glycoprotein galectin-3 binding protein (LGALS3BP was strongly enriched in EVs and it contained sialylated complex N-glycans. Lectin blotting with a panel of lectins showed that EVs had specific glycosignatures relative to MBs. Furthermore, the presence of glycoproteins bearing complex N-glycans with α2,3-linked sialic acid, fucose, bisecting-GlcNAc and LacdiNAc structures, and O-glycans with the T-antigen were detected. The inhibition of N-glycosylation processing from high mannose to complex glycans using kifunensine caused changes in the composition of EVs and induced a decrease of several glycoproteins. In conclusion, the results showed that glycosignatures of EVs were specific and altered glycosylation within the cell affected the composition and/or dynamics of EVs release. Furthermore, the identified glycosignatures of EVs could provide novel biomarkers for ovarian cancer.

  16. Distinct interactions select and maintain a specific cell fate.

    Science.gov (United States)

    Doncic, Andreas; Falleur-Fettig, Melody; Skotheim, Jan M

    2011-08-19

    The ability to specify and maintain discrete cell fates is essential for development. However, the dynamics underlying selection and stability of distinct cell types remain poorly understood. Here, we provide a quantitative single-cell analysis of commitment dynamics during the mating-mitosis switch in budding yeast. Commitment to division corresponds precisely to activating the G1 cyclin positive feedback loop in competition with the cyclin inhibitor Far1. Cyclin-dependent phosphorylation and inhibition of the mating pathway scaffold Ste5 are required to ensure exclusive expression of the mitotic transcriptional program after cell cycle commitment. Failure to commit exclusively results in coexpression of both cell cycle and pheromone-induced genes, and a morphologically mixed inviable cell fate. Thus, specification and maintenance of a cellular state are performed by distinct interactions, which are likely a consequence of disparate reaction rates and may be a general feature of the interlinked regulatory networks responsible for selecting cell fates. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Tissue-specific effector functions of innate lymphoid cells

    Science.gov (United States)

    Björkström, Niklas K; Kekäläinen, Eliisa; Mjösberg, Jenny

    2013-01-01

    Innate lymphoid cells (ILCs) is the collective term for a group of related innate lymphocytes, including natural killer (NK) cells and the more recently discovered non-NK ILCs, which all lack rearranged antigen receptors such as those expressed by T and B cells. Similar to NK cells, the newly discovered ILCs depend on the transcription factor Id2 and the common γ-chain of the interleukin-2 receptor for development. However, in contrast to NK cells, non-NK ILCs also require interleukin-7. In addition to the cytotoxic functions of NK cells, assuring protection against tumour development and viruses, new data indicate that ILCs contribute to a wide range of homeostatic and pathophysiological conditions in various organs via specialized cytokine production capabilities. Here we summarize current knowledge on ILCs with a particular emphasis on their tissue-specific effector functions, in the gut, liver, lungs and uterus. When possible, we try to highlight the role that these cells play in humans. PMID:23489335

  18. Correlation between maximum isometric strength variables and specific performance of Brazilian military judokas

    Directory of Open Access Journals (Sweden)

    Michel Moraes Gonçalves

    2017-06-01

    Full Text Available It was our objective to correlate specific performance in the Special Judo Fitness Test (SJFT and the maximum isometric handgrip (HGSMax, scapular traction (STSMax and lumbar traction (LTSMax strength tests in military judo athletes. Twenty-two military athletes from the judo team of the Brazilian Navy Almirante Alexandrino Instruction Centre, with average age of 26.14 ± 3.31 years old, and average body mass of 83.23 ± 14.14 kg participated in the study. Electronic dynamometry tests for HGSMax, STSMax and LTSMax were conducted. Then, after approximately 1 hour-interval, the SJFT protocol was applied. All variables were adjusted to the body mass of the athletes. Pearson correlation coefficient for statistical analysis was used. The results showed moderate negative correlation between the SJFT index and STSMax (r= -0.550, p= 0.008, strong negative correlations between the SJFT index and HGSMax (r= -0.706, p< 0.001, SJFT index and LTSMax (r= -0.721; p= 0.001, besides the correlation between the sum of the three maximum isometric strength tests and the SJFT index (r= -0.786, p< 0.001. This study concludes that negative correlations occur between the SJFT index and maximum isometric handgrip, shoulder and lumbar traction strength and the sum of the three maximum isometric strength tests in military judokas.

  19. Cell-specific synaptic plasticity induced by network oscillations.

    Science.gov (United States)

    Zarnadze, Shota; Bäuerle, Peter; Santos-Torres, Julio; Böhm, Claudia; Schmitz, Dietmar; Geiger, Jörg Rp; Dugladze, Tamar; Gloveli, Tengis

    2016-05-24

    Gamma rhythms are known to contribute to the process of memory encoding. However, little is known about the underlying mechanisms at the molecular, cellular and network levels. Using local field potential recording in awake behaving mice and concomitant field potential and whole-cell recordings in slice preparations we found that gamma rhythms lead to activity-dependent modification of hippocampal networks, including alterations in sharp wave-ripple complexes. Network plasticity, expressed as long-lasting increases in sharp wave-associated synaptic currents, exhibits enhanced excitatory synaptic strength in pyramidal cells that is induced postsynaptically and depends on metabotropic glutamate receptor-5 activation. In sharp contrast, alteration of inhibitory synaptic strength is independent of postsynaptic activation and less pronounced. Further, we found a cell type-specific, directionally biased synaptic plasticity of two major types of GABAergic cells, parvalbumin- and cholecystokinin-expressing interneurons. Thus, we propose that gamma frequency oscillations represent a network state that introduces long-lasting synaptic plasticity in a cell-specific manner.

  20. Generation of Transplantable Beta Cells for Patient-Specific Cell Therapy

    Directory of Open Access Journals (Sweden)

    Xiaojie Wang

    2012-01-01

    Full Text Available Islet cell transplantation offers a potential cure for type 1 diabetes, but it is challenged by insufficient donor tissue and side effects of current immunosuppressive drugs. Therefore, alternative sources of insulin-producing cells and isletfriendly immunosuppression are required to increase the efficiency and safety of this procedure. Beta cells can be transdifferentiated from precursors or another heterologous (non-beta-cell source. Recent advances in beta cell regeneration from somatic cells such as fibroblasts could circumvent the usage of immunosuppressive drugs. Therefore, generation of patient-specific beta cells provides the potential of an evolutionary treatment for patients with diabetes.

  1. Missing data and technical variability in single-cell RNA-sequencing experiments.

    Science.gov (United States)

    Hicks, Stephanie C; Townes, F William; Teng, Mingxiang; Irizarry, Rafael A

    2017-11-06

    Until recently, high-throughput gene expression technology, such as RNA-Sequencing (RNA-seq) required hundreds of thousands of cells to produce reliable measurements. Recent technical advances permit genome-wide gene expression measurement at the single-cell level. Single-cell RNA-Seq (scRNA-seq) is the most widely used and numerous publications are based on data produced with this technology. However, RNA-seq and scRNA-seq data are markedly different. In particular, unlike RNA-seq, the majority of reported expression levels in scRNA-seq are zeros, which could be either biologically-driven, genes not expressing RNA at the time of measurement, or technically-driven, genes expressing RNA, but not at a sufficient level to be detected by sequencing technology. Another difference is that the proportion of genes reporting the expression level to be zero varies substantially across single cells compared to RNA-seq samples. However, it remains unclear to what extent this cell-to-cell variation is being driven by technical rather than biological variation. Furthermore, while systematic errors, including batch effects, have been widely reported as a major challenge in high-throughput technologies, these issues have received minimal attention in published studies based on scRNA-seq technology. Here, we use an assessment experiment to examine data from published studies and demonstrate that systematic errors can explain a substantial percentage of observed cell-to-cell expression variability. Specifically, we present evidence that some of these reported zeros are driven by technical variation by demonstrating that scRNA-seq produces more zeros than expected and that this bias is greater for lower expressed genes. In addition, this missing data problem is exacerbated by the fact that this technical variation varies cell-to-cell. Then, we show how this technical cell-to-cell variability can be confused with novel biological results. Finally, we demonstrate and discuss how batch

  2. Sources of variability in cytosolic calcium transients triggered by stimulation of homogeneous uro-epithelial cell monolayers.

    Science.gov (United States)

    Appleby, Peter A; Shabir, Saqib; Southgate, Jennifer; Walker, Dawn

    2015-04-06

    Epithelial tissue structure is the emergent outcome of the interactions between large numbers of individual cells. Experimental cell biology offers an important tool to unravel these complex interactions, but current methods of analysis tend to be limited to mean field approaches or representation by selected subsets of cells. This may result in bias towards cells that respond in a particular way and/or neglect local, context-specific cell responses. Here, an automated algorithm was applied to examine in detail the individual calcium transients evoked in genetically homogeneous, but asynchronous populations of cultured non-immortalized normal human urothelial cells when subjected to either the global application of an external agonist or a localized scratch wound. The recorded calcium transients were classified automatically according to a set of defined metrics and distinct sub-populations of cells that responded in qualitatively different ways were observed. The nature of this variability in the homogeneous cell population was apportioned to two sources: intrinsic variation in individual cell responses and extrinsic variability due to context-specific factors of the environment, such as spatial heterogeneity. Statistically significant variation in the features of the calcium transients evoked by scratch wounding according to proximity to the wound edge was identified. The manifestation of distinct sub-populations of cells is considered central to the coordination of population-level response resulting in wound closure.

  3. Endoglin is a novel endothelial cell specification gene.

    Science.gov (United States)

    Banerjee, Saswati; Dhara, Sujoy K; Bacanamwo, Methode

    2012-01-01

    Endothelial cells (EC) are important in vasculogenesis and organogenesis during development and in the pathogenesis of cancer and cardiovascular diseases. However, few EC specification factors are known and primary EC production remains inefficient. Based on recent studies implicating endoglin (Eng) in early vascular development and angiogenesis, we hypothesized that Eng may be an EC specification gene. Mouse embryonic stem cells (ESC) were treated with recombinant Eng or a plasmid expressing the Eng ORF, and differentiated in the presence or absence of bone morphogenic protein 4 (BMP4). Expression of the mesoderm and EC marker genes, the known mediators of EC specification and their downstream targets was monitored by quantitative PCR, western blot, immunocytochemistry, and flow cytometry. Functionality of the differentiated EC was assessed by in vitro angiogenesis assay and the induction of Icam1 expression in response to TNF-α treatment. Both recombinant Eng and forced Eng expression increased the number of functional EC expressing the EC marker genes VE-cadherin, vWF, and Tie2, and enhanced the effect of BMP4. The Eng-induced EC differentiation was independent of known mediators of EC specification such as Indian Hedgehog (IHH) and BMP4 or of BMP4/Smad1/5/8 signaling. These studies suggest that Eng is a novel EC specification gene. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  5. Increased sequence diversity coverage improves detection of HIV-Specific T cell responses

    DEFF Research Database (Denmark)

    Frahm, N.; Kaufmann, D.E.; Yusim, K.

    2007-01-01

    The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss...... assay, these "toggled" peptides detected HIV-specific CD4(+) and CD8(+) T cell responses of significantly higher breadth and magnitude than matched consensus peptides. The observed increases were explained by a closer match of the toggled peptides to the autologous viral sequence. Toggled peptides...... responses to regions where test set and infecting virus differ. In this study, we report the design of a peptide test set with significantly increased coverage of HIV sequence diversity by including alternative amino acids at variable positions during the peptide synthesis step. In an IFN-gamma ELISpot...

  6. HIV-1 Epitope Variability Is Associated with T Cell Receptor Repertoire Instability and Breadth.

    Science.gov (United States)

    Balamurugan, Arumugam; Claiborne, Deon; Ng, Hwee L; Yang, Otto O

    2017-08-15

    Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of epitope varied considerably in stability over time, although clonal stability (Sorensen index) was not significantly time dependent within this interval. However, TCR stability inversely correlated with epitope variability in the Los Alamos HIV-1 Sequence Database, consistent with TCR evolution being driven by epitope variation. Finally, a robust inverse correlation of TCR breadth against each epitope versus epitope variability further suggested that this variability drives TCR repertoire diversification. In the context of studies demonstrating rapidly shifting HIV-1 sequences in vivo, our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier to effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here, we demonstrate that the clonal stability and number of clones for the CTL response against an epitope are inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV-1 escape may require coordinated direction of the CTL clonal repertoire to

  7. Prediction of persistent post-operative pain: Pain-specific psychological variables compared with acute post-operative pain and general psychological variables.

    Science.gov (United States)

    Horn-Hofmann, C; Scheel, J; Dimova, V; Parthum, A; Carbon, R; Griessinger, N; Sittl, R; Lautenbacher, S

    2018-01-01

    Psychological variables and acute post-operative pain are of proven relevance for the prediction of persistent post-operative pain. We aimed at investigating whether pain-specific psychological variables like pain catastrophizing add to the predictive power of acute pain and more general psychological variables like depression. In all, 104 young male patients undergoing thoracic surgery for pectus excavatum correction were studied on the pre-operative day (T0) and 1 week (T1) and 3 months (T2) after surgery. They provided self-report ratings (pain-related: Pain Catastrophizing Scale, Pain Anxiety Symptoms Scale = PASS, Pain Vigilance and Awareness Questionnaire = PVAQ; general psychological: Screening for Somatoform Symptoms, State-Anxiety Inventory-X1, Center for Epidemiologic Studies Depression Scale = CES-D). Additional predictors (T1) as well as criterion variables (T2) were pain intensity (Numerical Rating Scale) and pain disability (Pain Disability Index). Three months after surgery, 25% of the patients still reported clinically relevant pain (pain intensity ≥3) and over 50% still reported pain-related disability. Acute post-operative pain as well as general psychological variables did not allow for a significant prediction of persistent post-operative pain; in contrast, pain-related psychological variables did. The best single predictors were PASS for pain intensity and PVAQ for pain disability. Pain-related psychological variables derived from the fear-avoidance model contributed significantly to the prediction of persistent post-operative pain. The best possible compilation of these measures requires further research. More general psychological variables may become relevant predictors later in the medical history. Our results suggest that pain-specific psychological variables such as pain anxiety and pain hypervigilance add significantly to the prediction of persistent post-operative pain and might even outperform established predictors such as

  8. Cell number as an important variable in optimising inoculum age ...

    African Journals Online (AJOL)

    The growth lag phase during microbial fermentation reduced productivity and equipment efficiency. Inoculum age and size were the two important variables considered in fermentation optimisation. Previous studies investigated these two variables separately, in contrast to the orthogonal or use of response surface ...

  9. Modelling epigenetic regulation of gene expression in 12 human cell types reveals combinatorial patterns of cell-type-specific genes.

    Science.gov (United States)

    Lu, Yiming; Qu, Wubin; Min, Bo; Liu, Zheyan; Chen, Changsheng; Zhang, Chenggang

    2014-06-01

    The maintenance of the diverse cell types in a multicellular organism is one of the fundamental mysteries of biology. Modelling the dynamic regulatory relationships between the histone modifications and the gene expression across the diverse cell types is essential for the authors to understand the mechanisms of the epigenetic regulation. Here, the authors thoroughly assessed the histone modification enrichment profiles at the promoters and constructed quantitative models between the histone modification abundances and the gene expression in 12 human cell types. The author's results showed that the histone modifications at the promoters exhibited remarkably cell-type-dependent variability in the cell-type-specific (CTS) genes. They demonstrated that the variable profiles of the modifications are highly predictive for the dynamic changes of the gene expression across all the cell types. Their findings revealed the close relationship between the combinatorial patterns of the histone modifications and the CTS gene expression. They anticipate that the findings and the methods they used in this study could provide useful information for the future studies of the regulatory roles of the histone modifications in the CTS genes.

  10. Transcription Factor Antagonism Controls Enteroendocrine Cell Specification from Intestinal Stem Cells.

    Science.gov (United States)

    Li, Yumei; Pang, Zhimin; Huang, Huanwei; Wang, Chenhui; Cai, Tao; Xi, Rongwen

    2017-04-20

    The balanced maintenance and differentiation of local stem cells is required for Homeostatic renewal of tissues. In the Drosophila midgut, the transcription factor Escargot (Esg) maintains undifferentiated states in intestinal stem cells, whereas the transcription factors Scute (Sc) and Prospero (Pros) promote enteroendocrine cell specification. However, the mechanism through which Esg and Sc/Pros coordinately regulate stem cell differentiation is unknown. Here, by combining chromatin immunoprecipitation analysis with genetic studies, we show that both Esg and Sc bind to a common promoter region of pros. Moreover, antagonistic activity between Esg and Sc controls the expression status of Pros in stem cells, thereby, specifying whether stem cells remain undifferentiated or commit to enteroendocrine cell differentiation. Our study therefore reveals transcription factor antagonism between Esg and Sc as a novel mechanism that underlies fate specification from intestinal stem cells in Drosophila.

  11. Statistical Physics of T-Cell Development and Pathogen Specificity

    Science.gov (United States)

    Košmrlj, Andrej; Kardar, Mehran; Chakraborty, Arup K.

    2013-04-01

    In addition to an innate immune system that battles pathogens in a nonspecific fashion, higher organisms, such as humans, possess an adaptive immune system to combat diverse (and evolving) microbial pathogens. Remarkably, the adaptive immune system mounts pathogen-specific responses, which can be recalled upon reinfection with the same pathogen. It is difficult to see how the adaptive immune system can be preprogrammed to respond specifically to a vast and unknown set of pathogens. Although major advances have been made in understanding pertinent molecular and cellular phenomena, the precise principles that govern many aspects of an immune response are largely unknown. We discuss complementary approaches from statistical mechanics and cell biology that can shed light on how key components of the adaptive immune system, T cells, develop to enable pathogen-specific responses against many diverse pathogens. The mechanistic understanding that emerges has implications for how host genetics may influence the development of T cells with differing responses to the human immunodeficiency virus (HIV) infection.

  12. A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups

    Directory of Open Access Journals (Sweden)

    Matthew Emerson Randolph

    2015-10-01

    Full Text Available The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies, such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some muscular dystrophies. The biology of muscle stem cells varies depending on their embryologic origins and the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease.

  13. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

    Science.gov (United States)

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E; Oltz, Eugene M; Jarvis, James N; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F; Wang, Ting

    2016-04-07

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. Copyright © 2016 Gu et al.

  14. Comparing measured and modelled soil carbon: which site-specific variables are linked to high stability?

    Science.gov (United States)

    Robertson, Andy; Schipanski, Meagan; Ma, Liwang; Ahuja, Lajpat; McNamara, Niall; Smith, Pete; Davies, Christian

    2016-04-01

    Changes in soil carbon (C) stocks have been studied in depth over the last two decades, as net greenhouse gas (GHG) sinks are highlighted to be a partial solution to the causes of climate change. However, the stability of this soil C is often overlooked when measuring these changes. Ultimately a net sequestration in soils is far less beneficial if labile C is replacing more stable forms. To date there is no accepted framework for measuring soil C stability, and as a result there is considerable uncertainty associated with the simulated impacts of land management and land use change when using process-based systems models. However, a recent effort to equate measurable soil C fractions to model pools has generated data that help to assess the impacts of land management, and can ultimately help to reduce the uncertainty of model predictions. Our research compiles this existing fractionation data along with site metadata to create a simplistic statistical model able to quantify the relative importance of different site-specific conditions. Data was mined from 23 published studies and combined with original data to generate a dataset of 100+ land use change sites across Europe. For sites to be included they required soil C fractions isolated using the Zimmermann et al. (2007) method and specific site metadata (mean annual precipitation, MAP; mean annual temperature, MAT; soil pH; land use; altitude). Of the sites, 75% were used to develop a generalized linear mixed model (GLMM) to create coefficients where site parameters can be used to predict influence on the measured soil fraction C stocks. The remaining 25% of sites were used to evaluate uncertainty and validate this empirical model. Further, four of the aforementioned sites were used to simulate soil C dynamics using the RothC, DayCent and RZWQM2 models. A sensitivity analysis (4096 model runs for each variable applying Latin hypercube random sampling techniques) was then used to observe whether these models place

  15. Dual specificity phosphatase 15 regulates Erk activation in Schwann cells.

    Science.gov (United States)

    Rodríguez-Molina, José F; Lopez-Anido, Camila; Ma, Ki H; Zhang, Chongyu; Olson, Tyler; Muth, Katharina N; Weider, Matthias; Svaren, John

    2017-02-01

    Schwann cells and oligodendrocytes are the myelinating cells of the peripheral and central nervous system, respectively. Despite having different myelin components and different transcription factors driving their terminal differentiation there are shared molecular mechanisms between the two. Sox10 is one common transcription factor required for several steps in development of myelinating glia. However, other factors are divergent as Schwann cells need the transcription factor early growth response 2/Krox20 and oligodendrocytes require Myrf. Likewise, some signaling pathways, like the Erk1/2 kinases, are necessary in both cell types for proper myelination. Nonetheless, the molecular mechanisms that control this shared signaling pathway in myelinating cells remain only partially characterized. The hypothesis of this study is that signaling pathways that are similarly regulated in both Schwann cells and oligodendrocytes play central roles in coordinating the differentiation of myelinating glia. To address this hypothesis, we have used genome-wide binding data to identify a relatively small set of genes that are similarly regulated by Sox10 in myelinating glia. We chose one such gene encoding Dual specificity phosphatase 15 (Dusp15) for further analysis in Schwann cell signaling. RNA interference and gene deletion by genome editing in cultured RT4 and primary Schwann cells showed Dusp15 is necessary for full activation of Erk1/2 phosphorylation. In addition, we show that Dusp15 represses expression of several myelin genes, including myelin basic protein. The data shown here support a mechanism by which early growth response 2 activates myelin genes, but also induces a negative feedback loop through Dusp15 to limit over-expression of myelin genes. © 2016 International Society for Neurochemistry.

  16. Variable TERRA abundance and stability in cervical cancer cells.

    Science.gov (United States)

    Oh, Bong-Kyeong; Keo, Ponnarath; Bae, Jaeman; Ko, Jung Hwa; Choi, Joong Sub

    2017-06-01

    Telomeres are transcribed into long non-coding RNA, referred to as telomeric repeat-containing RNA (TERRA), which plays important roles in maintaining telomere integrity and heterochromatin formation. TERRA has been well characterized in HeLa cells, a type of cervical cancer cell. However, TERRA abundance and stability have not been examined in other cervical cancer cells, at least to the best of our knowledge. Thus, in this study, we measured TERRA levels and stability, as well as telomere length in 6 cervical cancer cell lines, HeLa, SiHa, CaSki, HeLa S3, C-33A and SNU-17. We also examined the association between the TERRA level and its stability and telomere length. We found that the TERRA level was several fold greater in the SiHa, CaSki, HeLa S3, C-33A and SNU-17 cells, than in the HeLa cells. An RNA stability assay of actinomycin D-treated cells revealed that TERRA had a short half-life of ~4 h in HeLa cells, which was consistent with previous studies, but was more stable with a longer half-life (>8 h) in the other 5 cell lines. Telomere length varied from 4 to 9 kb in the cells and did not correlate significantly with the TERRA level. On the whole, our data indicate that TERRA abundance and stability vary between different types of cervical cancer cells. TERRA degrades rapidly in HeLa cells, but is maintained stably in other cervical cancer cells that accumulate higher levels of TERRA. TERRA abundance is associated with the stability of RNA in cervical cancer cells, but is unlikely associated with telomere length.

  17. Bioaccumulation of photoprotective compounds in copepods: environmental triggers and sources of intra-specific variability

    Science.gov (United States)

    Zagarese, H. E.; García, P.; Diéguez, M. D.; Ferraro, M. A.

    2012-12-01

    Ultraviolet radiation (UVR) and temperature are two globally important abiotic factors affecting freshwater ecosystems. Planktonic organisms have developed a battery of counteracting mechanisms to minimize the risk of being damaged by UVR, which respond to three basic principles: avoid, protect, repair. Copepods are among the most successful zooplankton groups. They are highly adaptable animals, capable of displaying flexible behaviors, physiologies, and life strategies. In particular, they are well equipped to cope with harmful UVR. Their arsenal includes vertical migration, accumulation of photoprotective compounds, and photorepair. The preference for a particular strategy is affected by a plethora of environmental (extrinsic) parameters, such as the existence of a depth refuge, the risk of visual predation, and temperature. Temperature modifies the environment (e.g. the lake thermal structure), and animal metabolism (e.g., swimming speed, bioaccumulation of photoprotective compounds). In addition, the relative weight of UVR-coping strategies is also influenced by the organism (intrinsic) characteristics (e.g., inter- and intra-specific variability). The UV absorbing compounds, mycosporine-like amino acids (MAAs), are widely distributed among freshwater copepods. Animals are unable to synthesize MAAs, and therefore depend on external sources for accumulating these compounds. Although copepods may acquire MAAs from their food, for the few centropagic species investigated so far, the main source of MAAs are microbial (most likely prokaryotic) organisms living in close association with the copepods. Boeckella gracilipes is a common centropagic copepod in Patagonian lakes. We suspected that its occurrence in different types of lakes, hydrologically unconnected, but within close geographical proximity, could have resulted in different microbial-copepod associations (i.e., different MAAs sources) that could translate into intra-specific differences in the accumulation

  18. A cancer cell-specific fluorescent probe for imaging Cu2 + in living cancer cells

    Science.gov (United States)

    Wang, Chao; Dong, Baoli; Kong, Xiuqi; Song, Xuezhen; Zhang, Nan; Lin, Weiying

    2017-07-01

    Monitoring copper level in cancer cells is important for the further understanding of its roles in the cell proliferation, and also could afford novel copper-based strategy for the cancer therapy. Herein, we have developed a novel cancer cell-specific fluorescent probe for the detecting Cu2 + in living cancer cells. The probe employed biotin as the cancer cell-specific group. Before the treatment of Cu2 +, the probe showed nearly no fluorescence. However, the probe can display strong fluorescence at 581 nm in response to Cu2 +. The probe exhibited excellent sensitivity and high selectivity for Cu2 + over the other relative species. Under the guidance of biotin group, could be successfully used for detecting Cu2 + in living cancer cells. We expect that this design strategy could be further applied for detection of the other important biomolecules in living cancer cells.

  19. Variable rate application of nematicides on cotton fields: a promising site-specific management strategy.

    Science.gov (United States)

    Ortiz, Brenda V; Perry, Calvin; Sullivan, Dana; Lu, Ping; Kemerait, Robert; Davis, Richard F; Smith, Amanda; Vellidis, George; Nichols, Robert

    2012-03-01

    specific application of nematicides based on management zones, although this approach might not be economically beneficial in fields with little variability in soil texture.

  20. Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

    Science.gov (United States)

    Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2014-01-01

    A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116

  1. Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Sachiko Ishida

    Full Text Available A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3 concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+ oscillations in terms of the time constant of Ca(2+ spike amplitude decay and the Ca(2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca(2+ oscillations, such as the time constant of the temporal changes in the Ca(2+ spike amplitude and the Ca(2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+ release, can cause cell-to-cell variability in the patterns of Ca(2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca(2+ signals evoked by G protein-coupled receptor stimulation.

  2. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials

    National Research Council Canada - National Science Library

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    .... Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63...

  3. Gene expression analyses reveal metabolic specifications in acute O2 -sensing chemoreceptor cells.

    Science.gov (United States)

    Gao, Lin; Bonilla-Henao, Victoria; García-Flores, Paula; Arias-Mayenco, Ignacio; Ortega-Sáenz, Patricia; López-Barneo, José

    2017-09-15

    Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2 -sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2 -sensitive, with superior cervical ganglion neurons, which are practically O2 -insensitive. In O2 -sensitive cells, we found a characteristic prolyl hydroxylase 3 down-regulation and hypoxia inducible factor 2α up-regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a 'signature metabolic profile'. Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2 -sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2 -sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable

  4. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    Science.gov (United States)

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  5. Pancreatic α-cell specific deletion of mouse Arx leads to α-cell identity loss.

    Science.gov (United States)

    Wilcox, Crystal L; Terry, Natalie A; Walp, Erik R; Lee, Randall A; May, Catherine Lee

    2013-01-01

    The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, Aristaless-related homeobox gene (Arx) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of Arx in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of Arx, we have generated mice in which the Arx gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of Arx in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express β-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.

  6. Tract specific analysis in patients with sickle cell disease

    Science.gov (United States)

    Chai, Yaqiong; Coloigner, Julie; Qu, Xiaoping; Choi, Soyoung; Bush, Adam; Borzage, Matt; Vu, Chau; Lepore, Natasha; Wood, John

    2015-12-01

    Sickle cell disease (SCD) is a hereditary blood disorder in which the oxygen-carrying hemoglobin molecule in red blood cells is abnormal. It affects numerous people in the world and leads to a shorter life span, pain, anemia, serious infections and neurocognitive decline. Tract-Specific Analysis (TSA) is a statistical method to evaluate white matter alterations due to neurocognitive diseases, using diffusion tensor magnetic resonance images. Here, for the first time, TSA is used to compare 11 major brain white matter (WM) tracts between SCD patients and age-matched healthy subjects. Alterations are found in the corpus callosum (CC), the cortico-spinal tract (CST), inferior fronto-occipital fasciculus (IFO), inferior longitudinal fasciculus (ILF), superior longitudinal fasciculus (SLF), and uncinated fasciculus (UNC). Based on previous studies on the neurocognitive functions of these tracts, the significant areas found in this paper might be related to several cognitive impairments and depression, both of which are observed in SCD patients.

  7. Gene-regulatory interactions in embryonic stem cells represent cell-type specific gene regulatory programs.

    Science.gov (United States)

    Ha, Misook; Hong, Soondo

    2017-10-13

    Pluripotency, the ability of embryonic stem cells to differentiate into specialized cell types, is determined by ESC-specific gene regulators such as transcription factors and chromatin modification factors. It is not well understood how ESCs are poised for differentiation, however, and methods are needed for prognosis of the molecular changes in the differentiation of ESCs into specific organs. We describe a new approach to infer cell-type specific gene regulatory programs based on gene regulatory interactions in ESCs. Our method infers the molecular logic of gene regulatory mechanisms by mapping the position-specific combinatory patterns of numerous regulators in ESCs into cell-type specific gene regulations. We validate the proposed approach by recapitulating the RNA-seq and microarray data of neuronal progenitor cells, adult liver cells, and ESCs from the integrated patterns of diverse gene regulators in ESCs. We find that the collective functions of diverse gene regulators in ESCs represent distinct gene regulatory programs in specialized cell types. Our new approach expands our understanding of the differential gene regulatory information in developments encoded in regulatory networks of ESCs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Damage-specific DNA-binding proteins from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1

  9. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain...... acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed...... that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF...

  10. Variable stretch reduces the pro-inflammatory response of alveolar epithelial cells.

    Science.gov (United States)

    Rentzsch, Ines; Santos, Cíntia L; Huhle, Robert; Ferreira, Jorge M C; Koch, Thea; Schnabel, Christian; Koch, Edmund; Pelosi, Paolo; Rocco, Patricia R M; Gama de Abreu, Marcelo

    2017-01-01

    Mechanical ventilation has the potential to increase inflammation in both healthy and injured lungs. Several animal studies have shown that variable ventilation recruits the lungs and reduces inflammation. However, it is unclear which cellular mechanisms are involved in those findings. We hypothesized that variable stretch of LPS-stimulated alveolar epithelial cells (AECs) reduces the production of pro-inflammatory cytokines compared to non-variable stretch. AECs were subjected to non-variable or variable cyclic stretch (sinusoidal pattern), with and without LPS stimulation. The expression and release of interleukin-6, CXCL-2 and CCL-2 mRNA were analyzed after 4 hours. The phosphorylation of the MAPKs ERK1/2 and SAPK/JNK was determined by Western Blot analysis at 0, 15, 30, 45 and 60 min of cyclic stretch. In LPS-stimulated AECs, variable cyclic cell stretching led to reduced cytokine expression and release compared to non-variable cell stretching. Furthermore, the phosphorylation of the MAPK ERK1/2 was increased after 30 minutes in non-variable stretched AECs, whereas variable stretched cells demonstrated only the non-stretched level of phosphorylation. After the 4h period of cyclic cell stretch and inhibition of the ERK1/2, but not the SAPK/JNK, signaling pathway, the gene expression of investigated cytokines increased in variable stretched, and decreased in non-variable stretched AECs. We conclude that in LPS-stimulated AECs, variable stretch reduced the pro-inflammatory response compared to non-variable stretch. This effect was mediated by the ERK1/2 signaling pathway, and might partly explain the findings of reduced lung inflammation during mechanical ventilation modes that enhance breath-by-breath variability of the respiratory pattern.

  11. Male Differentiation of Germ Cells Induced by Embryonic Age-Specific Sertoli Cells in Mice1

    Science.gov (United States)

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R.; Yamazaki, Yukiko

    2012-01-01

    ABSTRACT Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  12. The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification

    Science.gov (United States)

    Lotinun, Sutada; Akhavanfard, Sara; Coffman, Erik J.; Cook, Edward B.; Stoykova, Svetlana; Mukherjee, Siddhartha; Schoonmaker, Jesse A.; Burger, Alexa; Kim, Woo Jae; Kronenberg, Henry M.; Baron, Roland; Haber, Daniel A.; Bardeesy, Nabeel

    2014-01-01

    SUMMARY WTX is an X-linked tumor suppressor targeted by somatic mutations in Wilms tumor, a pediatric kidney cancer, and by germline inactivation in osteopathia striata with cranial sclerosis, a bone overgrowth syndrome. Here, we show that Wtx deletion in mice causes neonatal lethality, somatic overgrowth, and malformation of multiple mesenchyme-derived tissues, including bone, fat, kidney, heart, and spleen. Inactivation of Wtx at different developmental stages and in primary mesenchymal progenitor cells (MPCs) reveals that bone mass increase and adipose tissue deficiency are due to altered lineage fate decisions coupled with delayed terminal differentiation. Specification defects in MPCs result from aberrant β-catenin activation, whereas alternative pathways contribute to the subsequently delayed differentiation of lineage-restricted cells. Thus, Wtx is a regulator of MPC commitment and differentiation with stage-specific functions in inhibiting canonical Wnt signaling. Furthermore, the constellation of anomalies in Wtx null mice suggests that this tumor suppressor broadly regulates MPCs in multiple tissues. PMID:21571217

  13. Effect of Interaction between Chromatin Loops on Cell-to-Cell Variability in Gene Expression.

    Directory of Open Access Journals (Sweden)

    Tuoqi Liu

    2016-05-01

    Full Text Available According to recent experimental evidence, the interaction between chromatin loops, which can be characterized by three factors-connection pattern, distance between regulatory elements, and communication form, play an important role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effect that addresses the question of how changes in these factors affect variability at the expression level in a systematic rather than case-by-case fashion. Here we make such an effort, based on a mechanic model that maps three fundamental patterns for two interacting DNA loops into a 4-state model of stochastic transcription. We first show that in contrast to side-by-side loops, nested loops enhance mRNA expression and reduce expression noise whereas alternating loops have just opposite effects. Then, we compare effects of facilitated tracking and direct looping on gene expression. We find that the former performs better than the latter in controlling mean expression and in tuning expression noise, but this control or tuning is distance-dependent, remarkable for moderate loop lengths, and there is a limit loop length such that the difference in effect between two communication forms almost disappears. Our analysis and results justify the facilitated chromatin-looping hypothesis.

  14. Cell-specific modulation of surfactant proteins by ambroxol treatment.

    Science.gov (United States)

    Seifart, Carola; Clostermann, Ursula; Seifart, Ulf; Müller, Bernd; Vogelmeier, Claus; von Wichert, Peter; Fehrenbach, Heinz

    2005-02-15

    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  15. Rare earth fluorescent nanoparticles for specific cancer cell targeting

    Energy Technology Data Exchange (ETDEWEB)

    Stefanakis, Dimitrios; Ghanotakis, Demetrios F., E-mail: ghanotakis@uoc.gr [University of Crete, Department of Chemistry (Greece)

    2016-07-15

    Terbium layered hydroxide nanoparticles (Tb{sub 2}(OH){sub 5}NO{sub 3}) were synthesized by a one-pot coprecipitation method. The characterization of this preparation revealed highly oriented fluorescent nanoparticles. An attempt to improve the properties of Tb{sub 2}(OH){sub 5}NO{sub 3} resulted in the preparation of two optimized nanoparticles. In particular, Tb{sub 2}(OH){sub 5}NO{sub 3}:Eu and Tb{sub 2}(OH){sub 5}NO{sub 3}-FA were prepared when Tb{sub 2}(OH){sub 5}NO{sub 3} was doped with Europium and when the surface was modified with folic acid (FA), respectively. The size of the above nanoparticles was below 100 nm, and thus they have the potential to be used for biomedical applications. The interaction of nanoparticles with human cells was studied using confocal microscopy. This study revealed that only the nanoparticles modified with folic acid have the ability to be targeted to HeLa cells. This specific identification of cancer cells, in combination with the fluorescent properties of Tb{sub 2}(OH){sub 5}NO{sub 3}, could render these nanoparticles appropriate for biomedical applications.

  16. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  17. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

  18. The Effect of Listening to Specific Musical Genre Selections on Measures of Heart Rate Variability

    Science.gov (United States)

    Orman, Evelyn K.

    2011-01-01

    University students (N = 30) individually listened to the Billboard 100 top-ranked musical selection for their most and least liked musical genre. Two minutes of silence preceded each musical listening condition, and heart rate variability (HRV) was recorded throughout. All HRV measures decreased during music listening as compared with silence.…

  19. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Directory of Open Access Journals (Sweden)

    Ki Jung Lim

    Full Text Available Cell-penetrating peptides (CPPs have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2 was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57. The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv directed toward a mutated K-ras (G12V. BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  20. Distinguishing between stochasticity and determinism: Examples from cell cycle duration variability.

    Science.gov (United States)

    Pearl Mizrahi, Sivan; Sandler, Oded; Lande-Diner, Laura; Balaban, Nathalie Q; Simon, Itamar

    2016-01-01

    We describe a recent approach for distinguishing between stochastic and deterministic sources of variability, focusing on the mammalian cell cycle. Variability between cells is often attributed to stochastic noise, although it may be generated by deterministic components. Interestingly, lineage information can be used to distinguish between variability and determinism. Analysis of correlations within a lineage of the mammalian cell cycle duration revealed its deterministic nature. Here, we discuss the sources of such variability and the possibility that the underlying deterministic process is due to the circadian clock. Finally, we discuss the "kicked cell cycle" model and its implication on the study of the cell cycle in healthy and cancerous tissues. © 2015 WILEY Periodicals, Inc.

  1. Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

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    Laurence Rolland

    1992-06-01

    Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

  2. NOTCH SIGNALING ALTERS SENSORY OR NEURONAL CELL FATE SPECIFICATION OF INNER EAR STEM CELLS

    Science.gov (United States)

    Jeon, Sang-Jun; Fujioka, Masato; Kim, Shi-Chan; Edge, Albert S.B.

    2011-01-01

    Multipotent progenitor cells in the otic placode give rise to the specialized cell types of the inner ear, including neurons, supporting cells and hair cells. The mechanisms governing acquisition of specific fates by the cells that form the cochleovestibular organs remain poorly characterized. Here we show that whereas blocking Notch signaling with a γ-secretase inhibitor increased the conversion of inner ear stem cells to hair cells by a mechanism that involved the upregulation of bHLH transcription factor, Math1 (mouse Atoh1), differentiation to a neuronal lineage was increased by expression of the Notch intracellular domain. The shift to a neuronal lineage could be attributed in part to the continued cell proliferation in cells that did not undergo sensory cell differentiation due to the high Notch signaling, but also involved upregulation of Ngn1. The Notch intracellular domain influenced Ngn1 indirectly by upregulation of Sox2, a transcription factor expressed in many neural progenitor cells, and directly by an interaction with an RBP-J binding site in the Ngn1 promoter/enhancer. The induction of Ngn1 was blocked partially by mutation of the RBP-J site and nearly completely when the mutation was combined with inhibition of Sox2 expression. Thus Notch signaling had a significant role in the fate specification of neurons and hair cells from inner ear stem cells, and decisions about cell fate were mediated in part by a differential effect of combinatorial signaling by Notch and Sox2 on the expression of bHLH transcription factors. PMID:21653840

  3. Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes.

    Science.gov (United States)

    Lindgren, David; Eriksson, Pontus; Krawczyk, Krzysztof; Nilsson, Helén; Hansson, Jennifer; Veerla, Srinivas; Sjölund, Jonas; Höglund, Mattias; Johansson, Martin E; Axelson, Håkan

    2017-08-08

    Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. A Nonlinear Mixed Effects Approach for Modeling the Cell-To-Cell Variability of Mig1 Dynamics in Yeast.

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    Joachim Almquist

    Full Text Available The last decade has seen a rapid development of experimental techniques that allow data collection from individual cells. These techniques have enabled the discovery and characterization of variability within a population of genetically identical cells. Nonlinear mixed effects (NLME modeling is an established framework for studying variability between individuals in a population, frequently used in pharmacokinetics and pharmacodynamics, but its potential for studies of cell-to-cell variability in molecular cell biology is yet to be exploited. Here we take advantage of this novel application of NLME modeling to study cell-to-cell variability in the dynamic behavior of the yeast transcription repressor Mig1. In particular, we investigate a recently discovered phenomenon where Mig1 during a short and transient period exits the nucleus when cells experience a shift from high to intermediate levels of extracellular glucose. A phenomenological model based on ordinary differential equations describing the transient dynamics of nuclear Mig1 is introduced, and according to the NLME methodology the parameters of this model are in turn modeled by a multivariate probability distribution. Using time-lapse microscopy data from nearly 200 cells, we estimate this parameter distribution according to the approach of maximizing the population likelihood. Based on the estimated distribution, parameter values for individual cells are furthermore characterized and the resulting Mig1 dynamics are compared to the single cell times-series data. The proposed NLME framework is also compared to the intuitive but limited standard two-stage (STS approach. We demonstrate that the latter may overestimate variabilities by up to almost five fold. Finally, Monte Carlo simulations of the inferred population model are used to predict the distribution of key characteristics of the Mig1 transient response. We find that with decreasing levels of post-shift glucose, the transient

  5. Secretory products of breast cancer cells specifically affect human osteoblastic cells: partial characterization of active factors.

    Science.gov (United States)

    Siwek, B; Lacroix, M; De Pollak, C; Marie, P; Body, J J

    1997-04-01

    The pathogenesis of tumor-induced osteolysis (TIO) following breast cancer metastases in bone remains unclear. We postulated that osteoblasts could be target cells for the secretory products of breast cancer cells. We previously showed that serum-free conditioned medium (CM) of the breast cancer cell line MCF-7 inhibits DNA synthesis by 75% of control values in osteoblast-like cells SaOS-2 and that this effect is only in a minor part due to transforming growth factor beta secretion. To establish the specificity of our observations and to look for other biologically active factors, we have tested the effects of medium conditioned by several cancer and noncancer cell lines (breast, colon, placenta, or fibrosarcoma) on the proliferation of osteoblast-like cells (SaOS-2, MG-63), normal human osteoblasts, human fibrosarcoma cells, and normal human fibroblasts. Culture medium (1:2) of the breast cancer cell lines MCF-7, T-47D, MDA-MB-231, and SK-BR-3 inhibited by 25-50% the proliferation of osteoblast-like cells SaOS-2, MG-63, and normal osteoblasts as evaluated by the MTT survival test or [3H]thymidine incorporation. MCF-7 cells completely inhibited the proliferation of normal human osteoblasts in coculture. This inhibitory effect was reversible and not due to cytotoxicity. Moreover, the cyclic adenosine monophosphate (cAMP) response to parathyroid hormone (PTH) of osteoblast-like cells SaOS-2 was also increased by 100-240% by the same CM. Such activities were, however, not detected in medium from the breast noncancer cell line HBL-100 or in the medium conditioned by non-breast cancer cell lines (COLO 320DM, HT-29, JAR, or HT-1080). Medium from the breast cancer cells had no effect on normal human fibroblasts or fibrosarcoma cells (HT-1080), suggesting the specificity of their action on human osteoblasts. After partial purification by ultrafiltration and size-exclusion chromatography, we found that medium of T-47D cells contained at least three nonprostanoid factors of

  6. Langerhans Cells Prevent Autoimmunity via Expansion of Keratinocyte Antigen-Specific Regulatory T Cells

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    Daniela Y. Kitashima

    2018-01-01

    Full Text Available Langerhans cells (LCs are antigen-presenting cells in the epidermis whose roles in antigen-specific immune regulation remain incompletely understood. Desmoglein 3 (Dsg3 is a keratinocyte cell-cell adhesion molecule critical for epidermal integrity and an autoantigen in the autoimmune blistering disease pemphigus. Although antibody-mediated disease mechanisms in pemphigus are extensively characterized, the T cell aspect of this autoimmune disease still remains poorly understood. Herein, we utilized a mouse model of CD4+ T cell-mediated autoimmunity against Dsg3 to show that acquisition of Dsg3 and subsequent presentation to T cells by LCs depended on the C-type lectin langerin. The lack of LCs led to enhanced autoimmunity with impaired Dsg3-specific regulatory T cell expansion. LCs expressed the IL-2 receptor complex and the disruption of IL-2 signaling in LCs attenuated LC-mediated regulatory T cell expansion in vitro, demonstrating that direct IL-2 signaling shapes LC function. These data establish that LCs mediate peripheral tolerance against an epidermal autoantigen and point to langerin and IL-2 signaling pathways as attractive targets for achieving tolerogenic responses particularly in autoimmune blistering diseases such as pemphigus.

  7. Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

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    Takahiro Nagatake

    Full Text Available Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4, and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga and other enteroendocrine cell-related genes (Ffar1, Ffar4, Gpr119, and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1. A Cld4+UEA-1- cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip, thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg, pancreatic polypeptide-like peptide with N-terminal tyrosine amide (Pyy, cholecystokinin (Cck, secretin (Sct, and tryptophan hydroxylase 1 (Tph1. In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.

  8. Floral scent in food-deceptive orchids: species specificity and sources of variability.

    Science.gov (United States)

    Salzmann, C C; Cozzolino, S; Schiestl, F P

    2007-11-01

    One third of all orchid species are deceptive and do not reward their pollinators. Such deceptive orchids are often characterised by unusually high variation in floral signals such as colour and scent. In this study, we investigated the scent composition of two Mediterranean food-deceptive orchids Orchis mascula, Orchis pauciflora, and their hybrid, O. x colemanii. Scent was collected IN SITU by headspace sorption and was subsequently analysed with gas chromatography and gas chromatography-mass spectrometry. We compared variation of odour compounds within and between populations as well as species. We identified 35 floral scent compounds, mainly monoterpenes, which were shared by both species. Both quantitative and qualitative variability within and among populations was high. Many individuals within species could be classified to different "odour-types". In spite of high qualitative and quantitative intra- and inter-population variability, the species were clearly differentiated in their scent bouquets, whereas most hybrid individuals emitted an intermediate scent.

  9. Variability in sea ice cover and climate elicit sex specific responses in an Antarctic predator.

    Science.gov (United States)

    Labrousse, Sara; Sallée, Jean-Baptiste; Fraser, Alexander D; Massom, Rob A; Reid, Phillip; Hobbs, William; Guinet, Christophe; Harcourt, Robert; McMahon, Clive; Authier, Matthieu; Bailleul, Frédéric; Hindell, Mark A; Charrassin, Jean-Benoit

    2017-02-24

    Contrasting regional changes in Southern Ocean sea ice have occurred over the last 30 years with distinct regional effects on ecosystem structure and function. Quantifying how Antarctic predators respond to such changes provides the context for predicting how climate variability/change will affect these assemblages into the future. Over an 11-year time-series, we examine how inter-annual variability in sea ice concentration and advance affect the foraging behaviour of a top Antarctic predator, the southern elephant seal. Females foraged longer in pack ice in years with greatest sea ice concentration and earliest sea ice advance, while males foraged longer in polynyas in years of lowest sea ice concentration. There was a positive relationship between near-surface meridional wind anomalies and female foraging effort, but not for males. This study reveals the complexities of foraging responses to climate forcing by a poleward migratory predator through varying sea ice property and dynamic anomalies.

  10. Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells.

    Science.gov (United States)

    Hoff, Soren T; Salman, Ahmed M; Ruhwald, Morten; Ravn, Pernille; Brock, Inger; Elsheikh, Nabila; Andersen, Peter; Agger, Else Marie

    2015-01-01

    The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment. A candidate for such regulation could be the chemokine CXCL10. CXCL10 is mainly produced by human monocytes, but a few reports have also found CXCL10 production by human B cells. The objective of this study was to investigate CXCL10 production by human B cells in response to in vitro stimulation with Mtb antigens. We analyzed human blood samples from 30 volunteer donors using multiparameter flow cytometry, and identified a subgroup of B cells producing CXCL10 in response to in vitro stimulation with antigens. T cells did not produce CXCL10, but CXCL10 production by B cells appeared to be mediated via IFN-γ and dependent on contact with antigen-specific T cells recognizing the antigen. Human B cells are able to produce CXCL10 in an IFN-γ and T cell contact-dependent manner. The present findings suggest a possible mechanism through which B cells in part may influence granuloma formation in human tuberculosis (TB) and participate in infection control. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. A Comparison of Recurrent Non-specific Low Back Pain Patients and Healthy Subjects in Variability of Anticipatory Postural Adjustments

    Directory of Open Access Journals (Sweden)

    Rozita Hedayati

    2010-10-01

    Full Text Available Objective: The purpose of this study was to ivestigate of the postural strategies variability in low back pain patients, as a criterion in the adaptability evaluation of this system to the environmental demands and comparison of patients with healthy persons and also the importance of psychological factors associated with pain as an effective factor in postural disturbances was assessed. Materials & Methods: This analytical, cross-sectional and case-control study was performed on twenty one recurrent non-specific low back pain patients whom had been selected simply and conveniently and twenty one healthy persons whom had been matched with patients. The electromyographic activity of Deltoid, External Oblique, Transverse Abdominis / Internal Oblique and Erector Spinae muscles of each person was recorded in 75 rapid arm flexion with maximum acceleration. To study the patient’s avoidance belief and disability, two standard tools Fear-Avoidance Belief Questionnaire (FABQ and Roland-Morris Questionnaire were used respectively. Data were analyzed by MANOVA and Pearson correlation coefficient. Results: The results showed that chronic low back pain patients exhibit less variability in their anticipatory postural adjustments than the control group(P=0.047. However, the decrease in variability of the timing of anticipatory postural adjustments of External Oblique(P=0.45 and Erector Spinae(P=0.6 muscles was not significant. The correlation between the timing variability of anticipatory postural adjustments and fear-avoidance belief questionnaire scores was significant(P=0.006, however there was no significant correlation between this variable and disability(P=0.09 in the patients. Conclusion: There is a decrease in variability of postural control system of recurrent non-specific low back pain patients that can result in the persistence of pain and chronicity by decreasing the adaptability to environmental demands. The restoration of variability to

  12. Basal Cell Carcinoma : Evaluation Of Clinical And Histologic Variables

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    Mahanthesh Mamata

    2004-01-01

    Full Text Available A retrospective review of 32 patients with basal cell carcinoma was performed with aims to characterize the demographic profile and to analyze the clinical and histological parameters influencing the treatment of basal cell carcinoma. The maximum number occurred in the sixth decade with an unusual female preponderance. Hisologically, the classic type predominated while the other types included pigmented, morphoea, metatypical, adenoid, eccrine, firoepihelioma and superficial spreading type. All patients had excision of the tumour with involvement of surgical margin in three of them. None of the three patients belonged to aggressive histological types, such as morphoea or metatypical type. Two year follow-up showed no recurrence. Size of the tumor less than 2 cm, absence of ulceration and a good inflammatory response were probably other reasons for non-recurrence.

  13. Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells.

    Science.gov (United States)

    Jia, Bei; Chen, Shen; Zhao, Zhiju; Liu, Pengfei; Cai, Jinglei; Qin, Dajiang; Du, Juan; Wu, Changwei; Chen, Qianyu; Cai, Xiujuan; Zhang, Hui; Yu, Yanhong; Pei, Duanqing; Zhong, Mei; Pan, Guangjin

    2014-07-11

    Hemophilia A (HA) is a severe, congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years, traditional laboratory animals have been used to study HA and its therapies, although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction. In this study, we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4, SOX2, SV40LT and KLF4, and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells. HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining, in vitro assays and in vivo assays. Importantly, we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII, but otherwise functioned normally, recapitulating the phenotype of HA disease in vitro. HA-iPSCs, particular those generated from the urine using a non-viral approach, provide an efficient way for modeling HA in vitro. Furthermore, HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Influenza virus and endothelial cells: a species specific relationship

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    Kirsty Renfree Short

    2014-12-01

    Full Text Available Influenza A virus infection is an important cause of respiratory disease in humans. The original reservoirs of influenza A virus are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI. Part of this evolution involves increased viral tropism for endothelial cells. HPAI virus infections not only cause severe disease in chickens and other terrestrial poultry species but can also spread to humans and back to wild bird populations. Here, we review the role of the endothelium in the pathogenesis of influenza virus infection in wild birds, terrestrial poultry and humans with a particular focus on HPAI viruses. We demonstrate that whilst the endothelium is an important target of virus infection in terrestrial poultry and some wild bird species, in humans the endothelium is more important in controlling the local inflammatory milieu. Thus, the endothelium plays an important, but species-specific, role in the pathogenesis of influenza virus infection.

  15. Influenza virus and endothelial cells: a species specific relationship.

    Science.gov (United States)

    Short, Kirsty R; Veldhuis Kroeze, Edwin J B; Reperant, Leslie A; Richard, Mathilde; Kuiken, Thijs

    2014-01-01

    Influenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves increased viral tropism for endothelial cells. HPAI virus infections not only cause severe disease in chickens and other terrestrial poultry species but can also spread to humans and back to wild bird populations. Here, we review the role of the endothelium in the pathogenesis of influenza virus infection in wild birds, terrestrial poultry and humans with a particular focus on HPAI viruses. We demonstrate that whilst the endothelium is an important target of virus infection in terrestrial poultry and some wild bird species, in humans the endothelium is more important in controlling the local inflammatory milieu. Thus, the endothelium plays an important, but species-specific, role in the pathogenesis of influenza virus infection.

  16. Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types

    DEFF Research Database (Denmark)

    Ecker, Simone; Chen, Lu; Pancaldi, Vera

    2017-01-01

    Background: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic v...

  17. Maintenance of human embryonic stem cells in mesenchymal stem cell-conditioned media augments hematopoietic specification.

    Science.gov (United States)

    Ramos-Mejía, Verónica; Fernández, Agustín F; Ayllón, Verónica; Real, Pedro J; Bueno, Clara; Anderson, Per; Martín, Francisco; Fraga, Mario F; Menendez, Pablo

    2012-06-10

    The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.

  18. Sex-Specific Effects of Unpredictable Variable Prenatal Stress: Implications for Mammalian Developmental Programming During Spaceflight

    Science.gov (United States)

    Talyansky, Y.; Moyer, E. L.; Oijala, E.; Baer, L. A.; Ronca, A. E.

    2016-01-01

    During adaptation to the microgravity environment, adult mammals experience stress mediated by the Hypothalamic-Pituitary-Adrenal axis. In our previous studies of pregnant rats exposed to 2-g hypergravity via centrifugation, we reported decreased corticosterone and increased body mass and leptin in adult male, but not female, offspring. In this study, we utilized Unpredictable Variable Prenatal Stress to simulate the stressors of spaceflight by exposing dams to different stressors. Stress response modulation occurs via both positive and negative feedback in the hypothalamus, anterior pituitary gland, and adrenal cortex resulting in the differential release of corticosterone (CORT), a murine analog to human cortisol.

  19. Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation.

    Science.gov (United States)

    Meyer, Randall A; Sunshine, Joel C; Perica, Karlo; Kosmides, Alyssa K; Aje, Kent; Schneck, Jonathan P; Green, Jordan J

    2015-04-01

    Non-spherical nanodimensional artificial antigen presenting cells (naAPCs) offer the potential to systemically induce an effective antigen-specific immune response. In this report it is shown biodegradable ellipsoidal naAPCs mimic the T-Cell/APC interaction better than equivalent spherical naAPCs. In addition, it is demonstrated ellipsoidal naAPCs offer reduced non-specific cellular uptake and a superior pharmacokinetic profile compared to spherical naAPCs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Conserved and variable: Understanding mammary stem cells across species.

    Science.gov (United States)

    Rauner, Gat; Ledet, Melissa M; Van de Walle, Gerlinde R

    2018-01-01

    Postnatal mammary gland development requires the presence of mammary stem and progenitor cells (MaSC), which give rise to functional milk-secreting cells and regenerate the mammary epithelium with each cycle of lactation. These long-lived, tissue-resident MaSC are also targets for malignant transformation and may be cancer cells-of-origin. Consequently, MaSC are extensively researched in relation to their role and function in development, tissue regeneration, lactation, and breast cancer. The basic structure and function of the mammary gland are conserved among all mammalian species, from the most primitive to the most evolved. However, species vary greatly in their lactation strategies and mammary cancer incidence, making MaSC an interesting focus for comparative research. MaSC have been characterized in mice, to a lesser degree in humans, and to an even lesser degree in few additional mammals. They remain uncharacterized in most mammalian species, including "ancient" monotremes, marsupials, wild, and rare species, as well as in common and domestic species such as cats. Identification and comparison of MaSC across a large variety of species, particularly those with extreme lactational adaptations or low mammary cancer incidence, is expected to deepen our understanding of development and malignancy in the mammary gland. Here, we review the current status of MaSC characterization across species, and underline species variations in lactation and mammary cancer through which we may learn about the role of MaSC in these processes. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  1. A xylogalacturonan epitope is specifically associated with plant cell detachment

    DEFF Research Database (Denmark)

    Willats, William George Tycho; McCartney, L.; Steele-King, C.G.

    2004-01-01

    associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified...

  2. The variability of pulp-wood basic specific gravity of softwood

    Directory of Open Access Journals (Sweden)

    Jiří Holan

    2004-01-01

    Full Text Available In the present study, basic density of pulpwood of Norway spruce (Piceas abies /L./ Karst. and Scots pine (Pinus sylvestris L. was investigated. The variability of basic density and factors applied are out of concern. Factors are characterised by localization along the log, growing conditions defined by geographic area of Czech Republic, diameter of log, and the storing of logs within transport loading. Basic density was determined on cores taken by modified Pressler borer in the radial direction from bark to pith. The average basic density of Norway spruce is comparable to Scots pine, as well as the variation coefficient. Basic density of Norway spruce was found 443 kg.m-3 (Vx = 13.9% where as the Scots pine base density was 450 kg.m-3 (Vx = 15.5%. Basic density was significantly influenced by all factors assessed except the localization of cores along logs. The investigation demonstrated that basic density can be affected by locality of supply in comparison to within logs variability that have much lower importance.

  3. Intra-individual gait pattern variability in specific situations: Implications for forensic gait analysis.

    Science.gov (United States)

    Ludwig, Oliver; Dillinger, Steffen; Marschall, Franz

    2016-07-01

    In this study, inter- and intra-individual gait pattern differences are examined in various gait situations by means of phase diagrams of the extremity angles (cyclograms). 8 test subjects walked along a walking distance of 6m under different conditions three times each: barefoot, wearing sneakers, wearing combat boots, after muscular fatigue, and wearing a full-face motorcycle helmet restricting vision. The joint angles of foot, knee, and hip were recorded in the sagittal plane. The coupling of movements was represented by time-adjusted cyclograms, and the inter- and intra-individual differences were captured by calculating the similarity between different gait patterns. Gait pattern variability was often greater between the defined test situations than between the individual test subjects. The results have been interpreted considering neurophysiological regulation mechanisms. Footwear, masking, and fatigue were interpreted as disturbance parameters, each being a cause for gait pattern variability and complicating the inference of identity of persons in video recordings. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Phage exposure causes dynamic shifts in the expression states of specific phase-variable genes of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Aidley, Jack; Holst Sørensen, Martine C.; Bayliss, Christopher D.

    2017-01-01

    Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacter jejuni this reversible adaptive process is mediated by mutations...... in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase...... for this CPS modification, cj1421, and two other phase-variable CPS genes generated phage resistance in C. jejuni. Here we investigate the population dynamics of C. jejuni NCTC11168 when exposed to phage F336 in vitro using a newly described method - the 28-locus-CJ11168 PV analysis. Dynamic switching...

  5. T-cell numbers and antigen-specific T-cell function follow different circadian rhythms.

    Science.gov (United States)

    Kirsch, Sarah; Thijssen, Stephan; Alarcon Salvador, Susana; Heine, Gunnar H; van Bentum, Kai; Fliser, Danilo; Sester, Martina; Sester, Urban

    2012-12-01

    Circadian rhythms play an important role in modulating cellular immune responses. The present study was performed to characterise circadian variations in lymphocyte numbers and antigen-specific T-cell functionality in healthy individuals under physiological conditions. Blood leukocyte populations of six healthy volunteers were quantified over 24 h. In addition, antigen-specific T-cell functionality was analysed directly ex vivo from whole blood using flow cytometry based on intracellular cytokine induction after a 6-hour stimulation with adenovirus antigen and Staphylococcus aureus enterotoxin B (SEB), respectively. T-cell numbers and reactivity were stable during daytime, whereas a significant increase was observed during late evening and early morning hours. The percentage of T cells reacting towards adenovirus antigen and SEB showed a 1.76 ± 0.55-fold (p = 0.0002) and a 1.42 ± 0.33-fold (p = 0.0002) increase, respectively. Dynamics in T-cell reactivity were independent of the mode of antigen stimulation and inversely correlated with plasma levels of endogenous cortisol. Interestingly, peak frequencies of reactive T cells occurred late in the evening and did not directly coincide with peak numbers of bulk T cells that were observed in the early morning hours. Taken together, our data reveal a circadian regulation of T-cell immune responses in the peripheral blood of humans under physiological conditions. This knowledge may be of practical consequence for the timing of blood sampling for functional T-cell assays as well as for immunosuppressive drug intake after organ transplantation, where T-cell function may be influenced not only by drug-mediated inhibition but also by circadian fluctuations in T-cell reactivity.

  6. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Science.gov (United States)

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  7. Patterns and processes of habitat-specific demographic variability in exploited marine species

    NARCIS (Netherlands)

    Vasconcelos, R.P.; Eggleston, D.B.; Pape, le O.; Tulp, I.Y.M.

    2014-01-01

    Population dynamics are governed by four demographic rates: births, deaths, immigration, and emigration. Variation in these rates and processes underlying such variation can be used to prioritize habitat conservation and restoration as well as to parameterize models that predict habitat-specific

  8. Human adenovirus-specific T cells modulate HIV-specific T cell responses to an Ad5-vectored HIV-1 vaccine

    National Research Council Canada - National Science Library

    Frahm, Nicole; DeCamp, Allan C; Friedrich, David P; Carter, Donald K; Defawe, Olivier D; Kublin, James G; Casimiro, Danilo R; Duerr, Ann; Robertson, Michael N; Buchbinder, Susan P; Huang, Yunda; Spies, Gregory A; De Rosa, Stephen C; McElrath, M Juliana

    2012-01-01

    .... Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5...

  9. Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney.

    Science.gov (United States)

    Holthöfer, H

    1988-08-01

    The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.

  10. Effect of Specific Drills through Table Tennis Ball Feeding Machine on Selected Skill Performance Variables of Non- Table Tennis Players

    OpenAIRE

    Dr. M. Srinivasan; Ilangovan, P

    2017-01-01

    The purpose of the study was to determine the effect of effect of specific drills through table tennis ball feeding machine on selected skill performance variables of non- table tennis players. To achieve the purpose 30 men non-table tennis players from faculty of general and adapted physical education and yoga, Ramakrishna Mission Vivekananda University and Maruthi College of Physical Education, Coimbatore. The age of the subject’s was ranged from 23 to 28 years. the selected subjects were c...

  11. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...... programs. Functional analysis gathered insights in fate-specific candidates of interface functionalities. The non-transcriptionally regulated interface proteins were found to be highly regulated by post-translational ubiquitylation modification, which may synchronize the transition between cell...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...

  12. Specific targeting of whole lymphoma cells to dendritic cells ex vivo provides a potent antitumor vaccine

    Directory of Open Access Journals (Sweden)

    Mocikat Ralph

    2007-03-01

    Full Text Available Abstract Background Dendritic cells (DC pulsed with tumor-derived antigenic material have widely been used in antitumor vaccination protocols. However, the optimal strategy of DC loading has not yet been established. Our aim was to define requirements of optimal DC vaccines in terms of in vivo protection in a murine B-cell lymphoma model. Methods We compare various loading reagents including whole parental and modified tumor cells and a single tumor-specific antigen, namely the lymphoma idiotype (Id. Bone marrow-derived DC were pulsed in vitro and used for therapy of established A20 lymphomas. Results We show that a vaccine with superior antitumor efficacy can be generated when DC are loaded with whole modified tumor cells which provide both (i antigenic polyvalency and (ii receptor-mediated antigen internalization. Uptake of cellular material was greatly enhanced when the tumor cells used for DC pulsing were engineered to express an anti-Fc receptor immunoglobulin specificity. Upon transfer of these DC, established tumor burdens were eradicated in 50% of mice. By contrast, pulsing DC with unmodified lymphoma cells or with the lymphoma Id, even when it was endowed with the anti-Fc receptor binding arm, was far less effective. A specific humoral anti-Id response could be detected, particularly following delivery of Id protein-pulsed DC, but it was not predictive of tumor protection. Instead a T-cell response was pivotal for successful tumor protection. Interaction of the transferred DC with CD8+ T lymphocytes seemed to play a role for induction of the immune response but was dispensable when DC had received an additional maturation stimulus. Conclusion Our analyses show that the advantages of specific antigen redirection and antigenic polyvalency can be combined to generate DC-based vaccines with superior antitumor efficacy. This mouse model may provide information for the standardization of DC-based vaccination protocols.

  13. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  14. Long-term nonprogression and broad HIV-1-specific proliferative T-cell responses

    Directory of Open Access Journals (Sweden)

    Nesrina eImami

    2013-03-01

    Full Text Available Complex mechanisms underlying the maintenance of fully functional, proliferative, HIV-1-specific T-cell responses involve processes from early T-cell development through to the final stages of T-cell differentiation and antigen recognition. Virus-specific proliferative CD4 and CD8 T-cell responses, important for the control of infection, are observed in some HIV-1+ patients during early stages of disease, and are maintained in long-term nonprogressing subjects. In the vast majority of HIV-1+ patients, full immune functionality is lost when proliferative HIV-1-specific T-cell responses undergo a variable progressive decline throughout the course of chronic infection. This appears irreparable despite administration of potent combination antiretroviral therapy, which to date is non-curative, necessitating life-long administration and the development of effective, novel, therapeutic interventions. While a sterilising cure, involving clearance of virus from the host, remains a primary aim, a functional cure may be a more feasible goal with considerable impact on worldwide HIV-1 infection. Such an approach would enable long-term co-existence of host and virus in the absence of toxic and costly drugs. Effective immune homeostasis coupled with a balanced response appropriately targeting conserved viral antigens, in a manner that avoids hyperactivation and exhaustion, may prove to be the strongest correlate of durable viral control. This review describes novel concepts underlying full immune functionality in the context of HIV-1 infection, which may be utilised in future strategies designed to improve upon existing therapy. The aim will be to induce long-term nonprogressor or elite controller status in every infected host, through immune-mediated control of viraemia and reduction of viral reservoirs, leading to lower HIV-1 transmission rates.

  15. Parallel arrangements of positive feedback loops limit cell-to-cell variability in differentiation.

    Directory of Open Access Journals (Sweden)

    Anupam Dey

    Full Text Available Cellular differentiations are often regulated by bistable switches resulting from specific arrangements of multiple positive feedback loops (PFL fused to one another. Although bistability generates digital responses at the cellular level, stochasticity in chemical reactions causes population heterogeneity in terms of its differentiated states. We hypothesized that the specific arrangements of PFLs may have evolved to minimize the cellular heterogeneity in differentiation. In order to test this we investigated variability in cellular differentiation controlled either by parallel or serial arrangements of multiple PFLs having similar average properties under extrinsic and intrinsic noises. We find that motifs with PFLs fused in parallel to one another around a central regulator are less susceptible to noise as compared to the motifs with PFLs arranged serially. Our calculations suggest that the increased resistance to noise in parallel motifs originate from the less sensitivity of bifurcation points to the extrinsic noise. Whereas estimation of mean residence times indicate that stable branches of bifurcations are robust to intrinsic noise in parallel motifs as compared to serial motifs. Model conclusions are consistent both in AND- and OR-gate input signal configurations and also with two different modeling strategies. Our investigations provide some insight into recent findings that differentiation of preadipocyte to mature adipocyte is controlled by network of parallel PFLs.

  16. A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Seonmi Park

    2017-04-01

    Full Text Available Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs from patients of different ethnicities, β-globin gene (HBB haplotypes, and fetal hemoglobin (HbF levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS mutation.

  17. Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges.

    Science.gov (United States)

    Schmitt, Susanne; Tsai, Peter; Bell, James; Fromont, Jane; Ilan, Micha; Lindquist, Niels; Perez, Thierry; Rodrigo, Allen; Schupp, Peter J; Vacelet, Jean; Webster, Nicole; Hentschel, Ute; Taylor, Michael W

    2012-03-01

    Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world's oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.

  18. Fruit specific variability in capsaicinoid accumulation and transcription of structural and regulatory genes in Capsicum fruit

    Science.gov (United States)

    Keyhaninejad, Neda; Curry, Jeanne; Romero, Joslynn; O’Connell, Mary A.

    2013-01-01

    Accumulation of capsaicinoids in the placental tissue of ripening chile (Capsicum spp.) fruit follows the coordinated expression of multiple biosynthetic enzymes producing the substrates for capsaicin synthase. Transcription factors are likely agents to regulate expression of these biosynthetic genes. Placental RNAs from habanero fruit (C. chinense) were screened for expression of candidate transcription factors; with two candidate genes identified, both in the ERF family of transcription factors. Characterization of these transcription factors, Erf and Jerf, in nine chile cultivars with distinct capsaicinoid contents demonstrated a correlation of expression with pungency. Amino acid variants were observed in both ERF and JERF from different chile cultivars; none of these changes involved the DNA binding domains. Little to no transcription of Erf was detected in non-pungent C. annuum or C. chinense mutants. This correlation was characterized at an individual fruit level in a set of jalapeño (C. annuum) lines again with distinct and variable capsaicinoid contents. Both Erf and Jerf are expressed early in fruit development, 16–20 days post-anthesis, at times prior to the accumulation of capsaicinoids in the placental tissues. These data support the hypothesis that these two members of the complex ERF family participate in regulation of the pungency phenotype in chile. PMID:24388515

  19. IgH isotype-specific B cell receptor expression influences B cell fate.

    Science.gov (United States)

    Tong, Pei; Granato, Alessandra; Zuo, Teng; Chaudhary, Neha; Zuiani, Adam; Han, Seung Seok; Donthula, Rakesh; Shrestha, Akritee; Sen, Debattama; Magee, Jennifer M; Gallagher, Michael P; van der Poel, Cees E; Carroll, Michael C; Wesemann, Duane R

    2017-10-03

    Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

  20. Quantitative Age-specific Variability of Plasma Proteins in Healthy Neonates, Children and Adults.

    Science.gov (United States)

    Bjelosevic, Stefan; Pascovici, Dana; Ping, Hui; Karlaftis, Vasiliki; Zaw, Thiri; Song, Xiaomin; Molloy, Mark P; Monagle, Paul; Ignjatovic, Vera

    2017-05-01

    Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Specification of Transplantable Astroglial Subtypes from Human Pluripotent Stem Cells

    Science.gov (United States)

    Krencik, Robert; Weick, Jason P.; Liu, Yan; Zhang, Zhijian; Zhang, Su-Chun

    2011-01-01

    Functionally diversified neuronal populations have now been efficiently generated from human pluripotent stem cells (hPSCs). However, directed differentiation of hPSCs to functional astroglial subtypes remains elusive. In this study, hPSCs were successfully directed to nearly uniform populations of immature astrocytes in large quantities (>90% S100β+ and GFAP+). The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels following transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that carry distinct homeodomain transcription factors and display phenotypic differences. These human astroglial progenitors and immature astrocytes will be instrumental for studying astrocytes in brain development and function, for revealing their roles in disease processes, and for developing novel treatments for neurological disorders. PMID:21602806

  2. Exploring the contextual sensitivity of factors that determine cell-to-cell variability in receptor-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Suzanne Gaudet

    Full Text Available Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions.

  3. Reporting of sex as a variable in cardiovascular studies using cultured cells

    Directory of Open Access Journals (Sweden)

    Taylor K

    2011-11-01

    Full Text Available Abstract Background Chromosomal complement, including that provided by the sex chromosomes, influences expression of proteins and molecular signaling in every cell. However, less than 50% of the scientific studies published in 2009 using experimental animals reported sex as a biological variable. Because every cell has a sex, we conducted a literature review to determine the extent to which sex is reported as a variable in cardiovascular studies on cultured cells. Methods Articles from 10 cardiovascular journals with high impact factors (Circulation, J Am Coll Cardiol, Eur Heart J, Circ Res, Arterioscler Thromb Vasc Biol, Cardiovasc Res, J Mol Cell Cardiol, Am J Physiol Heart Circ Physiol, J Heart Lung Transplant and J Cardiovasc Pharmacol and published in 2010 were searched using terms 'cultured' and 'cells' in any order to determine if the sex of those cells was reported. Studies using established cell lines were excluded. Results Using two separate search strategies, we found that only 25 of 90 articles (28% and 20 of 101 articles (19.8% reported the sex of cells. Of those reporting the sex of cells, most (68.9%; n = 31 used only male cells and none used exclusively female cells. In studies reporting the sex of cells of cardiovascular origin, 40% used vascular smooth-muscle cells, and 30% used stem/progenitor cells. In studies using cells of human origin, 35% did not report the sex of those cells. None of the studies using neonatal cardiac myocytes reported the sex of those cells. Conclusions The complement of sex chromosomes in cells studied in culture has the potential to affect expression of proteins and 'mechanistic' signaling pathways. Therefore, consistent with scientific excellence, editorial policies should require reporting sex of cells used in in vitro experiments.

  4. How did variable NK-cell receptors and MHC class I ligands influence immunity, reproduction and human evolution?

    Science.gov (United States)

    Parham, Peter; Moffett, Ashley

    2014-01-01

    Preface Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, where they have progressively co-evolved with MHC class I molecules. The emergence of MHC-C in hominids drove the evolution of a system of MHC-C receptors that is most elaborate in chimpanzees. In contrast, the human system appears to have been subject to different and competing selection pressures that have acted on its immunological and reproductive functions. We suggest that this compromise facilitated development of the bigger brains that enabled archaic and modern humans to migrate out-of-Africa and populate other continents. PMID:23334245

  5. Innate cell communication kick-starts pathogen-specific immunity

    Science.gov (United States)

    Rivera, Amariliz; Siracusa, Mark C.; Yap, George S.; Gause, William C.

    2016-01-01

    Innate cells are responsible for the rapid recognition of infection and mediate essential mechanisms of pathogen elimination, and also facilitate adaptive immune responses. We review here the numerous intricate interactions among innate cells that initiate protective immunity. The efficient eradication of pathogens depends on the coordinated actions of multiple cells, including innate cells and epithelial cells. Rather than acting as isolated effector cells, innate cells are in constant communication with other responding cells of the immune system, locally and distally. These interactions are critically important for the efficient control of primary infections as well for the development of ‘trained’ innate cells that facilitate the rapid elimination of homologous or heterologous infections. PMID:27002843

  6. Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells.

    Science.gov (United States)

    Fish, Jennifer L; Kosodo, Yoichi; Enard, Wolfgang; Pääbo, Svante; Huttner, Wieland B

    2006-07-05

    The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.

  7. Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

    Science.gov (United States)

    Ruijtenberg, Suzan; van den Heuvel, Sander

    2016-01-01

    ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

  8. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

    Science.gov (United States)

    Ball, David A.

    2016-01-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947

  9. Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

    Science.gov (United States)

    Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

    2017-11-10

    Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the

  10. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  11. Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system.

    Science.gov (United States)

    Jakka, Gopinadh; Schuberth, Petra C; Thiel, Markus; Held, Gerhard; Stenner, Frank; Van Den Broek, Maries; Renner, Christoph; Mischo, Axel; Petrausch, Ulf

    2013-10-01

    Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8(+) T-cells. The anti-idiotypic Fab antibody A4 with specificity for HLA-A 0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A 0201/NY-ESO-1157-165 tetramer. HLA-A 0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A 0201/NY-ESO-1157-165-positive multiple myeloma cells. A4 demonstrated antigen-specific binding to HLA-A 0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A 0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. A4 expanded HLA-A 0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function.

  12. Cell type-specific responses to salinity - the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum.

    Science.gov (United States)

    Oh, Dong-Ha; Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar; Lee, Sang-Yeol; Bohnert, Hans J; Dassanayake, Maheshi

    2015-08-01

    Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms. No claim to original US government works. New Phytologist © 2015 New Phytologist Trust.

  13. Cell type specific alterations in interchromosomal networks across the cell cycle.

    Directory of Open Access Journals (Sweden)

    Andrew J Fritz

    2014-10-01

    Full Text Available The interchromosomal organization of a subset of human chromosomes (#1, 4, 11, 12, 16, 17, and 18 was examined in G1 and S phase of human WI38 lung fibroblast and MCF10A breast epithelial cells. Radial positioning of the chromosome territories (CTs was independent of gene density, but size dependent. While no changes in radial positioning during the cell cycle were detected, there were stage-specific differences between cell types. Each CT was in close proximity (interaction with a similar number of other CT except the gene rich CT17 which had significantly more interactions. Furthermore, CT17 was a member of the highest pairwise CT combinations with multiple interactions. Major differences were detected in the pairwise interaction profiles of MCF10A versus WI38 including cell cycle alterations from G1 to S. These alterations in interaction profiles were subdivided into five types: overall increase, overall decrease, switching from 1 to ≥2 interactions, vice versa, or no change. A global data mining program termed the chromatic median determined the most probable overall association network for the entire subset of CT. This probabilistic interchromosomal network was nearly completely different between the two cell lines. It was also strikingly altered across the cell cycle in MCF10A, but only slightly in WI38. We conclude that CT undergo multiple and preferred interactions with other CT in the nucleus and form preferred -albeit probabilistic- interchromosomal networks. This network of interactions is altered across the cell cycle and between cell types. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program across the cell cycle and in different cell types.

  14. Phloem differentiation: An integrative model for cell specification

    OpenAIRE

    Blob, Bernhard; Heo, Jung-ok; Helariutta, Yrjo Eero

    2018-01-01

    Plant vasculature consists of two major conductive cell types, xylem tracheary elements and phloem sieve elements (SEs). Both cell types undergo a highly specialized differentiation process. The root meristem of Arabidopsis displays a stereotypical anatomy in which the central vasculature is surrounded by concentric layers of outer tissues. Each cell file is derived from stem cells located in the root tip. A series of formative and proliferative divisions take place in the meristem; these are...

  15. Tissue-specific designs of stem cell hierarchies

    NARCIS (Netherlands)

    Visvader, Jane E; Clevers, Hans

    Recent work in the field of stem cell biology suggests that there is no single design for an adult tissue stem cell hierarchy, and that different tissues employ distinct strategies to meet their self-renewal and repair requirements. Stem cells may be multipotent or unipotent, and can exist in

  16. Cell specificity of the cytoplasmic Ca2+ response to tolbutamide is impaired in beta-cells from hyperglycemic mice

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Larsson-Nyrén, Gerd; Lindström, Per

    2006-01-01

    -cells and if so, whether it is disturbed in beta-cells from hyperglycemic ob/ob and db/db mice. Zn(2+) outflow measurements were used to study the correlation between Ca(2+) response and insulin secretion in individual beta-cells. Tolbutamide and arginine induced cell-specific Ca(2+) responses in lean mouse beta...... of Ca(2+) response was cell-specific, not the timing. The lag time of tolbutamide-induced insulin secretion was cell-specific in lean mouse beta-cells but not in ob/ob mouse cells. Therefore, cell specificity seems to be a robust mechanism, and probably important for an adequate beta-cell function...

  17. Phage exposure causes dynamic shifts in the expression states of specific phase-variable genes of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Aidley, Jack; Holst Sørensen, Martine C.; Bayliss, Christopher D.

    2017-01-01

    Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacter jejuni this reversible adaptive process is mediated by mutations in homop......Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacter jejuni this reversible adaptive process is mediated by mutations...... in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase...

  18. Specific suppression elicited by EL4 lymphoma cells in syngeneic mice. Specificity includes self-antigens on EL4.

    Science.gov (United States)

    Grooten, J; Leroux-Roels, G; Fiers, W

    1987-05-01

    In vivo, subclones derived from EL4 lymphoma cells generate suppressor T lymphocytes specific for anti-EL4 immune responses. Spleen cells of EL4-sensitized C57BL/6 mice down-regulate the in vitro induction of EL4-specific cytolytic T lymphocytes (CTL). In addition, EL4-sensitized spleen cells interfere with the antigen response of two T lymphocyte clones. These recognize, in an H-2b context, a self-antigen on spleen cells that is also expressed by transformed cells, including EL4. The simultaneous anti-self and anti-EL4 specificity of the helper and suppressor activities suggests, therefore, that they are the product of an in vivo autoimmune reaction to EL4. The anti-self suppression might aim to re-establish self-tolerance, at the same time down-regulating responses against immunogenic epitopes that are co-expressed with the self-antigen on the EL4 cells. This agrees well with our observation that suppressor T cells, apparently elicited by suppressogenic epitopes on non-immunogenic EL4 subclones, down-regulate the CTL response elicited by immunogenic EL4 subclones. The additional self-specificity of this suppression indicates that the suppressogenic epitopes at least in part represent EL4 self-antigens.

  19. Colony-specific investigations reveal highly variable responses among individual corals to ocean acidification and warming.

    Science.gov (United States)

    Kavousi, Javid; Reimer, James Davis; Tanaka, Yasuaki; Nakamura, Takashi

    2015-08-01

    As anthropogenic climate change is an ongoing concern, scientific investigations on its impacts on coral reefs are increasing. Although impacts of combined ocean acidification (OA) and temperature stress (T) on reef-building scleractinian corals have been studied at the genus, species and population levels, there are little data available on how individual corals respond to combined OA and anomalous temperatures. In this study, we exposed individual colonies of Acropora digitifera, Montipora digitata and Porites cylindrica to four pCO2-temperature treatments including 400 μatm-28 °C, 400 μatm-31 °C, 1000 μatm-28 °C and 1000 μatm-31 °C for 26 days. Physiological parameters including calcification, protein content, maximum photosynthetic efficiency, Symbiodinium density, and chlorophyll content along with Symbiodinium type of each colony were examined. Along with intercolonial responses, responses of individual colonies versus pooled data to the treatments were investigated. The main results were: 1) responses to either OA or T or their combination were different between individual colonies when considering physiological functions; 2) tolerance to either OA or T was not synonymous with tolerance to the other parameter; 3) tolerance to both OA and T did not necessarily lead to tolerance of OA and T combined (OAT) at the same time; 4) OAT had negative, positive or no impacts on physiological functions of coral colonies; and 5) pooled data were not representative of responses of all individual colonies. Indeed, the pooled data obscured actual responses of individual colonies or presented a response that was not observed in any individual. From the results of this study we recommend improving experimental designs of studies investigating physiological responses of corals to climate change by complementing them with colony-specific examinations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Cell-Specific Cre Strains For Genetic Manipulation in Salivary Glands.

    Directory of Open Access Journals (Sweden)

    Eri O Maruyama

    Full Text Available The secretory acinar cells of the salivary gland are essential for saliva secretion, but are also the cell type preferentially lost following radiation treatment for head and neck cancer. The source of replacement acinar cells is currently a matter of debate. There is evidence for the presence of adult stem cells located within specific ductal regions of the salivary glands, but our laboratory recently demonstrated that differentiated acinar cells are maintained without significant stem cell contribution. To enable further investigation of salivary gland cell lineages and their origins, we generated three cell-specific Cre driver mouse strains. For genetic manipulation in acinar cells, an inducible Cre recombinase (Cre-ER was targeted to the prolactin-induced protein (Pip gene locus. Targeting of the Dcpp1 gene, encoding demilune cell and parotid protein, labels intercalated duct cells, a putative site of salivary gland stem cells, and serous demilune cells of the sublingual gland. Duct cell-specific Cre expression was attempted by targeting the inducible Cre to the Tcfcp2l1 gene locus. Using the R26Tomato Red reporter mouse, we demonstrate that these strains direct inducible, cell-specific expression. Genetic tracing of acinar cells using PipGCE supports the recent finding that differentiated acinar cells clonally expand. Moreover, tracing of intercalated duct cells expressing DcppGCE confirms evidence of duct cell proliferation, but further analysis is required to establish that renewal of secretory acinar cells is dependent on stem cells within these ducts.

  1. High specific power, direct methanol fuel cell stack

    Science.gov (United States)

    Ramsey, John C [Los Alamos, NM; Wilson, Mahlon S [Los Alamos, NM

    2007-05-08

    The present invention is a fuel cell stack including at least one direct methanol fuel cell. A cathode manifold is used to convey ambient air to each fuel cell, and an anode manifold is used to convey liquid methanol fuel to each fuel cell. Tie-bolt penetrations and tie-bolts are spaced evenly around the perimeter to hold the fuel cell stack together. Each fuel cell uses two graphite-based plates. One plate includes a cathode active area that is defined by serpentine channels connecting the inlet manifold with an integral flow restrictor to the outlet manifold. The other plate includes an anode active area defined by serpentine channels connecting the inlet and outlet of the anode manifold. Located between the two plates is the fuel cell active region.

  2. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

    OpenAIRE

    Dubois, Nicole C.; Craft, April M; Sharma, Parveen; Elliott, David A.; Stanley, Edouard G.; Elefanty, Andrew G.; Gramolini, Anthony; Keller, Gordon

    2011-01-01

    To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody...

  3. Characterization of structural variability sheds light on the specificity determinants of the interaction between effector domains and histone tails.

    Science.gov (United States)

    Lois, Sergi; Akizu, Naiara; de Xaxars, Gemma Mas; Vázquez, Iago; Martínez-Balbás, Marian; de la Cruz, Xavier

    2010-02-16

    Structural characterization of the interaction between histone tails and effector modules (bromodomains, chromodomains, PHD fingers, etc.) is fundamental to understand the mechanistic aspects of epigenetic regulation of gene expression. In recent years many researchers have applied this approach to specific systems, thus providing a valuable but fragmentary view of the histone-effector interaction. In our work we use this information to characterize the structural features of the two main components of this interaction, histone peptides and the binding site of effector domains (focusing on those which target modified lysines), and increase our knowledge on its specificity determinants. Our results show that the binding sites of effectors are structurally variable, but some clear trends allow their classification in three main groups: flat-groove, narrow-groove and cavity-insertion. In addition, we found that even within these classes binding site variability is substantial. These results in context with the work from other researchers indicate that the there are at least two determinants of binding specificity in the binding site of effector modules. Finally, our analysis of the histone peptides sheds light on the structural transition experienced by histone tails upon effector binding, showing that it may vary depending on the local properties of the sequence stretch considered, thus allowing us to identify an additional specificity determinant for this interaction. Overall, the results of our analysis contribute to clarify the origins of specificity: different regions of the binding site and, in particular, differences in the disorder-order transitions experienced by different histone sequence stretches upon binding.

  4. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    Science.gov (United States)

    Sinang, S. C.; Reichwaldt, E. S.; Ghadouani, A.

    2015-05-01

    Toxic cyanobacterial blooms in urban lakes present serious health hazards to humans and animals and require effective management strategies. Managing such blooms requires a sufficient understanding of the controlling environmental factors. A range of them has been proposed in the literature as potential triggers for cyanobacterial biomass development and cyanotoxin (e.g. microcystin) production in freshwater systems. However, the environmental triggers of cyanobacteria and microcystin variability remain a subject of debate due to contrasting findings. This issue has raised the question of whether the relevance of environmental triggers may depend on site-specific combinations of environmental factors. In this study, we investigated the site-specificity of environmental triggers for cyanobacterial bloom and microcystin dynamics in three urban lakes in Western Australia. Our study suggests that cyanobacterial biomass, cyanobacterial dominance and cyanobacterial microcystin content variability were significantly correlated to phosphorus and iron concentrations. However, the correlations were different between lakes, thus suggesting a site-specific effect of these environmental factors. The discrepancies in the correlations could be explained by differences in local nutrient concentration. For instance, we found no correlation between cyanobacterial fraction and total phosphorous (TP) in the lake with the highest TP concentration, while correlations were significant and negative in the other two lakes. In addition, our study indicates that the difference of the correlation between total iron (TFe) and the cyanobacterial fraction between lakes might have been a consequence of differences in the cyanobacterial community structure, specifically the presence or absence of nitrogen-fixing species. In conclusion, our study suggests that identification of significant environmental factors under site-specific conditions is an important strategy to enhance successful outcomes

  5. Spontaneous presence of FOXO3-specific T cells in cancer patients

    DEFF Research Database (Denmark)

    Larsen, Stine Kiaer; Ahmad, Shamaila Munir; Idorn, Manja

    2014-01-01

    ) were able to specifically recognize and kill both FOXO3-expressing cancer cells as well as dendritic cells. Thus, FOXO3 was processed and presented by HLA-A2 on the cell surface of both immune cells and cancer cells. As FOXO3 programs TADCs to become tolerogenic, FOXO3 signaling thereby comprises...... a significant immunosuppressive mechanism, such that FOXO3 targeting by means of specific T cells is an attractive clinical therapy to boost anticancer immunity. In addition, the natural occurrence of FOXO3-specific CTLs in the periphery suggests that these T cells hold a function in the complex network...... of immune regulation in cancer patients....

  6. β-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development.

    Directory of Open Access Journals (Sweden)

    Ahmed M Abdellatif

    Full Text Available The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.

  7. Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.

    Directory of Open Access Journals (Sweden)

    Ekaterina Y Lukianova-Hleb

    Full Text Available The limited specificity of nanoparticle (NP uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

  8. Population of computational rabbit-specific ventricular action potential models for investigating sources of variability in cellular repolarisation.

    Science.gov (United States)

    Gemmell, Philip; Burrage, Kevin; Rodriguez, Blanca; Quinn, T Alexander

    2014-01-01

    Variability is observed at all levels of cardiac electrophysiology. Yet, the underlying causes and importance of this variability are generally unknown, and difficult to investigate with current experimental techniques. The aim of the present study was to generate populations of computational ventricular action potential models that reproduce experimentally observed intercellular variability of repolarisation (represented by action potential duration) and to identify its potential causes. A systematic exploration of the effects of simultaneously varying the magnitude of six transmembrane current conductances (transient outward, rapid and slow delayed rectifier K(+), inward rectifying K(+), L-type Ca(2+), and Na(+)/K(+) pump currents) in two rabbit-specific ventricular action potential models (Shannon et al. and Mahajan et al.) at multiple cycle lengths (400, 600, 1,000 ms) was performed. This was accomplished with distributed computing software specialised for multi-dimensional parameter sweeps and grid execution. An initial population of 15,625 parameter sets was generated for both models at each cycle length. Action potential durations of these populations were compared to experimentally derived ranges for rabbit ventricular myocytes. 1,352 parameter sets for the Shannon model and 779 parameter sets for the Mahajan model yielded action potential duration within the experimental range, demonstrating that a wide array of ionic conductance values can be used to simulate a physiological rabbit ventricular action potential. Furthermore, by using clutter-based dimension reordering, a technique that allows visualisation of multi-dimensional spaces in two dimensions, the interaction of current conductances and their relative importance to the ventricular action potential at different cycle lengths were revealed. Overall, this work represents an important step towards a better understanding of the role that variability in current conductances may play in experimentally

  9. Phloem differentiation: an integrative model for cell specification.

    Science.gov (United States)

    Blob, Bernhard; Heo, Jung-Ok; Helariutta, Yka

    2018-01-01

    Plant vasculature consists of two major conductive cell types, xylem tracheary elements and phloem sieve elements (SEs). Both cell types undergo a highly specialized differentiation process. The root meristem of Arabidopsis displays a stereotypical anatomy in which the central vasculature is surrounded by concentric layers of outer tissues. Each cell file is derived from stem cells located in the root tip. A series of formative and proliferative divisions take place in the meristem; these are followed by cell expansion and differentiation. Protophloem differentiation is unique in being complete only 20-25 cells away from the first stem cell, and during the differentiation process the cells lose several organelles, including the nucleus, while the remaining organelles are rearranged. Defects in SE development have been shown to result in impaired auxin transport and response and therefore systemically affect root growth. Although a few genes have been demonstrated to function in phloem development, detailed analyses and a comprehensive understanding of sieve element development (i.e. how often the stem cells divide, how frequently enucleation takes place, and how SE development is coordinated between cell division and differentiation on a molecular level) are still lacking. Advanced live-imaging techniques which enable prolonged time-lapse captures of root tip growth as well as single-cell transcriptomic analysis of the 20-25 cells in the SE file could help resolve these questions. In addition, understanding the interplay between the PLETHORA (PLT) gradient, which is known to govern the root zonation, and phloem development within the root meristem could shed light on the rapidity of SE differentiation and its importance to the meristem.

  10. Improving Cytomegalovirus-Specific T Cell Reconstitution after Haploidentical Stem Cell Transplantation

    Science.gov (United States)

    Chang, Ying-Jun; Huang, Xiao-Jun

    2014-01-01

    Cytomegalovirus (CMV) infection and delayed immune reconstitution (IR) remain serious obstacles for successful haploidentical stem cell transplantation (haplo-SCT). CMV-specific IR varied according to whether patients received manipulated/unmanipulated grafts or myeloablative/reduced intensity conditioning. CMV infection commonly occurs following impaired IR of T cell and its subsets. Here, we discuss the factors that influence IR based on currently available evidence. Adoptive transfer of donor T cells to improve CMV-specific IR is discussed. One should choose grafts from CMV-positive donors for transplant into CMV-positive recipients (D+/R+) because this will result in better IR than would grafts from CMV-negative donors (D−/R+). Stem cell source and donor age are other important factors. Posttransplant complications, including graft-versus-host disease and CMV infection, as well as their associated treatments, should also be considered. The effects of varying degrees of HLA disparity and conditioning regimens are more controversial. As many of these factors and strategies are considered in the setting of haplo-SCT, it is anticipated that haplo-SCT will continue to advance, further expanding our understanding of IR and CMV infection. PMID:24864269

  11. Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues.

    Science.gov (United States)

    Laitinen, L

    1987-04-01

    The binding of Griffonia simplicifolia agglutinin-I (GSA-I) and the isolectins GSA-I-AB3 and GSA-I-B4, having affinity for some alpha-D-galactosyl and N-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver and kidney cortex the GSA-I-conjugates selectively reacted with endothelial cells of the sinusoids and with intertubular and glomerular capillaries, respectively. However, a strong reactivity with the GSA-I-conjugates was additionally seen in the acinar cells of the pancreas, in the stratified squamous epithelia of skin and tongue, and in transitional epithelium. SDS-PAGE electrophoresis combined with the lectin-blotting technique indicated that a similar set of glycoproteins are responsible for the GSA-I binding, even in different tissues. Another lectin with specificity for alpha-D-galactose, the Maclura pomifera agglutinin, displayed a distinctly different distribution of binding sites, mainly in the basement membranes, of all mouse tissues studied. The results suggest that some alpha-D-galactosyl residues, recognized by the binding of GSA-I lectins, are preferentially expressed in endothelial cells of mouse tissues, and also provide further evidence that endothelial cells can present a highly specific surface glycosylation pattern.

  12. Single cell adhesion strength assessed with variable-angle total internal reflection fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Marcelina Cardoso Dos Santos

    2017-06-01

    Full Text Available We propose a new strategy to evaluate adhesion strength at the single cell level. This approach involves variable-angle total internal reflection fluorescence microscopy to monitor in real time the topography of cell membranes, i.e. a map of the membrane/substrate separation distance. According to the Boltzmann distribution, both potential energy profile and dissociation energy related to the interactions between the cell membrane and the substrate were determined from the membrane topography. We have highlighted on glass substrates coated with poly-L-lysine and fibronectin, that the dissociation energy is a reliable parameter to quantify the adhesion strength of MDA-MB-231 motile cells.

  13. Study of process variables associated with manufacturing hermetically-sealed nickel-cadmium cells

    Science.gov (United States)

    Miller, L.; Doan, D. J.; Carr, E. S.

    1971-01-01

    A program to determine and study the critical process variables associated with the manufacture of aerospace, hermetically-sealed, nickel-cadmium cells is described. The determination and study of the process variables associated with the positive and negative plaque impregnation/polarization process are emphasized. The experimental data resulting from the implementation of fractional factorial design experiments are analyzed by means of a linear multiple regression analysis technique. This analysis permits the selection of preferred levels for certain process variables to achieve desirable impregnated plaque characteristics.

  14. Allergen-specific Th2 cells as targets for immune intervention in allergic disease

    Directory of Open Access Journals (Sweden)

    Jan E. de Vries

    1996-01-01

    Finally, it is shown that IL-4-driven allergen-specific Th2 cell differentiation can be redirected into a Th0 and Thl cell differentiation pathway by stimulating these IL-4-driven allergen-specific Th cell populations in the presence of IL-12, or by co-stimulating these cells via a novel T cell receptor, designated signalling lymphocyte activation molecule (SLAM. The clinical implications of these approaches are discussed.

  15. Prestalk and prespore differentiation in Dictyostelium as detected by cell type-specific monoclonal antibodies

    OpenAIRE

    Tasaka, Masao; Noce, Toshiaki; Takeuchi, Ikuo

    1983-01-01

    Monoclonal antibodies specifically reactive against prestalk and prespore cells of the cellular slime mold Dictyostelium discoideum were obtained. By the use of these antibodies, we examined processes of differentiation of the two cell types during development. Cells stained with prespore-specific antibodies first appeared after 12-14 hr of starvation within cell aggregates with tips, coincidentally with the appearance of other prespore markers. The number of prespore cells then increased to ...

  16. Identification of α-fetoprotein-specific T cell receptors for hepatocellular carcinoma immunotherapy.

    Science.gov (United States)

    Zhu, Wei; Peng, Yibing; Wang, Lan; Hong, Yuan; Jiang, Xiaotao; Li, Qi; Liu, Heping; Huang, Lei; Wu, Juan; Celis, Esteban; Merchen, Todd; Kruse, Edward; He, Yukai

    2018-02-14

    Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T cell receptors (TCR) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, by using lentivector and peptide immunization, we identified a population of CD8-T cells in HLA-A2 transgenic AAD mice that recognized AFP 158 epitope on human HCC cells. Adoptive transfer of the AFP 158 -specific mouse CD8-T cells eradicated HepG2 tumor xenografts as large as 2cm in diameter in immunocompromised NSG mice. We then established T cell hybridoma clones from the AFP 158 -specific mouse CD8-T cells and identified three sets of paired TCR genes out of 5 hybridomas. Expression of the murine TCR genes redirected primary human T cells to bind HLA-A2/AFP 158 tetramer. The TCR gene-engineered human T cells (TCR-T) also specifically recognized HLA-A2 + AFP + HepG2 HCC tumor cells and produced effector cytokines. Importantly, the TCR-T cells could specifically kill HLA-A2 + AFP + HepG2 tumor cells without significant toxicity to normal primary hepatocytes in vitro. Adoptive transfer of the AFP-specific human TCR-T cells could eradicate HepG2 tumors in NSG mice. We have identified novel AFP-specific murine TCR genes that can redirect human T cells to specifically recognize and kill HCC tumor cells, and those AFP 158 -specific TCRs have a great potential to engineer a patient's autologous T cells to treat HCC tumors. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  17. Mirror-drawing skill in children with specific language impairment: Improving generalization by incorporating variability into the practice session.

    Science.gov (United States)

    Desmottes, Lise; Maillart, Christelle; Meulemans, Thierry

    2017-05-01

    This study aimed to investigate the procedural deficit hypothesis (PDH) in children with specific language impairment (SLI) by using a mirror-drawing task, a sensorimotor adaptation paradigm that does not involve sequence learning and has never before been used in SLI. A total of 30 school-aged children with SLI matched to 30 typically developing (TD) control children had to trace several figures seen only in mirror-reversed view in two practice sessions separated by a one-week interval. Two practice conditions were compared: a constant condition in which children had to trace the same figure throughout the learning trials, and a variable one in which they had to trace different figures in each trial. The results revealed a similar learning pattern between SLI and TD children in both practice conditions, suggesting that initial learning for a non-sequential procedural task is preserved in SLI. However, the children with SLI generalized the mirror-drawing skill in the same way as the TD children only if there was variability in the way the material was trained (variable practice). No significant schedule effects were observed in the control group.

  18. High specific energy Lithium Sulfur cell for space application

    Directory of Open Access Journals (Sweden)

    Samaniego Bruno

    2017-01-01

    Airbus DS has been testing and characterizing prototype Li-S cells manufactured by OXIS Energy Ltd. since 2014, demonstrating the potential and fast evolution of the cells performance. This paper presents the last test results on a set of different batches provided by OXIS and performed at Airbus DS premises in the frame of an ESA Innovation Triangle Initiative (ITI.

  19. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    Science.gov (United States)

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  20. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    Directory of Open Access Journals (Sweden)

    Mohammad Soltani

    2016-08-01

    Full Text Available Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i stochastic expression; ii partitioning errors at the time of cell division and iii random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  1. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    Science.gov (United States)

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  2. Cytotoxic T lymphocytes specific for self tumor immunoglobulin express T cell receptor delta chain.

    Science.gov (United States)

    Wright, A; Lee, J E; Link, M P; Smith, S D; Carroll, W; Levy, R; Clayberger, C; Krensky, A M

    1989-05-01

    CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy.

  3. Dissection of the functional structure of aptamer17, which specifically recognizes differentiated PC12 cells.

    Science.gov (United States)

    Liu, Jiao-Jiao; Wang, Cheng-Long; Xi, Qing; Xu, Juan; Deng, Bin; Ding, Hong-Mei; Chu, Bingfeng; Su, Dong-Hua

    2011-06-01

    A specific single-stranded DNA (ssDNA) aptamer (aptamer17) that specifically recognizes differentiated PC12 cells had been previously obtained after 6 rounds of whole cell-based subtractive systematic evolution of ligands by exponential enrichment selection from a random ssDNA library. To further investigate the relationship between the structure and function of this aptamer, 3 truncated ssDNA aptamers were designed according to the predicted secondary structure of aptamer17. Our results show that the stem-loop is the core structure of the aptamers required for specific binding to differentiated PC12 cells, specifically loops I and II. Aptamer17 and the truncated aptamers with this basic structure could bind specifically to differentiated PC12 cells and identify these cells from a mixture of differentiated and undifferentiated PC12 cells. Therefore, truncated forms of aptamer17 may be useful in the clinic to identify undifferentiated and differentiated PC12 cells from a mixture of cells.

  4. Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

    Science.gov (United States)

    Montanucci, Pia; Terenzi, Silvia; Pennoni, Ilaria; Basta, Giuseppe; Calafiore, Riccardo

    2015-01-01

    Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation, in vitro and in vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules' basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others. PMID:26078974

  5. Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Pia Montanucci

    2015-01-01

    Full Text Available Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation, in vitro and in vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

  6. Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid

    Directory of Open Access Journals (Sweden)

    Christopher J. Petzold

    2013-10-01

    Full Text Available Cell density is the critical parameter controlling tendon morphogenesis. Knowing its neighbors allows a cell to regulate correctly its proliferation and collagen production. A missing link to understanding this process is a molecular description of the sensing mechanism. Previously, this mechanism was shown in cell culture to rely on a diffusible factor (SNZR [sensor] with an affinity for the cell layer. This led to purifying conditioned medium over 4 columns and analyzing the final column fractions for band intensity on SDS gels versus biological activity – a 16 kD band strongly correlated between assays. N-terminal sequencing – EPLAVVDL – identified a large gene (424 AA, extremely conserved between chicken and human. In this paper we probe whether this is the correct gene. Can the predicted large protein be cleaved to a smaller protein? EPLAVVDL occurs towards the C-terminus and cleavage would create a small 94 AA protein. This protein would run at ∼10 kD, so what modifications or cofactor binding accounts for its running at 16 kD on SDS gels? This protein has no prominent hydrophobic regions, so can it be secreted? To validate its role, the chicken cDNA for this gene was tagged with myc and his and transfected into a human osteosarcoma cell line (U2OS. U2OS cells expressed the gene but not passively: differentiating into structures resembling spongy bone and expressing alkaline phosphatase, an early bone marker. Intracellularly, two bands were observed by Western blotting: the full length protein and a smaller form (26 kD. Outside the cell, a small band (28 kD was detected, although it was 40% larger than expected, as well as multiple larger bands. These larger forms could be converted to the predicted smaller protein (94 AA + tags by changing salt concentrations and ultrafiltering – releasing a cofactor to the filtrate while leaving a protein factor in the retentate. Using specific degradative enzymes and mass spectrometry, the

  7. Fatigue in patients with ankylosing spondylitis: prevalence and relationships with disease-specific variables, psychological status, and sleep disturbance.

    Science.gov (United States)

    Aissaoui, N; Rostom, S; Hakkou, J; Berrada Ghziouel, K; Bahiri, R; Abouqal, R; Hajjaj-Hassouni, N

    2012-07-01

    This study aims to evaluate the frequency of fatigue in Moroccan patients with ankylosing spondylitis (AS), and its relationships with disease-specific variables, psychological status, and sleep disturbance. A cross-sectional study included patients fulfilled the modified New York classification criteria for ankylosing spondylitis. To assess fatigue, the first item of Bath ankylosing spondylitis disease activity index (BASDAI) and the multidimensional assessment of fatigue (MAF) was used. The evaluation included the activity of the disease (BASDAI), global well-being (Bath ankylosing spondylitis global index), functional status (Bath ankylosing spondylitis functional index), metrologic measurements (Bath ankylosing spondylitis metrological index), and visual analog scale of axial or joint pain. The erythrocyte sedimentation rate and C-reactive protein were measured. To assess psychological status, the hospital anxiety and depression scale (HADS) was used. Sleep disturbance was assessed by the fourth item of Hamilton anxiety scale. One hundred and ten patients were included, of average age 38.0 years ± 12.6. In our data, 66.4% experienced severe fatigue (BASDAI fatigue ≥ 5). The mean total score of MAF was 26 ± 12.77. The disease-specific variables contributed significantly with both BASDAI fatigue and MAF as dependent variables, accounting for 71.3 and 65.6% of the variance, respectively. The contribution of the depression, anxiety, and sleep disturbance were 24.9, 18.4 and 15.4%, respectively. This study state the importance of fatigue in AS patients. Even though disease activity was the most powerful predictor of fatigue, the effects of psychogenic factors and sleep disturbance, should be taken into consideration in the management of AS.

  8. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  9. The Diversity of Nanos Expression in Echinoderm Embryos Supports Different Mechanisms in Germ Cell Specification

    Science.gov (United States)

    Fresques, Tara; Swartz, S. Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M.

    2016-01-01

    Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32–128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. PMID:27402572

  10. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells

    Science.gov (United States)

    Hepworth, Matthew R.; Fung, Thomas C.; Masur, Samuel H.; Kelsen, Judith R.; McConnell, Fiona M.; Dubrot, Juan; Withers, David R.; Hugues, Stephanie; Farrar, Michael A.; Reith, Walter; Eberl, Gerard; Baldassano, Robert N.; Laufer, Terri M.; Elson, Charles O.; Sonnenberg, Gregory F.

    2015-01-01

    Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on human colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine, and suggest that this process is dysregulated in human IBD. PMID:25908663

  11. T-cell responses to allergens: epitope-specificity and clinical relevance

    NARCIS (Netherlands)

    van Neerven, R. J.; Ebner, C.; Yssel, H.; Kapsenberg, M. L.; Lamb, J. R.

    1996-01-01

    Allergen-specific T cells play an important role in the pathophysiology of atopic allergies. Recently, cDNAs that encode many important allergens have been cloned and their amino acid sequences deduced, thus allowing the elucidation of the epitope-specificity of allergen-specific T cells. Here,

  12. Minimization of CD34+ cell enumeration variability using the ProCOUNT standardized methodology.

    Science.gov (United States)

    McNiece, I; Kern, B; Zilm, K; Brunaud, C; Dziem, G; Briddell, R

    1998-12-01

    The dose of cells expressing the surface antigen CD34 (CD34+) has been shown to be a reliable predictor of the time to engraftment following transplantation of PBPC to support high-dose chemotherapy. However, evaluation of rare cells is complicated by a number of factors, including the variability in operator and technical procedures. Recently, Becton Dickinson Immunocytometry Systems introduced a new CD34+ cell analysis system, the ProCOUNT cell enumeration kit, which automates the analysis of CD34+ cells and minimizes the variabilities of this procedure. We have evaluated the ProCOUNT system in comparison to a standard CD34 cell analysis (based on the Milan approach) using leukapheresis products from patients and normal donors mobilized with chemotherapy plus recombinant human G-CSF (rhG-CSF) or with rhG-CSF alone. In addition, we compared these analyses using CD34+ cell-selected mobilized leukapheresis products with purities of 75% or greater. The standard CD34 cell analysis methodology quantitated the frequency of cells identified as CD45+, low side scatter, and CD34+. A high correlation coefficient was obtained between the ProCOUNT methodology and the standard CD34 cell analysis methodology for cells obtained from leukapheresis products mobilized with chemotherapy plus rhG-CSF (r = 0.98), rhG-CSF alone (r = 0.96), and CD34+-selected mobilized leukapheresis products (r = 0.83). A comparison was also made between technicians using both analysis methods. Whereas the correlation coefficient between two technicians using the standard methodology was r = 0.77, the correlation coefficient was much higher when using ProCOUNT (r = 0.99). These data demonstrate that the use of ProCOUNT is associated with less variability between data analyzed by different operators. Also, ProCOUNT is consistent with existing CD34+ cellular analysis methodologies. An additional advantage is the ability to determine the absolute concentration of CD34+ cells, thereby allowing calculation

  13. Specification of regional intestinal stem cell identity during Drosophila metamorphosis

    National Research Council Canada - National Science Library

    Driver, Ian; Ohlstein, Benjamin

    2014-01-01

    In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR...

  14. Aspm Specifically Maintains Symmetric Proliferative Divisions of Neuroepithelial Cells

    National Research Council Canada - National Science Library

    Jennifer L. Fish; Yoichi Kosodo; Wolfgang Enard; Svante Pääbo; Wieland B. Huttner

    2006-01-01

    The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been...

  15. Probing the effector and suppressive functions of human T cell subsets using antigen-specific engineered T cell receptors.

    Science.gov (United States)

    Wan, Qi; Kozhaya, Lina; Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

    2013-01-01

    Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8(+) and CD4(+) T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.

  16. Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate.

    Directory of Open Access Journals (Sweden)

    Karen G Dowell

    Full Text Available Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.

  17. Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate.

    Science.gov (United States)

    Dowell, Karen G; Simons, Allen K; Wang, Zack Z; Yun, Kyuson; Hibbs, Matthew A

    2013-01-01

    Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC) self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org) to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.

  18. Generation of polyclonal CMV-specific T cells for the adoptive immunotherapy of glioblastoma.

    Science.gov (United States)

    Ghazi, Alexia; Ashoori, Aidin; Hanley, Patrick J; Brawley, Vita S; Shaffer, Donald R; Kew, Yvonne; Powell, Suzanne Z; Grossman, Robert; Grada, Zakaria; Scheurer, Michael E; Hegde, Meenakshi; Leen, Ann M; Bollard, Catherine M; Rooney, Cliona M; Heslop, Helen E; Gottschalk, Stephen; Ahmed, Nabil

    2012-01-01

    Glioblastoma (GBM) is the most common primary brain cancer in adults and is virtually incurable. Recent studies have shown that cytomegalovirus (CMV) is present in majority of GBMs. To evaluate whether the CMV antigens pp65 and IE1, which are expressed in GBMs, could be targeted by CMV-specific T cells, we measured the frequency of T cells targeting pp65 and IE1 in the peripheral blood of a cohort of 11 sequentially diagnosed CMV-seropositive GBM patients, and evaluated whether it was feasible to expand autologous CMV-specific T cells for future clinical studies. All 11 CMV-seropositive GBM patients had T cells specific for pp65 and IE1 in their peripheral blood assessed by IFNγ enzyme-linked immunospot assay. However, the precursor frequency of pp65-specific T cells was decreased in comparison with healthy donors (P=0.001). We successfully reactivated and expanded CMV-specific T cells from 6 out of 6 GBM patients using antigen-presenting cells transduced with an adenoviral vector encoding pp65 and IE1. CMV-specific T-cell lines contained CD4 as well as CD8 T cells, recognized pp65 and IE1 targets and killed CMV-infected autologous GBM cells. Infusion of such CMV-specific T-cell lines may extend the benefits of T-cell therapy to patients with CMV GBMs.

  19. Inter- and intra-laboratory variability of CD4 cell counts in Swaziland

    African Journals Online (AJOL)

    2012-06-01

    Jun 1, 2012 ... variation between CD4 cell count results conducted using flow cytometers of different ages in service.7 ... quantified the inter- and intra-laboratory variability in CD4 count measurements. In addition, the possible ... South African laboratory commonly used as standard in clinical practice across southern.

  20. Cell type-specific neuroprotective activity of untranslocated prion protein.

    Directory of Open Access Journals (Sweden)

    Elena Restelli

    2010-10-01

    Full Text Available A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

  1. Monitoring of pathogen-specific T-cell immune reconstitution after allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Shigeo eFuji

    2013-09-01

    Full Text Available The clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT has been significantly improved during the last decades with regard to the reduction in organ failure, infection, and severe acute graft-versus-host disease (GVHD. However, severe complications due to infectious diseases are still one of the major causes of morbidity and mortality after allogeneic HSCT, in particular in patients receiving haploidentical HSCT or cord blood transplant due to a slow and often incomplete immune reconstitution. In order to improve the immune control of pathogens without an increased risk of alloreactivity, adoptive immunotherapy using highly enriched pathogen-specific T cells offers a promising approach. In order to identify patients who are at high risk for infectious diseases, several monitoring assays have been developed with potential for the guidance of immunosuppressive drugs and adoptive immunotherapy in clinical practice. In this article, we aim to give a comprehensive overview regarding current developments of T-cell monitoring techniques focusing on T cells against viruses and fungi. In particular, we will focus on rather simple, fast, non-labor-intensive, cellular assays which could be integrated in routine clinical screening approaches.

  2. Selection of a set of specific primers for the identification of Tuber rufum: a truffle species with high genetic variability.

    Science.gov (United States)

    Iotti, Mirco; Amicucci, Antonella; Bonito, Gregory; Bonuso, Enrico; Stocchi, Vilberto; Zambonelli, Alessandra

    2007-12-01

    Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.

  3. Specific aspects of cognitive and language proficiency account for variability in neural indices of semantic and syntactic processing in children.

    Science.gov (United States)

    Hampton Wray, Amanda; Weber-Fox, Christine

    2013-07-01

    The neural activity mediating language processing in young children is characterized by large individual variability that is likely related in part to individual strengths and weakness across various cognitive abilities. The current study addresses the following question: How does proficiency in specific cognitive and language functions impact neural indices mediating language processing in children? Thirty typically developing seven- and eight-year-olds were divided into high-normal and low-normal proficiency groups based on performance on nonverbal IQ, auditory word recall, and grammatical morphology tests. Event-related brain potentials (ERPs) were elicited by semantic anomalies and phrase structure violations in naturally spoken sentences. The proficiency for each of the specific cognitive and language tasks uniquely contributed to specific aspects (e.g., timing and/or resource allocation) of neural indices underlying semantic (N400) and syntactic (P600) processing. These results suggest that distinct aptitudes within broader domains of cognition and language, even within the normal range, influence the neural signatures of semantic and syntactic processing. Furthermore, the current findings have important implications for the design and interpretation of developmental studies of ERPs indexing language processing, and they highlight the need to take into account cognitive abilities both within and outside the classic language domain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Perforin and Fas in murine gammaherpesvirus-specific CD8(+) T cell control and morbidity

    DEFF Research Database (Denmark)

    Topham, D J; Cardin, R C; Christensen, Jan Pravsgaard

    2001-01-01

    The immune system uses both virus-specific T cells and B cells to control the acute and latent phases of respiratory infection with the murine gammaherpesvirus 68 (gammaHV-68). We sought to further define the important effector mechanisms for CD8(+) T cells. First, depletion of the CD4(+) T cells...

  5. Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

    NARCIS (Netherlands)

    Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

    1985-01-01

    T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

  6. Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate

    Science.gov (United States)

    Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell ...

  7. Influenza virus and endothelial cells: A species specific relationship

    NARCIS (Netherlands)

    K.R. Short (Kirsty); E.J.B. Veldhuis Kroeze (Edwin); L.A. Reperant (Leslie); M. Richard (Mathilde); T. Kuiken (Thijs)

    2014-01-01

    textabstractInfluenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target

  8. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.

    2009-01-01

    Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous...

  9. Cardiomyocyte expression and cell-specific processing of procholecystokinin

    DEFF Research Database (Denmark)

    Gøtze, Jens P.; Johnsen, Anders H.; Kistorp, Caroline

    2015-01-01

    Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptide...

  10. Targeting inflammation with autoantigen-specific T cells

    NARCIS (Netherlands)

    Guichelaar, T.

    2008-01-01

    Chronic autoimmune diseases are driven by cells that respond to tissue components of the body. Inflammation in diseases like rheumatoid arthritis, diabetes or multiple sclerosis, can be suppressed by drug therapy. However, the broad range of immunosuppressive action of these drugs often does not

  11. Frequency and specificity of red blood cell alloantibodies among ...

    African Journals Online (AJOL)

    Background: Blood transfusion usually results in production of alloantibody against one or more foreign red blood cell antigens which may complicate subsequent transfusions. The probability of alloimmunization depends on number and frequency of transfusion, antigen immunogenicity, recipient immune response and ...

  12. CD117 immunoexpression in canine mast cell tumours: correlations with pathological variables and proliferation markers

    Directory of Open Access Journals (Sweden)

    Pires Maria A

    2007-08-01

    Full Text Available Abstract Background Cutaneous mast cell tumours are one of the most common neoplasms in dogs and show a highly variable biologic behaviour. Several prognosis tools have been proposed for canine mast cell tumours, including histological grading and cell proliferation markers. CD117 is a receptor tyrosine kinase thought to play a key role in human and canine mast cell neoplasms. Normal (membrane-associated and aberrant (cytoplasmic, focal or diffuse CD117 immunoexpression patterns have been identified in canine mast cell tumours. Cytoplasmic CD117 expression has been found to correlate with higher histological grade and with a worsened post-surgical prognosis. This study addresses the role of CD117 in canine mast cell tumours by studying the correlations between CD117 immunoexpression patterns, two proliferation markers (Ki67 and AgNORs histological grade, and several other pathological variables. Results Highly significant (p Conclusion These findings highlight the key role of CD117 in the biopathology of canine MCTs and confirm the relationship between aberrant CD117 expression and increased cell proliferation and higher histological grade. Further studies are needed to unravel the cellular mechanisms underlying focal and diffuse cytoplasmic CD117 staining patterns, and their respective biopathologic relevance.

  13. Hyper-variable regions in 18S rDNA of Strongyloides spp. as markers for species-specific diagnosis.

    Science.gov (United States)

    Hasegawa, Hideo; Hayashida, Shotaro; Ikeda, Yatsukaho; Sato, Hiroshi

    2009-03-01

    Four hyper-variable regions (HVR-I to -IV) found in 18S ribosomal DNA sequences were compared among 34 isolates of 15 species of the genus Strongyloides to evaluate their diagnostic value. HVR-I to -III were short, and plural species exhibit the same nucleotide arrangement. Meanwhile, HVR-IV had 23 to 39 nucleotides, showing species-specific arrangements, except Strongyloides ransomi and Strongyloides venezuelensis, which had the same nucleotide sequence in HVR-IV but were readily distinguished by the difference in HVR-I and -III. Isolates of Strongyloides stercoralis from humans of USA, Japan, and Philippines, chimpanzees, and dogs had an identical sequence in this region. Meanwhile, intraspecific polymorphism in HVR-IV nucleotide arrangement was observed among isolates of Strongyloides fuelleborni and Strongyloides callosciureus, presumably reflecting process of geographical dispersal and adaptation to the hosts.

  14. Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

    OpenAIRE

    Qi Wan; Lina Kozhaya; Keren Imberg; Frances Mercer; Shi Zhong; Michelle Krogsgaard; Derya Unutmaz

    2013-01-01

    Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either ...

  15. Association of Seasonal Climate Variability and Age-Specific Mortality in Northern Sweden before the Onset of Industrialization

    Directory of Open Access Journals (Sweden)

    Joacim Rocklöv

    2014-07-01

    Full Text Available Background and aims: Little is known about health impacts of climate in pre-industrial societies. We used historical data to investigate the association of temperature and precipitation with total and age-specific mortality in Skellefteå, northern Sweden, between 1749 and 1859. Methods: We retrieved digitized aggregated population data of the Skellefteå parish, and monthly temperature and precipitation measures. A generalized linear model was established for year to year variability in deaths by annual and seasonal average temperature and cumulative precipitation using a negative binomial function, accounting for long-term trends in population size. The final full model included temperature and precipitation of all four seasons simultaneously. Relative risks (RR with 95% confidence intervals (CI were calculated for total, sex- and age-specific mortality. Results: In the full model, only autumn precipitation proved statistically significant (RR 1.02; CI 1.00–1.03, per 1cm increase of autumn precipitation, while winter temperature (RR 0.98; CI 0.95–1.00, per 1 °C increase in temperature and spring precipitation (RR 0.98; CI 0.97–1.00 per 1 cm increase in precipitation approached significance. Similar effects were observed for men and women. The impact of climate variability on mortality was strongest in children aged 3–9, and partly also in older children. Infants, on the other hand, appeared to be less affected by unfavourable climate conditions. Conclusions: In this pre-industrial rural region in northern Sweden, higher levels of rain during the autumn increased the annual number of deaths. Harvest quality might be one critical factor in the causal pathway, affecting nutritional status and susceptibility to infectious diseases. Autumn rain probably also contributed to the spread of air-borne diseases in crowded living conditions. Children beyond infancy appeared most vulnerable to climate impacts.

  16. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders

    2014-01-01

    Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development......, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94...... positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies...

  17. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E

    2014-01-01

    ), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. DESIGN AND METHODS: Here, we used an array of different human leukocyte antigen(HLA)-B*15:03 and HLA-B*42:01 tetramers to characterize inhibitory receptor expression as a function...... by effector memory CD8+ T cells. CONCLUSION: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels...

  18. Microglia specific fluorescent probes for live cell imaging.

    Science.gov (United States)

    Leong, Cheryl; Lee, Sung Chan; Ock, Jiyeon; Li, Xin; See, Peter; Park, Sung Jin; Ginhoux, Florent; Yun, Seong-Wook; Chang, Young-Tae

    2014-02-04

    Small molecule fluorescent probes offer significant advantages over conventional antibody and fluorescent protein labeling techniques. Here we present and , dyes that label live microglia specifically. They may be applied to the isolation and imaging of live microglia when investigating their role in neuroinflammatory diseases.

  19. A xylogalacturonan epitope is specifically associated with plant cell detachment.

    NARCIS (Netherlands)

    Willats, W.G.T.; McCartney, L.; Steele-King, C.G.; Marcus, S.E.; Mort, A.J.; Huisman, M.M.H.; Alebeek, van G.J.W.M.; Schols, H.A.; Voragen, A.G.J.; Goff, le A.; Bonnin, E.; Thibault, J.F.; Knox, J.P.

    2004-01-01

    A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope

  20. The role of auxin in cell specification during arabidopsis embryogenesis

    NARCIS (Netherlands)

    Lokerse, A.S.

    2011-01-01

    Auxin is a structurally simple molecule, yet it elicits many different responses in plants. In Chapter 1 we have reviewed how specificity in the output of auxin signaling could be generated by distinct regulation and the unique properties of the members of the Aux/IAA and ARF transcription factor

  1. Modeling dynamics of cell-to-cell variability in TRAIL-induced apoptosis explains fractional killing and predicts reversible resistance.

    Directory of Open Access Journals (Sweden)

    François Bertaux

    2014-10-01

    Full Text Available Isogenic cells sensing identical external signals can take markedly different decisions. Such decisions often correlate with pre-existing cell-to-cell differences in protein levels. When not neglected in signal transduction models, these differences are accounted for in a static manner, by assuming randomly distributed initial protein levels. However, this approach ignores the a priori non-trivial interplay between signal transduction and the source of this cell-to-cell variability: temporal fluctuations of protein levels in individual cells, driven by noisy synthesis and degradation. Thus, modeling protein fluctuations, rather than their consequences on the initial population heterogeneity, would set the quantitative analysis of signal transduction on firmer grounds. Adopting this dynamical view on cell-to-cell differences amounts to recast extrinsic variability into intrinsic noise. Here, we propose a generic approach to merge, in a systematic and principled manner, signal transduction models with stochastic protein turnover models. When applied to an established kinetic model of TRAIL-induced apoptosis, our approach markedly increased model prediction capabilities. One obtains a mechanistic explanation of yet-unexplained observations on fractional killing and non-trivial robust predictions of the temporal evolution of cell resistance to TRAIL in HeLa cells. Our results provide an alternative explanation to survival via induction of survival pathways since no TRAIL-induced regulations are needed and suggest that short-lived anti-apoptotic protein Mcl1 exhibit large and rare fluctuations. More generally, our results highlight the importance of accounting for stochastic protein turnover to quantitatively understand signal transduction over extended durations, and imply that fluctuations of short-lived proteins deserve particular attention.

  2. Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    Science.gov (United States)

    Zhang, Xiaochang; Chen, Ming Hui; Wu, Xuebing; Kodani, Andrew; Fan, Jean; Doan, Ryan; Ozawa, Manabu; Ma, Jacqueline; Yoshida, Nobuaki; Reiter, Jeremy F.; Black, Douglas L.; Kharchenko, Peter V.; Sharp, Phillip A.; Walsh, Christopher A.

    2017-01-01

    SUMMARY Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains—especially in cytoskeletal proteins—and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. While Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1 binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development. PMID:27565344

  3. Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

    Science.gov (United States)

    Zhang, Xiaochang; Chen, Ming Hui; Wu, Xuebing; Kodani, Andrew; Fan, Jean; Doan, Ryan; Ozawa, Manabu; Ma, Jacqueline; Yoshida, Nobuaki; Reiter, Jeremy F; Black, Douglas L; Kharchenko, Peter V; Sharp, Phillip A; Walsh, Christopher A

    2016-08-25

    Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. In vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis roots.

    Science.gov (United States)

    Long, Yuchen; Stahl, Yvonne; Weidtkamp-Peters, Stefanie; Postma, Marten; Zhou, Wenkun; Goedhart, Joachim; Sánchez-Pérez, María-Isabel; Gadella, Theodorus W J; Simon, Rüdiger; Scheres, Ben; Blilou, Ikram

    2017-08-03

    During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.

  5. Target-specific activation of mast cells by immunoglobulin E reactive with a renal cell carcinoma-associated antigen

    NARCIS (Netherlands)

    Luiten, R. M.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    Immunoglobulin E (IgE) that specifically binds to antigens present on carcinoma cells may represent a useful tool to combat carcinomas. Induction of an inflammatory response at the tumor site by tumor-specific IgE may result in reduced tumor growth and tumor regression. Local mast cells may be

  6. Influenza virus and endothelial cells: a species specific relationship

    OpenAIRE

    Kirsty Renfree Short; Edwin eVeldhuis Kroeze; Leslie A Reperant; Mathilde eRichard; Thijs eKuiken

    2014-01-01

    Influenza A virus infection is an important cause of respiratory disease in humans. The original reservoirs of influenza A virus are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves...

  7. Influenza virus and endothelial cells: a species specific relationship

    OpenAIRE

    Short, Kirsty R; Veldhuis Kroeze, Edwin J. B.; Leslie A Reperant; Richard, Mathilde; Kuiken, Thijs

    2014-01-01

    Influenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves increas...

  8. Cell Type-Specific Effects of Adenosine on Cortical Neurons

    Science.gov (United States)

    van Aerde, Karlijn I.; Qi, Guanxiao; Feldmeyer, Dirk

    2015-01-01

    The neuromodulator adenosine is widely considered to be a key regulator of sleep homeostasis and an indicator of sleep need. Although the effect of adenosine on subcortical areas has been previously described, the effects on cortical neurons have not been addressed systematically to date. To that purpose, we performed in vitro whole-cell patch-clamp recordings and biocytin staining of pyramidal neurons and interneurons throughout all layers of rat prefrontal and somatosensory cortex, followed by morphological analysis. We found that adenosine, via the A1 receptor, exerts differential effects depending on neuronal cell type and laminar location. Interneurons and pyramidal neurons in layer 2 and a subpopulation of layer 3 pyramidal neurons that displayed regular spiking were insensitive to adenosine application, whereas other pyramidal cells in layers 3–6 were hyperpolarized (range 1.2–10.8 mV). Broad tufted pyramidal neurons with little spike adaptation showed a small adenosine response, whereas slender tufted pyramidal neurons with substantial adaptation showed a bigger response. These studies of the action of adenosine at the postsynaptic level may contribute to the understanding of the changes in cortical circuit functioning that take place between sleep and awakening. PMID:24108800

  9. T cells specific for different latent and lytic viral proteins efficiently control Epstein-Barr virus-transformed B cells.

    Science.gov (United States)

    Nowakowska, Justyna; Stuehler, Claudia; Egli, Adrian; Battegay, Manuel; Rauser, Georg; Bantug, Glenn Robert; Brander, Christian; Hess, Christoph; Khanna, Nina

    2015-09-01

    Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) belong to the most dreaded complications of immunosuppression. The efficacy of EBV-specific T-cell transfer for PTLD has been previously shown, yet the optimal choice of EBV-derived antigens inducing polyclonal CD4(+) and CD8(+) T cells that cover a wide range of human leukocyte antigen types and efficiently control PTLD remains unclear. A pool of 125 T-cell epitopes from seven latent and nine lytic EBV-derived proteins (EBVmix) and peptide pools of EBNA1, EBNA3c, LMP2a and BZLF1 were used to determine T-cell frequencies and to isolate T cells through the use of the interferon (IFN)-γ cytokine capture system. We further evaluated the phenotype and functionality of the generated T-cell lines in vitro. EBVmix induced significantly higher T-cell frequencies and allowed selecting more CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells than single peptide pools. T cells of all specificities expanded similarly in vitro, recognized cognate antigen, and, to a lower extent, EBV-infected cells, exerted moderate cytotoxicity and showed reduced alloreactivity. However, EBVmix-specific cells most efficiently controlled EBV-infected lymphoblastoid cell lines (LCLs). This control was mainly mediated by EBV-specific CD8(+) cells with an oligoclonal epitope signature covering both latent and lytic viral proteins. Notably, EBV-specific CD4(+) cells unable to control LCLs produced significantly less perforin and granzyme B, probably because of limited LCL epitope presentation. EBVmix induces a broader T-cell response, probably because of its coverage of latent and lytic EBV-derived proteins that may be important to control EBV-transformed B cells and might offer an improvement of T-cell therapies. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. The circulating T helper subsets and regulatory T cells in patients with common variable immunodeficiency with no known monogenic disease.

    Science.gov (United States)

    Azizi, G; Mirshafiey, A; Abolhassani, H; Yazdani, R; Ansariha, F J; Shaghaghi, M; Mortazavi-Jahromi, S S; Noorbakhsh, F; Rezaei, N; Aghamohammadi, A

    2018-01-18

    Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID), characterized by heterogeneous clinical manifestations and defects in B- and T- cells. In the present study, we investigated the T helper (Th) cell subsets and regulatory T (Treg) cells, and their related cytokines and transcription factors in the CVID patients with no definite genetic diagnosis. The study population comprised 13 CVID patients and 13 healthy controls (HC). Mutation analysis was done by whole exome sequencing in CVID patients to rule out monogenic PIDs. Th subsets and Treg were examined by flow cytometry. The expression of determinant cytokines (IFN-γ, IL-17, IL-22, and IL-10), and cell subset specific transcription factors were evaluated before and after stimulation. The main clinical presentations of these patients were infections only and lymphoproliferations phenotypes, but no autoimmune and allergy phenotype were recorded. The frequencies of CD4+ T cells, Th17, and Treg cells were significantly reduced in CVID patients, however the subsets of Th1, Th1-like Th17 and Th22 cells were normal. After stimulation, retinoic-acid-orphan-receptor-C (RORC), and runt-related transcription factor 1 (RUNX1), IL17, and IL10 genes' expression in CVID patients were significantly lower, in comparison to the HC. Moreover, there was a lower concentration of IL-17 and IL-10 in cell culture supernatants of stimulated CD4+ T cells of CVID patients than HC. Our findings demonstrate that the imbalance of Th17 and Tregs could be an associated with infections only and lymphoproliferations phenotype in CVID patients without monogenic disorders.

  11. Genome flux in tomato cell clones cultured in vitro in different physiological equilibria. II. A RAPD analysis of variability.

    Science.gov (United States)

    Bogani, P; Simoni, A; Lio', P; Scialpi, A; Buiatti, M

    1996-10-01

    An analysis of the effect of changing physiological conditions on genome evolution in tomato cell populations has been carried out on long-term in vitro cultured clones grown on different auxin-cytokinin equilibria or selected for low-high competence for active defense against Fusarium oxysporum f.sp. lycopersici. RAPD analysis, confirmed through pattern rehybridization, was used as a random tool to measure the genetic variability. Through the use of a modified ANOVA, variation was shown to depend on both the initial genotype and the physiological conditions. Pattern correlation analysis through a mutual information algorithm suggested the fixation of RAPD patterns specific to physiological equilibria. The results are discussed in view of the possible relevance for evolution at hierarchical levels higher than cell populations. Key words : tomato clones, somaclonal variation, RAPD, coadaptation.

  12. Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

    Science.gov (United States)

    Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

    2013-01-01

    Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

  13. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  14. Rapid generation of NY-ESO-1-specific CD4(+) THELPER1 cells for adoptive T-cell therapy.

    Science.gov (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias

    2015-05-01

    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4(+), IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4(+) TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4(+) T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8(+) T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4(+) T cells showed strong specific TH1-responses with IFNγ(+), TNFα(+), IL-2(+) and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4(+) TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients.

  15. Rapid generation of NY-ESO-1-specific CD4+ THELPER1 cells for adoptive T-cell therapy

    Science.gov (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias

    2015-01-01

    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+ TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+ T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+ T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+ T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+ and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+ TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients. PMID:26155389

  16. Understanding the Molecular Circuitry of Cell Lineage Specification in the Early Mouse Embryo

    Science.gov (United States)

    Bergsmedh, Anna; Donohoe, Mary E.; Hughes, Rebecca-Ayme; Hadjantonakis, Anna-Katerina

    2011-01-01

    Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells. Here we provide an overview of cell lineage specification in the early (preimplantation) mouse embryo focusing on the transcriptional circuitry and epigenetic marks necessary for successive differentiation events leading to the formation of the blastocyst. PMID:24710206

  17. Use of Multicolor Flow Cytometry for Isolation of Specific Cell Populations Deriving from Differentiated Human Embryonic Stem Cells

    NARCIS (Netherlands)

    Mengarelli, Isabella; Fryga, Andrew; Barberi, Tiziano

    2016-01-01

    Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specific types of cells from a heterogeneous population of live cells. Here we describe a multicolor flow cytometry technique that uses five distinct cell surface antigens to isolate four live populations with

  18. PET probes for distinct metabolic pathways have different cell specificities during immune responses in mice

    OpenAIRE

    Nair-Gill, Evan; Wiltzius, Stephanie M.; Wei, Xiao X.; Cheng, Donghui; Riedinger, Mireille; Radu, Caius G.; Witte, Owen N.

    2010-01-01

    Clinical tools that measure changes in immune cell metabolism would improve the diagnosis and treatment of immune dysfunction. PET, utilizing probes for specific metabolic processes, detects regions of immune activation in vivo. In this study we investigated the immune cell specificity of PET probes for two different metabolic pathways: [18F]–2-fluorodeoxyglucose ([18F]-FDG) for glycolysis and ...

  19. Accessing complexity: the dynamics of virus-specific T cell responses

    DEFF Research Database (Denmark)

    Doherty, P C; Christensen, Jan Pravsgaard

    2000-01-01

    -specific CD8(+ )T cells. Analysis to date with both naturally acquired and experimentally induced infections has established that the numbers of virus-specific CD8(+) T cells present during both the acute and memory phases of the host response are more than tenfold in excess of previously suspected values...

  20. HIV-specific CD4+ T cells and viremia: who's in control?

    NARCIS (Netherlands)

    Jansen, Christine A.; van Baarle, Debbie; Miedema, Frank

    2006-01-01

    It has been proposed that HIV-specific CD4+ T cells with a central memory phenotype might be involved in controlling HIV replication. Based on recent data (lack of protective effects of HIV-specific CD4+ T-cell responses in acutely infected patients undergoing treatment interruptions; loss of

  1. Abbreviation as a Reflection of Terms Variability in Language for Specific Purposes: Translational Features (Terminology Case Study in German, English, Kazakh, and Russia)

    Science.gov (United States)

    Beisembayeva, Gulshat Z.; Yeskindirova, Manshuk Z.; Tulebayeva, Samal A.

    2016-01-01

    The range of modern dynamic social changes, globalization of world powers' economic cooperation, acceleration of technocratic processes have widespread impact on term systems' variability in language, in particular, on terminological variability for specific purposes. This globalized extra-linguistic factor provokes avalanche growth of…

  2. Daughter-specific transcription factors regulate cell size control in budding yeast.

    Directory of Open Access Journals (Sweden)

    Stefano Di Talia

    2009-10-01

    Full Text Available In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.

  3. High-level HIV-1 viremia suppresses viral antigen-specific CD4+ T cell proliferation

    OpenAIRE

    McNeil, Andrew C.; Shupert, W. Lesley; Iyasere, Christiana A.; Hallahan, Claire W.; Mican, JoAnn; Davey, Richard T.; Connors, Mark

    2001-01-01

    In chronic viral infections of humans and experimental animals, virus-specific CD4+ T cell function is believed to be critical for induction and maintenance of host immunity that mediates effective restriction of viral replication. Because in vitro proliferation of HIV-specific memory CD4+ T cells is only rarely demonstrable in HIV-infected individuals, it is presumed that HIV-specific CD4+ T cells are killed upon encountering the virus, and maintenance of CD4+ T cell responses in some patien...

  4. Induction of delayed-type hypersensitivity by the T cell line specific to bacterial peptidoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Katsuki, M.; Kakimoto, K.; Kawata, S.; Kotani, S.; Koga, T.

    1987-12-01

    A T cell line specific for the chemically well-defined peptidoglycan of bacterial cell wall, disaccharide tetrapeptide, was established from Lewis rats immunized with the antigen covalently linked to the autologous rat serum albumin. The antigen specificity was examined with various analogues or derivatives of the peptidoglycan. The cell line was reactive to analogues with the COOH-terminal D-amino acid, but least reactive to those with L-amino acid as COOH terminus. Transferring of the T cell line into X-irradiated normal Lewis rats induced delayed-type hypersensitivity in an antigen specific manner.

  5. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

    Directory of Open Access Journals (Sweden)

    William H Thiel

    Full Text Available The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX with high-throughput sequencing (HTS and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs. Several rounds of positive (VSMCs and negative (endothelial cells; ECs selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1 metrics of selection enrichment; and (2 pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  6. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

    Science.gov (United States)

    Thiel, William H; Bair, Thomas; Peek, Andrew S; Liu, Xiuying; Dassie, Justin; Stockdale, Katie R; Behlke, Mark A; Miller, Francis J; Giangrande, Paloma H

    2012-01-01

    The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  7. OptMAVEn--a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn. OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced

  8. SPECIES-SPECIFIC FOREST VARIABLE ESTIMATION USING NON-PARAMETRIC MODELING OF MULTI-SPECTRAL PHOTOGRAMMETRIC POINT CLOUD DATA

    Directory of Open Access Journals (Sweden)

    J. Bohlin

    2012-07-01

    Full Text Available The recent development in software for automatic photogrammetric processing of multispectral aerial imagery, and the growing nation-wide availability of Digital Elevation Model (DEM data, are about to revolutionize data capture for forest management planning in Scandinavia. Using only already available aerial imagery and ALS-assessed DEM data, raster estimates of the forest variables mean tree height, basal area, total stem volume, and species-specific stem volumes were produced and evaluated. The study was conducted at a coniferous hemi-boreal test site in southern Sweden (lat. 58° N, long. 13° E. Digital aerial images from the Zeiss/Intergraph Digital Mapping Camera system were used to produce 3D point-cloud data with spectral information. Metrics were calculated for 696 field plots (10 m radius from point-cloud data and used in k-MSN to estimate forest variables. For these stands, the tree height ranged from 1.4 to 33.0 m (18.1 m mean, stem volume from 0 to 829 m3 ha-1 (249 m3 ha-1 mean and basal area from 0 to 62.2 m2 ha-1 (26.1 m2 ha-1 mean, with mean stand size of 2.8 ha. Estimates made using digital aerial images corresponding to the standard acquisition of the Swedish National Land Survey (Lantmäteriet showed RMSEs (in percent of the surveyed stand mean of 7.5% for tree height, 11.4% for basal area, 13.2% for total stem volume, 90.6% for pine stem volume, 26.4 for spruce stem volume, and 72.6% for deciduous stem volume. The results imply that photogrammetric matching of digital aerial images has significant potential for operational use in forestry.

  9. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy.

    Science.gov (United States)

    Cardoso Dos Santos, Marcelina; Déturche, Régis; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-09-20

    We propose an improved version of variable-angle total internal reflection fluorescence microscopy (vaTIRFM) adapted to modern TIRF setup. This technique involves the recording of a stack of TIRF images, by gradually increasing the incident angle of the light beam on the sample. A comprehensive theory was developed to extract the membrane/substrate separation distance from fluorescently labeled cell membranes. A straightforward image processing was then established to compute the topography of cells with a nanometric axial resolution, typically 10-20 nm. To highlight the new opportunities offered by vaTIRFM to quantify adhesion process of motile cells, adhesion of MDA-MB-231 cancer cells on glass substrate coated with fibronectin was examined. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Notch signaling in the pancreas: patterning and cell fate specification.

    Science.gov (United States)

    Afelik, Solomon; Jensen, Jan

    2013-07-01

    Notch signaling is an evolutionarily conserved mechanism adapted to control binary fate decisions. The first evidence of Notch in pancreatic development focused on its critical role in controlling endocrine fate decisions. Since then, we have come to understand that this signaling system operates iteratively in the pancreas, and is not limited to the control of endocrine fate decision. Notch appears to play a role in early organ development, then during organ domain patterning, and only during a final refinement process, in the control of terminal cell fates. In so doing, Notch receptors and their ligands are under the influence of a wealth of genetic components that together help orchestrate the building of a complex, glandular organ. Copyright © 2012 Wiley Periodicals, Inc.

  11. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  12. Effective variables to control the fill factor of organic photovoltaic cells.

    Science.gov (United States)

    Kim, Myung-Su; Kim, Bong-Gi; Kim, Jinsang

    2009-06-01

    Effective cell design variables having a large impact on the fill factor (FF) of organic photovoltaic cells (OPVCs) were systematically identified using a general device structure of ITO/PEDOT:PSS/P3HT + PCBM/LiF/Al. The results show that the characteristic properties of the organic layer, such as morphology and thickness, the regioregularity of the conjugated polymer, and the two interfaces between the electrodes and the blend layer have a large influence on the FF by affecting the series resistance (R(s)) and the shunt resistance (R(sh)). The systematic investigation described in this contribution provides a comprehensive understanding of the correlation between the device variables and R(s) and R(sh) and a way to control FF, which is critically important to achieving a high-performance OPVC.

  13. Modeling a snap-action, variable-delay switch controlling extrinsic cell death.

    Directory of Open Access Journals (Sweden)

    John G Albeck

    2008-12-01

    Full Text Available When exposed to tumor necrosis factor (TNF or TNF-related apoptosis-inducing ligand (TRAIL, a closely related death ligand and investigational therapeutic, cells enter a protracted period of variable duration in which only upstream initiator caspases are active. A subsequent and sudden transition marks activation of the downstream effector caspases that rapidly dismantle the cell. Thus, extrinsic apoptosis is controlled by an unusual variable-delay, snap-action switch that enforces an unambiguous choice between life and death. To understand how the extrinsic apoptosis switch functions in quantitative terms, we constructed a mathematical model based on a mass-action representation of known reaction pathways. The model was trained against experimental data obtained by live-cell imaging, flow cytometry, and immunoblotting of cells perturbed by protein depletion and overexpression. The trained model accurately reproduces the behavior of normal and perturbed cells exposed to TRAIL, making it possible to study switching mechanisms in detail. Model analysis shows, and experiments confirm, that the duration of the delay prior to effector caspase activation is determined by initiator caspase-8 activity and the rates of other reactions lying immediately downstream of the TRAIL receptor. Sudden activation of effector caspases is achieved downstream by reactions involved in permeabilization of the mitochondrial membrane and relocalization of proteins such as Smac. We find that the pattern of interactions among Bcl-2 family members, the partitioning of Smac from its binding partner XIAP, and the mechanics of pore assembly are all critical for snap-action control.

  14. [Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus].

    Science.gov (United States)

    Béchet, J M; Montagnier, L; Latarjet, R

    1975-01-13

    RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.

  15. Streptamer versus tetramer-based selection of functional cytomegalovirus-specific T cells

    Directory of Open Access Journals (Sweden)

    Xin-Chao Wang

    2013-06-01

    Conclusion: Our study demonstrated that both tetra- and streptamer technologies can be used to purify CMV-specific cytotoxic CD8+ T cells for adoptive T-cell transfer. Both multimer technologies did not have any negative influence on the proliferation of selected T cells. Importantly, streptamer technology is available at good manufacturing practice level.

  16. A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals

    NARCIS (Netherlands)

    Heidt, S.; Roelen, D.L.; Vaal, Y.J. de; Kester, M.G.; Eijsink, C.; Thomas, S.; Besouw, N.M. van; Volk, H.D.; Weimar, W.; Claas, F.H.; Mulder, A.

    2012-01-01

    Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells

  17. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

    DEFF Research Database (Denmark)

    Kalis, Martins; Bolmeson, Caroline; Esguerra, Jonathan L.S.

    2011-01-01

    (¿/¿) mice showed ultrastructurally normal ß-cells and intact insulin secretion. In conclusion, we have demonstrated that a ß-cell specific disruption of the miRNAs network, although allowing for apparently normal ß-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis...

  18. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-06-01

    Full Text Available Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

  19. Differentiation of Induced Pluripotent Stem Cells to Lentoid Bodies Expressing a Lens Cell-Specific Fluorescent Reporter.

    Directory of Open Access Journals (Sweden)

    Taruna Anand

    Full Text Available Curative approaches for eye cataracts and other eye abnormalities, such as myopia and hyperopia currently suffer from a lack of appropriate models. Here, we present a new approach for in vitro growth of lentoid bodies from induced pluripotent stem (iPS cells as a tool for ophthalmological research. We generated a transgenic mouse line with lens-specific expression of a fluorescent reporter driven by the alphaA crystallin promoter. Fetal fibroblasts were isolated from transgenic fetuses, reprogrammed to iPS cells, and differentiated to lentoid bodies exploiting the specific fluorescence of the lens cell-specific reporter. The employment of cell type-specific reporters for establishing and optimizing differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations.

  20. Allergen specificity of IgG(4)-expressing B cells in patients with grass pollen allergy undergoing immunotherapy.

    Science.gov (United States)

    James, Louisa K; Bowen, Holly; Calvert, Rosaleen A; Dodev, Tihomir S; Shamji, Mohamed H; Beavil, Andrew J; McDonnell, James M; Durham, Stephen R; Gould, Hannah J

    2012-09-01

    Serum IgG(4) responses to allergen immunotherapy are well documented as blocking allergen binding to receptor-bound IgE on antigen-presenting cells and effector cells, but the molecular characteristics of treatment-induced IgG(4), particularly in relation to expressed antibody, are poorly defined. We aimed to clone and express recombinant IgG(4) from patients receiving grass pollen immunotherapy using single B cells to obtain matched heavy- and light-chain pairs. IgG(4)(+) B cells were enriched from blood samples taken from 5 patients receiving grass pollen immunotherapy. Matched heavy- and light-chain variable-region sequences were amplified from single IgG(4)(+) B cells. Variable regions were cloned and expressed as recombinant IgG(4). Binding analysis of grass pollen-specific IgG(4) was performed by using surface plasmon resonance. Functional assays were used to determine IgE blocking activity. In a separate experiment grass pollen-specific antibodies were depleted from serum samples to determine the proportion of grass pollen-specific IgG(4) within total IgG(4). Depletion of grass pollen-specific antibodies from serum led to a modest reduction in total IgG(4) levels. Matched heavy- and light-chain sequences were cloned from single IgG(4)(+) B cells and expressed as recombinant IgG(4). We identified an IgG(4) that binds with extremely high affinity to the grass pollen allergen Phl p 7. Furthermore, we found that a single specific mAb can block IgE-mediated facilitated allergen presentation, as well as IgE-mediated basophil activation. Although increases in IgG(4) levels cannot be wholly accounted for within the allergen-specific fraction, allergen immunotherapy might result in the production of high-affinity allergen-specific blocking IgG(4). Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  1. Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo.

    Science.gov (United States)

    Daley, William P; Matsumoto, Kazue; Doyle, Andrew D; Wang, Shaohe; DuChez, Brian J; Holmbeck, Kenn; Yamada, Kenneth M

    2017-06-15

    Branching morphogenesis of developing organs requires coordinated but poorly understood changes in epithelial cell-cell adhesion and cell motility. We report that Btbd7 is a crucial regulator of branching morphogenesis in vivo. Btbd7 levels are elevated in peripheral cells of branching epithelial end buds, where it enhances cell motility and cell-cell adhesion dynamics. Genetic ablation of Btbd7 in mice disrupts branching morphogenesis of salivary gland, lung and kidney. Btbd7 knockout results in more tightly packed outer bud cells, which display stronger E-cadherin localization, reduced cell motility and decreased dynamics of transient cell separations associated with cleft formation; inner bud cells remain unaffected. Mechanistic analyses using in vitro MDCK cells to mimic outer bud cell behavior establish that Btbd7 promotes loss of E-cadherin from cell-cell adhesions with enhanced migration and transient cell separation. Btbd7 can enhance E-cadherin ubiquitination, internalization, and degradation in MDCK and peripheral bud cells for regulating cell dynamics. These studies show how a specific regulatory molecule, Btbd7, can function at a local region of developing organs to regulate dynamics of cell adhesion and motility during epithelial branching morphogenesis. © 2017. Published by The Company of Biologists Ltd.

  2. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    Science.gov (United States)

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  3. Probing the effector and suppressive functions of human T cell subsets using antigen-specific engineered T cell receptors.

    Directory of Open Access Journals (Sweden)

    Qi Wan

    Full Text Available Activation of T cells through the engagement of the T cell receptors (TCRs with specific peptide-MHC complexes on antigen presenting cells (APCs is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8(+ and CD4(+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.

  4. Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy

    Science.gov (United States)

    Zhou, Jiehua; Rossi, John J.

    2014-01-01

    One hundred years ago, Dr. Paul Ehrlich popularized the “magic bullet” concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, “targeted therapy” that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges. PMID:24936916

  5. Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8(+) T cells.

    Science.gov (United States)

    Pollack, Seth M; Jones, Robin L; Farrar, Erik A; Lai, Ivy P; Lee, Sylvia M; Cao, Jianhong; Pillarisetty, Venu G; Hoch, Benjamin L; Gullett, Ashley; Bleakley, Marie; Conrad, Ernest U; Eary, Janet F; Shibuya, Kendall C; Warren, Edus H; Carstens, Jason N; Heimfeld, Shelly; Riddell, Stanley R; Yee, Cassian

    2014-01-01

    Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL). CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties. Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1. This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.

  6. A validated regulatory network for Th17 cell specification

    Science.gov (United States)

    Ciofani, Maria; Madar, Aviv; Galan, Carolina; Sellars, Maclean; Mace, Kieran; Pauli, Florencia; Agarwal, Ashish; Huang, Wendy; Parkhurst, Christopher N.; Muratet, Michael; Newberry, Kim M.; Meadows, Sarah; Greenfield, Alex; Yang, Yi; Jain, Preti; Kirigin, Francis F.; Birchmeier, Carmen; Wagner, Erwin F.; Murphy, Kenneth M.; Myers, Richard M.; Bonneau, Richard; Littman, Dan R.

    2012-01-01

    Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively-bound BATF and IRF4 contribute to initial chromatin accessibility, and with STAT3 initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple datasets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease. PMID:23021777

  7. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.

    Science.gov (United States)

    Gomes-Silva, Diogo; Srinivasan, Madhuwanti; Sharma, Sandhya; Lee, Ciaran M; Wagner, Dimitrios L; Davis, Timothy H; Rouce, Rayne H; Bao, Gang; Brenner, Malcolm K; Mamonkin, Maksim

    2017-07-20

    Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7(+)) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies. © 2017 by The American Society of Hematology.

  8. Functional killer Ig-like receptors on human memory CD4+ T cells specific for cytomegalovirus.

    Science.gov (United States)

    van Bergen, Jeroen; Kooy-Winkelaar, Engelina M C; van Dongen, Henrike; van Gaalen, Floris A; Thompson, Allan; Huizinga, Tom W J; Feltkamp, Mariet C W; Toes, René E M; Koning, Frits

    2009-04-01

    Although very few CD4(+) T cells express killer Ig receptors (KIR), a large proportion of CD4(+) T cells with a late memory phenotype, characterized by the absence of CD28, does express KIR. Here, we show that KIR expression on CD4(+) T cells is also associated with memory T cell function, by showing that the frequency of CMV-specific cells is higher in CD4(+)KIR(+) than CD4(+)KIR(-) T cells. In addition, engagement of an inhibitory KIR inhibited the CMV-specific proliferation of these CD4(+)KIR(+) memory T cells, but had no detectable effect on cytokine production. Our data reveal that, in marked contrast with CD8(+) T cells, the activity of a subset of CMV-specific CD4(+) T cells is modulated by HLA class I-specific KIR. Thus, the CMV-induced down-regulation of HLA class I may in fact enhance memory CMV-specific CD4(+) T cell responses restricted by HLA class II.

  9. Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system.

    Science.gov (United States)

    Singh, Harjeet; Manuri, Pallavi R; Olivares, Simon; Dara, Navid; Dawson, Margaret J; Huls, Helen; Hackett, Perry B; Kohn, Donald B; Shpall, Elizabeth J; Champlin, Richard E; Cooper, Laurence J N

    2008-04-15

    Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.

  10. Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients

    National Research Council Canada - National Science Library

    Baitsch, Lukas; Baumgaertner, Petra; Devêvre, Estelle; Raghav, Sunil K; Legat, Amandine; Barba, Leticia; Wieckowski, Sébastien; Bouzourene, Hanifa; Deplancke, Bart; Romero, Pedro; Rufer, Nathalie; Speiser, Daniel E

    2011-01-01

    ... differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion...

  11. Mechanisms of Intrinsic Beating Variability in Cardiac Cell Cultures and Model Pacemaker Networks

    Science.gov (United States)

    Ponard, Julien G. C.; Kondratyev, Aleksandar A.; Kucera, Jan P.

    2007-01-01

    Heart rate variability (HRV) exhibits fluctuations characterized by a power law behavior of its power spectrum. The interpretation of this nonlinear HRV behavior, resulting from interactions between extracardiac regulatory mechanisms, could be clinically useful. However, the involvement of intrinsic variations of pacemaker rate in HRV has scarcely been investigated. We examined beating variability in spontaneously active incubating cultures of neonatal rat ventricular myocytes using microelectrode arrays. In networks of mathematical model pacemaker cells, we evaluated the variability induced by the stochastic gating of transmembrane currents and of calcium release channels and by the dynamic turnover of ion channels. In the cultures, spontaneous activity originated from a mobile focus. Both the beat-to-beat movement of the focus and beat rate variability exhibited a power law behavior. In the model networks, stochastic fluctuations in transmembrane currents and stochastic gating of calcium release channels did not reproduce the spatiotemporal patterns observed in vitro. In contrast, long-term correlations produced by the turnover of ion channels induced variability patterns with a power law behavior similar to those observed experimentally. Therefore, phenomena leading to long-term correlated variations in pacemaker cellular function may, in conjunction with extracardiac regulatory mechanisms, contribute to the nonlinear characteristics of HRV. PMID:17325022

  12. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Science.gov (United States)

    Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

    2014-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  13. Tissue-specific B-cell dysfunction and generalized memory B-cell loss during acute SIV infection.

    Directory of Open Access Journals (Sweden)

    Sandrine Peruchon

    Full Text Available BACKGROUND: Primary HIV-infected patients display severe and irreversible damage to different blood B-cell subsets which is not restored by highly efficient anti-retroviral therapy (HAART. Because longitudinal investigations of primary HIV-infection is limited by the availability of lymphoid organs, we studied the tissue-specific B-cell dysfunctions in acutely simian immunodeficiency virus (SIV mac251-infected Cynomolgus macaques. METHODS AND FINDINGS: Experiments were performed on three groups of macaques infected for 14, 21 or 28 days and on three groups of animals treated with HAART for two-weeks either initiated at 4 h, 7 or 14 days post-infection (p.i.. We have simultaneously compared changes in B-cell phenotypes and functions and tissue organization of B-cell areas in various lymphoid organs. We showed that SIV induced a steady decline in SIgG-expressing memory (SIgD(-CD27(+ B-cells in spleen and lymph nodes during the first 4 weeks of infection, concomitant to selective homing/sequestration of B-cells to the small intestine and spleen. SIV non-specific Ig production was transiently increased before D14p.i., whereas SIV-specific Ig production was only detectable after D14p.i., coinciding with the presence of CD8(+ T-cells and IgG-expressing plasma cells within germinal centres. Transient B-cell apoptosis on D14p.i. and commitment to terminal differentiation contributed to memory B-cell loss. HAART abrogated B-cell apoptosis, homing to the small intestine and SIV-specific Ig production but had minimal effect on early Ig production, increased B-cell proportions in spleen and loss of memory B-cells. Therefore, virus-B-cell interactions and SIV-induced inflammatory cytokines may differently contribute to early B-cell dysfunction and impaired SIV/HIV-specific antibody response. CONCLUSIONS: These data establish tissue-specific impairments in B-cell trafficking and functions and a generalized and steady memory B-cell loss in secondary lymphoid

  14. Sleeping beauty system to redirect T-cell specificity for human applications.

    Science.gov (United States)

    Maiti, Sourindra N; Huls, Helen; Singh, Harjeet; Dawson, Margaret; Figliola, Matthew; Olivares, Simon; Rao, Pullavathi; Zhao, Yi Jue; Multani, Asha; Yang, Ge; Zhang, Ling; Crossland, Denise; Ang, Sonny; Torikai, Hiroki; Rabinovich, Brian; Lee, Dean A; Kebriaei, Partow; Hackett, Perry; Champlin, Richard E; Cooper, Laurence J N

    2013-02-01

    The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

  15. Human embryonic stem cells differentiated to lung lineage-specific cells ameliorate pulmonary fibrosis in a xenograft transplant mouse model.

    Directory of Open Access Journals (Sweden)

    Ena Ray Banerjee

    Full Text Available Our aim was to differentiate human (h embryonic stem (ES cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI and type II (AEII cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF. A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.Omission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC(-/- mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.This study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.

  16. Variability in Beta-Adrenergic Receptor Population in Cultured Chicken Muscle Cells

    Science.gov (United States)

    Young, Ronald B; Bridge, Kristin Y.; Vaughn, Jeffrey R.

    1998-01-01

    Investigations into expression of the beta-adrenergic receptor (bAR) in chicken skeletal muscle cells in culture were initiated because several beta-adrenergic receptor agonists are known to increase skeletal muscle protein deposition in avian and mammalian species. During initial attempts to study the bAR population on the surface of chicken skeletal muscle cells, we observed a high degree of variability that was later found to be the result of using different batches of horse serum in the cell culture media. The separation between total binding and nonspecific binding in cells grown in two serum samples was approximately two-fold The number of nuclei within multinucleated myotubes was not significantly different in cells grown in the two serum samples. To investigate whether these two sera had an effect on coupling efficiency between bAR population and cAMP production, the ability of these cells to synthesize cAMP was also assessed. Despite the two-fold difference in receptor population, the ability of these cells to synthesize cAMP was not significantly different. Because of the possible link between bAR population and muscle protein, we also determined if the quantity of the major skeletal muscle protein, myosin, was affected by conditions that so drastically affected the bAR population. The quantity of myosin heavy chain was not significantly different.

  17. Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.

    Science.gov (United States)

    Droms, K; Sueoka, N

    1987-01-01

    This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP. This indicates that conversion of RT4-AC to the derivative cell types is also a branch point for the regulation of cell-type-specific properties whose expression is responsive to cAMP. Thus, the potential for maturation in response to increased cAMP is a property that segregates in a cell-type-specific manner and is activated at the determinational level in this system. Images PMID:3029777

  18. T helper cell subsets specific for Pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    Hannah K Bayes

    Full Text Available We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis.Peripheral blood human memory CD4(+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines.Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4(+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6(+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood.Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions.

  19. Quantifying biomass changes of single CD8+ T cells during antigen specific cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Thomas A Zangle

    Full Text Available Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI, a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20-60% over 1-4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2-3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

  20. Influence of cell-to-cell variability on spatial pattern formation

    NARCIS (Netherlands)

    Greese, B.; Wester, K.; Bensch, R.; Ronneberger, O.; Timmer, J.; Huulskamp, M.; Fleck, C.

    2012-01-01

    Many spatial patterns in biology arise through differentiation of selected cells within a tissue, which is regulated by a genetic network. This is specified by its structure, parameterisation and the noise on its components and reactions. The latter, in particular, is not well examined because it is

  1. Cadherin-mediated cell sorting not determined by binding or adhesion specificity

    OpenAIRE

    Niessen, Carien M; Gumbiner, Barry M.

    2002-01-01

    Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domai...

  2. Primed antigen-specific CD4+ T cells are required for NK cell activation in vivo upon Leishmania major infection.

    Science.gov (United States)

    Bihl, Franck; Pecheur, Julien; Bréart, Béatrice; Poupon, Gwenola; Cazareth, Julie; Julia, Valérie; Glaichenhaus, Nicolas; Braud, Véronique M

    2010-08-15

    The ability of NK cells to rapidly produce IFN-gamma is an important innate mechanism of resistance to many pathogens including Leishmania major. Molecular and cellular components involved in NK cell activation in vivo are still poorly defined, although a central role for dendritic cells has been described. In this study, we demonstrate that Ag-specific CD4(+) T cells are required to initiate NK cell activation early on in draining lymph nodes of L. major-infected mice. We show that early IFN-gamma secretion by NK cells is controlled by IL-2 and IL-12 and is dependent on CD40/CD40L interaction. These findings suggest that newly primed Ag-specific CD4(+) T cells could directly activate NK cells through the secretion of IL-2 but also indirectly through the regulation of IL-12 secretion by dendritic cells. Our results reveal an unappreciated role for Ag-specific CD4(+) T cells in the initiation of NK cell activation in vivo upon L. major infection and demonstrate bidirectional regulations between innate and adaptive immunity.

  3. Neuroantigen-specific autoregulatory CD8+ T cells inhibit autoimmune demyelination through modulation of dendritic cell function.

    Directory of Open Access Journals (Sweden)

    Venkatesh P Kashi

    Full Text Available Experimental autoimmune encephalomyelitis (EAE is a well-established murine model of multiple sclerosis, an immune-mediated demyelinating disorder of the central nervous system (CNS. We have previously shown that CNS-specific CD8+ T cells (CNS-CD8+ ameliorate EAE, at least in part through modulation of CNS-specific CD4+ T cell responses. In this study, we show that CNS-CD8+ also modulate the function of CD11c+ dendritic cells (DC, but not other APCs such as CD11b+ monocytes or B220+ B cells. DC from mice receiving either myelin oligodendrocyte glycoprotein-specific CD8+ (MOG-CD8+ or proteolipid protein-specific CD8+ (PLP-CD8+ T cells were rendered inefficient in priming T cell responses from naïve CD4+ T cells (OT-II or supporting recall responses from CNS-specific CD4+ T cells. CNS-CD8+ did not alter DC subset distribution or MHC class II and CD86 expression, suggesting that DC maturation was not affected. However, the cytokine profile of DC from CNS-CD8+ recipients showed lower IL-12 and higher IL-10 production. These functions were not modulated in the absence of immunization with CD8-cognate antigen, suggesting an antigen-specific mechanism likely requiring CNS-CD8-DC interaction. Interestingly, blockade of IL-10 in vitro rescued CD4+ proliferation and in vivo expression of IL-10 was necessary for the suppression of EAE by MOG-CD8+. These studies demonstrate a complex interplay between CNS-specific CD8+ T cells, DC and pathogenic CD4+ T cells, with important implications for therapeutic interventions in this disease.

  4. Detection and quantification of drug-specific T cells in penicillin allergy.

    Science.gov (United States)

    Rozieres, A; Hennino, A; Rodet, K; Gutowski, M-C; Gunera-Saad, N; Berard, F; Cozon, G; Bienvenu, J; Nicolas, J-F

    2009-04-01

    Drug allergic reactions presenting as maculo-papular exanthema (MPE) are mediated by drug-specific T cells. In this study, the frequency of circulating specific T cells was analyzed by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay in 22 patients with an allergic MPE to amoxicillin (amox). Amox-specific circulating T cells were detected in 20/22 patients with frequencies ranging from 1 : 8000 to 1 : 30 000 circulating leucocytes. No reactivity was observed in 46 control patients, including 15 patients with immunoglobulin E-mediated allergy to amoxicillin, 11 patients with a history of drug-induced MPE but tolerant to amoxicillin and 20 healthy individuals. Furthermore, amox-specific T cells were still detectable several years after the occurrence of the allergic reaction even after strict drug avoidance. Finally, analysis of drug-specific T cells in one patient allergic to ticarcillin (a penicillin antibiotic distinct from amox) revealed the presence of IFN-gamma-producing T cells reactive to ticarcillin and several other betalactam antibiotics, suggesting that the IFN-gamma ELISPOT assay is able to detect T cell cross-reactivity against chemically related drugs. These findings confirm that drug-induced MPE is associated with the presence of specific T cells in blood and further suggest that the IFN-gamma ELISPOT is a sensitive assay which could improve the diagnosis of betalactam allergy.

  5. Virus-specific T cells as correlate of (cross-)protective immunity against influenza.

    Science.gov (United States)

    Altenburg, Arwen F; Rimmelzwaan, Guus F; de Vries, Rory D

    2015-01-15

    Since inactivated influenza vaccines mainly confer protective immunity by inducing strain-specific antibodies to the viral hemagglutinin, these vaccines only afford protection against infection with antigenically matching influenza virus strains. Due to the continuous emergence of antigenic drift variants of seasonal influenza viruses and the inevitable future emergence of pandemic influenza viruses, there is considerable interest in the development of influenza vaccines that induce broader protective immunity. It has long been recognized that influenza virus-specific CD8(+) T cells directed to epitopes located in the relatively conserved internal proteins can cross-react with various subtypes of influenza A virus. This implies that these CD8(+) T cells, induced by prior influenza virus infections or vaccinations, could afford heterosubtypic immunity. Furthermore, influenza virus-specific CD4(+) T cells have been shown to be important in protection from infection, either via direct cytotoxic effects or indirectly by providing help to B cells and CD8(+) T cells. In the present paper, we review the induction of virus-specific T cell responses by influenza virus infection and the role of virus-specific CD4(+) and CD8(+) T cells in viral clearance and conferring protection from subsequent infections with homologous or heterologous influenza virus strains. Furthermore, we discuss vector-based vaccination strategies that aim at the induction of a cross-reactive virus-specific T cell response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Specific gravity of hybrid poplars in the north-central region, USA: within-tree variability and site × genotype effects

    Science.gov (United States)

    William L. Headlee; Ronald S. Jr. Zalesny; Richard B. Hall; Edmund O. Bauer; Bradford Bender; Bruce A. Birr; Raymond O. Miller; Jesse A. Randall; Adam H. Wiese

    2013-01-01

    Specific gravity is an important consideration for traditional uses of hybrid poplars for pulp and solid wood products, as well as for biofuels and bioenergy production. While specific gravity has been shown to be under strong genetic control and subject to within-tree variability, the role of genotype × environment interactions is poorly understood. Most...

  7. L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

    Science.gov (United States)

    Hong, Hao; Brown, Christine E; Ostberg, Julie R; Priceman, Saul J; Chang, Wen-Chung; Weng, Lihong; Lin, Paul; Wakabayashi, Mark T; Jensen, Michael C; Forman, Stephen J

    2016-01-01

    New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.

  8. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

    Science.gov (United States)

    Lin, Weiming; Modiano, Jaime F; Ito, Daisuke

    2017-03-30

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1(-) and SSEA-1(+) cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

  9. Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand

    Directory of Open Access Journals (Sweden)

    Li Bin

    2011-01-01

    Full Text Available Abstract Objective To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells. Experimental Design The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging. Results PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control. Conclusions and Clinical Relevance PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

  10. A mouse variable gene fragment binds to DNA independently of the BCR context: a possible role for immature B-cell repertoire establishment.

    Directory of Open Access Journals (Sweden)

    Andrea Queiroz Maranhão

    Full Text Available B-cell maturation occurs in several steps and requires constant stimulus for its continuing development. From the emergence of the pre-B-cell receptor, signal transduction stimulates and supports B-cell development. Current viewpoints indicate that both positive selection pressure for autoantigens and tonic signaling constitutively stimulate B-cell maturation. In this work, we tested for the presence of a putative DNA binding site in a variable gene segment in a germline configuration, independently of VDJ recombination. After a survey of the public antibody databases, we chose a single mouse heavy variable gene segment that is highly represented in anti-nucleic acid antibodies and tested it for ssDNA binding. A phage display approach was used to search for intrinsic binding to oligo deoxythymidine. The results revealed that binding to an antigen can be influenced by the use of a specific DNA binding V[Formula: see text] gene segment. Our data support the idea that some variable genes have intrinsic reactivity towards specific types of endogenous autoantigens, and this property may contribute to the establishment of the immature B-cell repertoire.

  11. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice.

    Science.gov (United States)

    Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

    2016-02-09

    A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy.

  12. Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Schlimper

    2012-01-01

    Full Text Available Adoptive therapy of malignant diseases with cytokine-induced killer (CIK cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR with an antibody-defined specificity for carcinoembryonic antigen (CEA. CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

  13. The Natural Selection of Herpesviruses and Virus-Specific NK Cell Receptors

    Directory of Open Access Journals (Sweden)

    Joseph C. Sun

    2009-10-01

    Full Text Available During the co-evolution of cytomegalovirus (CMV and natural killer (NK cells, each has evolved specific tactics in an attempt to prevail. CMV has evolved multiple immune evasion mechanisms to avoid detection by NK cells and other immune cells, leading to chronic infection. Meanwhile, the host has evolved virus-specific receptors to counter these evasion strategies. The natural selection of viral genes and host receptors allows us to observe a unique molecular example of "survival of the fittest", as virus and immune cells try to out-maneuver one another or for the virus to achieve détente for optimal dissemination in the population.

  14. Near infrared photoimmunotherapy rapidly elicits specific host immunity against cancer cells (Conference Presentation)

    Science.gov (United States)

    Kobayashi, Hisataka

    2017-02-01

    Near infrared photoimmunotherapy (NIR-PIT) is a new molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (mAb) targeting cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic/immunogenic cell death (ICD) only in target-positive, mAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent target-negative cells are unharmed. Dynamic 3D-microscopy of live tumor cells undergoing NIR-PIT showed rapid swelling in treated cells immediately after light exposure, followed by irreversible morphologic changes such as bleb formation, and rupture of vesicles within several minutes. Furthermore, biological markers of ICD including relocation of HSP70/90 and calreticulin, and release of ATP and High Mobility Group Box 1 (HMGB1), were clearly detected immediately after NIR-PIT. When NIR-PIT was performed in a mixture of cancer cells and immature dendritic cells, maturation of immature dendritic cells was strongly induced rapidly after NIR-PIT. Alternatively, NIR-PIT can also target negative regulatory immune cells such as Treg only in the tumor bed. Treg targeting NIR-PIT against CD25 can deplete >80% of Treg in tumor bed within 20 min that induces activation of tumor cell-specific CD8+-T and NK cells within 1.5 hour, and then these activated cells killed cancer cells in local tumor within 1 day and also in distant tumors of the same cell origin within 2 days. In summary, cancer cell-targeting and immuno-suppressor cell-targeting NIR-PITs effectively induce innate and acquired immunity specifically against cancer cells growing in patients, respectively.

  15. Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes

    Science.gov (United States)

    Hazan-Halevy, Inbal; Rosenblum, Daniel; Weinstein, Shiri; Bairey, Osnat; Raanani, Pia; Peer, Dan

    2015-01-01

    Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib®), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities are needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients’ exosomes were taken up by both healthy and patients’ B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies. PMID:25933830

  16. Specific and Complex Reprogramming of Cellular Metabolism in Myeloid Cells during Innate Immune Responses.

    Science.gov (United States)

    Stienstra, Rinke; Netea-Maier, Romana T; Riksen, Niels P; Joosten, Leo A B; Netea, Mihai G

    2017-07-05

    Renewed interest in immune cell metabolism has led to the emergence of a research field aimed at studying the importance of metabolic processes for an effective immune response. In addition to the adaptive immune system, cells of the myeloid lineage have been shown to undergo robust metabolic changes upon activation. Whereas the specific metabolic requirements of myeloid cells after lipopolysaccharide/TLR4 stimulation have been extensively studied, recent evidence suggested that this model does not represent a metabolic blueprint for activated myeloid cells. Instead, different microbial stimuli, pathogens, or tissue microenvironments lead to specific and complex metabolic rewiring of myeloid cells. Here we present an overview of the metabolic heterogeneity in activated myeloid cells during health and disease. Directions for future research are suggested to ultimately provide new therapeutic opportunities. The uniqueness of metabolic signatures accompanying different conditions will require tailor-made interventions to ultimately modulate aberrant myeloid cell activation during disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. HaloTag protein-mediated specific labeling of living cells with quantum dots.

    Science.gov (United States)

    So, Min-kyung; Yao, Hequan; Rao, Jianghong

    2008-09-26

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging.

  18. Specification of neuronal and glial subtypes from human pluripotent stem cells

    Science.gov (United States)

    Liu, Huisheng; Zhang, Su-Chun

    2011-01-01

    Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a dynamic tool for revealing early embryonic development, modeling pathological processes, and developing therapeutics through drug discovery and potential cell replacement. The first step toward the utilities of human PSCs is directed differentiation to functionally specialized cell tissue types. Following developmental principles, human ESCs, and lately iPSCs, have been effectively differentiated to region-and/or transmitter-specific neuronal and glial types, including cerebral glutamatergic, striatal γ-aminobutyric acid (GABA)-ergic, forebrain cholinergic, midbrain dopaminergic, and spinal motor neurons, as well as astrocytes and oligodendrocytes. These studies also reveal unique aspects of human cell biology, including intrinsically programmed developmental course, differential uses of transcription factors for neuroectoderm specification, and distinct responses to extracellular signals in regulating cell fate. Such information will be instrumental for translating biological findings to therapeutic development. PMID:21786144

  19. Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways.

    Science.gov (United States)

    Suhovskih, Anastasia V; Kashuba, Vladimir I; Klein, George; Grigorieva, Elvira V

    2017-01-02

    Microenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

  20. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

  1. Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.

    OpenAIRE

    Droms, K; Sueoka, N

    1987-01-01

    This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of...

  2. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    Science.gov (United States)

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko

    2010-12-15

    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

  3. Influence of host genetic variation on rubella-specific T cell cytokine responses following rubella vaccination.

    Science.gov (United States)

    Ovsyannikova, Inna G; Ryan, Jenna E; Vierkant, Robert A; O'Byrne, Megan M; Pankratz, V Shane; Jacobson, Robert M; Poland, Gregory A

    2009-05-26

    The variability of immune response modulated by immune response gene polymorphisms is a significant factor in the protective effect of vaccines. We studied the association between cellular (cytokine) immunity and HLA genes among 738 schoolchildren (396 males and 342 females) between the ages of 11 and 19 years, who received two doses of rubella vaccine (Merck). Cytokine secretion levels in response to rubella virus stimulation were determined in PBMC cultures by ELISA. Cell supernatants were assayed for Th1 (IFN-gamma, IL-2, and IL-12p40), Th2 (IL-4, IL-5, and IL-10), and innate/proinflammatory (TNF-alpha, GM-CSF, and IL-6) cytokines. We found a strong association between multiple alleles of the HLA-DQA1 (global p-value 0.022) and HLA-DQB1 (global p-value 0.007) loci and variations in rubella-specific IL-2 cytokine secretion. Additionally, the relationships between alleles of the HLA-A (global p-value 0.058), HLA-B (global p-value 0.035), and HLA-C (global p-value 0.023) loci and TNF-alpha secretion suggest the importance of HLA class I molecules in innate/inflammatory immune response. Better characterization of these genetic profiles could help to predict immune responses at the individual and population level, provide data on mechanisms of immune response development, and further inform vaccine development and vaccination policies.

  4. Methodologies for the analysis of HCV-specific CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Megha eLokhande

    2015-02-01

    Full Text Available Virus-specific CD4+ T cells play a major role in viral infections, such as hepatitis C virus (HCV. Viral clearance is associated with vigorous and multispecific CD4+ T cell responses, while chronic infection has been shown to be associated with weak or absent T cell responses. Most of these studies have used functional assays to analyse virus-specific CD4+ T cell responses; however, these and other detection methods have various limitations. Therefore, the important question of whether virus-specific CD4+ T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analysed in detail. A novel assay, in which virus-specific CD4+ T cell frequencies can be determined by de novo CD154 (CD40 ligand expression in response to viral antigens, can help to overcome some of the limitations of functional assays and restrictions of multimer-based methods. This and other current established methods for the detection of HCV-specific CD4+ T cells will be discussed in this review.

  5. NK cells controlling virus-specific T cells: Rheostats for acute vs. persistent infections.

    Science.gov (United States)

    Welsh, Raymond M; Waggoner, Stephen N

    2013-01-05

    Viral infections characteristically induce a cytokine-driven activated natural killer (NK) cell response that precedes an antigen-driven T cell response. These NK cells can restrain some but not all viral infections by attacking virus-infected cells and can thereby provide time for an effective T cell response to mobilize. Recent studies have revealed an additional immunoregulatory role for the NK cells, where they inhibit the size and functionality of the T cell response, regardless of whether the viruses are themselves sensitive to NK cells. This subsequent change in T cell dynamics can alter patterns of immunopathology and persistence and implicates NK cells as rheostat-like regulators of persistent infections. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Mónica Serrano

    2015-04-01

    Full Text Available Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

  7. HIV-specific CD8+ T cells: serial killers condemned to die?

    Science.gov (United States)

    Petrovas, Constantinos; Mueller, Yvonne M; Katsikis, Peter D

    2004-04-01

    An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.

  8. Production of antigen-specific contrasuppressor cells and factor, and their use in augmentation of cell-mediated immunity.

    Science.gov (United States)

    Ptak, W; Bereta, M; Marcinkiewicz, J; Gershon, R K; Green, D R

    1984-08-01

    A single injection of TNP-labeled mouse gamma-globulin (TNP-IgG) can render the contact sensitivity response of mice resistant to suppressor cells (Tsc) and their biologically active cellfree products (TsF). Lyt-1 T cells of mice treated with TNP-IgG can protect the adoptive contact sensitivity response of immune cells from the antigen-specific suppressive effect produced by the addition of antigen-specific TsF or Tsc. When T cells of TNP-IgG-treated mice are put into culture, they produce an antigen-specific contrasuppressor factor (TcsF) that can replace the activity of the cells. When immune cells are preincubated in vitro with TcsF, they become refractory to Tsc and TsF added subsequently. The TcsF, however, has no ability to restore responsiveness to immune cells that had been previously exposed to TsF. The TcsF binds specifically to TNP, expresses an I-J-controlled determinant, and does not express standard determinants found on mouse Ig. The treatment that primes the contrasuppressor system to protect the contact sensitivity response also reportedly renders the antibody-producing system tolerant, (i.e., produces so called "split tolerance"). These results are discussed in light of the possibility that the contrasuppressor system can be responsible for so called isotype-specific immunity by rendering one arm of the immune system resistant to generalized suppressive mechanisms.

  9. Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes.

    Science.gov (United States)

    Ma, Hailun; Sumbilla, Carlota M; Farrance, Iain K G; Klein, Michael G; Inesi, Giuseppe

    2004-03-01

    We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+ -ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.

  10. Age related differences in dynamics of specific memory B cell populations after clinical pertussis infection.

    Directory of Open Access Journals (Sweden)

    Inonge van Twillert

    Full Text Available For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (Bmem cells were differentiated into IgG antibody secreting cells (ASC by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx, filamentous haemagglutinin (FHA and pertactin (Prn. Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and Bmem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of Bmem cell populations; higher levels of Bmem cells specific for Ptx and FHA were reached in adults and (pre- elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects' levels of specific Bmem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific Bmem cell and plasma IgG levels.

  11. The Influence of T Cell Development on Pathogen Specificity and Autoreactivity

    Science.gov (United States)

    Košmrlj, Andrej; Kardar, Mehran; Chakraborty, Arup K.

    2012-10-01

    T cells orchestrate adaptive immune responses upon activation. T cell activation requires sufficiently strong binding of T cell receptors on their surface to short peptides derived from foreign proteins bound to protein products of the major histocompatibility (MHC) gene products, which are displayed on the surface of antigen presenting cells. T cells can also interact with peptide-MHC complexes, where the peptide is derived from host (self) proteins. A diverse repertoire of relatively self-tolerant T cell receptors is selected in the thymus. We study a model, computationally and analytically, to describe how thymic selection shapes the repertoire of T cell receptors, such that T cell receptor recognition of pathogenic peptides is both specific and degenerate. We also discuss the escape probability of autoimmune T cells from the thymus.

  12. Cadherin-mediated cell sorting not determined by binding or adhesion specificity

    Science.gov (United States)

    Niessen, Carien M.; Gumbiner, Barry M.

    2002-01-01

    Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity. PMID:11790800

  13. MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells

    DEFF Research Database (Denmark)

    Labuda, Tord; Christensen, Jan Pravsgaard; Rasmussen, Susanne

    2006-01-01

    in the generation of a virus-specific immune response. Mekk1(DeltaKD) mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8(+) T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8(+) T cell expansion in Mekk1......(DeltaKD) mice following VSV infection is virus-specific and the frequency of virus-specific T cells is significantly higher (more than threefold) in Mekk1(DeltaKD) as compared to WT animals. Moreover, the hyper-expansion of T cells seen in Mekk1(DeltaKD) mice after VSV infection is a result of increased...... proliferation, since a significantly higher percentage of virus-specific Mekk1(DeltaKD) CD8(+) T cells incorporated BrdU as compared to virus-specific WT CD8(+) T cells. In contrast, similar levels of apoptosis were detected in Mekk1(DeltaKD) and WT T cells following VSV infection. These results strongly...

  14. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

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    Kerstin Trautwein-Weidner

    2015-10-01

    Full Text Available 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.

  15. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

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    Chenggang Lu

    2015-12-01

    Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  16. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Science.gov (United States)

    Lu, Chenggang; Fuller, Margaret T

    2015-12-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  17. Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS.

    Science.gov (United States)

    Yamamoto, Kotaro; Takahashi, Katsutoshi; Mizuno, Hajime; Anegawa, Aya; Ishizaki, Kimitsune; Fukaki, Hidehiro; Ohnishi, Miwa; Yamazaki, Mami; Masujima, Tsutomu; Mimura, Tetsuro

    2016-04-05

    Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

  18. Chromatin states modify network motifs contributing to cell-specific functions

    Science.gov (United States)

    Zhao, Hongying; Liu, Tingting; Liu, Ling; Zhang, Guanxiong; Pang, Lin; Yu, Fulong; Fan, Huihui; Ping, Yanyan; Wang, Li; Xu, Chaohan; Xiao, Yun; Li, Xia

    2015-01-01

    Epigenetic modification can affect many important biological processes, such as cell proliferation and apoptosis. It can alter chromatin conformation and contribute to gene regulation. To investigate how chromatin states associated with network motifs, we assembled chromatin state-modified regulatory networks by combining 269 ChIP-seq data and chromatin states in four cell types. We found that many chromatin states were significantly associated with network motifs, especially for feedforward loops (FFLs). These distinct chromatin state compositions contribute to different expression levels and translational control of targets in FFLs. Strikingly, the chromatin state-modified FFLs were highly cell-specific and, to a large extent, determined cell-selective functions, such as the embryonic stem cell-specific bivalent modification-related FFL with an important role in poising developmentally important genes for expression. Besides, comparisons of chromatin state-modified FFLs between cancerous/stem and primary cell lines revealed specific type of chromatin state alterations that may act together with motif structural changes cooperatively contribute to cell-to-cell functional differences. Combination of these alterations could be helpful in prioritizing candidate genes. Together, this work highlights that a dynamic epigenetic dimension can help network motifs to control cell-specific functions. PMID:26169043

  19. Identification of T-cell stimulating antigens from Giardia lamblia by using Giardia-specific T-cell hybridomas.

    Science.gov (United States)

    Astiazaran-Garcia, H; Quintero, J; Vega, R; Briceño, P; Oviedo, C; Rascon, L; Garibay-Escobar, A; Castillo-Yañez, F J; Robles-Zepeda, R; Hernandez, J; Velazquez, C

    2009-03-01

    T-cell immune response plays an important role in controlling Giardia lamblia infections. Little is known about the G. lamblia-specific antigens that stimulate a cell-mediated immune response. The aim of the present study was to identify T-cell stimulating G. lamblia antigens. For this purpose, we generated a group of Giardia-specific T-cell hybridomas (2F9, 4D5, 6D10, 8B9, 9B10, 10F7 and 10G5). Hybridomas were screened for reactivity with G. lamblia protein extract by the CTLL bioassay. These T-cell hybridomas did not exhibit any significant activation either in the absence of G. lamblia protein extract or in the presence of irrelevant antigen (hen white egg lysozyme). To further characterize the T-cell hybridomas generated, we selected three hybridomas (10G5, 4D5 and 9B10). Giardia lamblia proteins of 90-110, 65-77 and 40-64 kDa showed T-cell stimulating activity for the hybridomas 10G5, 4D5 and 9B10, respectively, in a concentration-dependent manner. Protein extract obtained from different G. lamblia strains (GS/M-83-H7, WB C6 and a clinical isolate (YJJ)) stimulated all T-cell hybridomas, indicating that T-cell-stimulating antigens are expressed among different G. lamblia strains. In conclusion, we identified T-cell stimulating G. lamblia antigens by using Giardia-specific T-cell hybridomas. To our knowledge, these hybridomas are the first-described T-cell hybridomas specific for G. lamblia.

  20. Dendritic cells recognize tumor-specific glycosylation of carcinoembryonic antigen on colorectal cancer cells through dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin

    NARCIS (Netherlands)

    van Gisbergen, Klaas P. J. M.; Aarnoudse, Corlien A.; Meijer, Gerrit A.; Geijtenbeek, Teunis B. H.; van Kooyk, Yvette

    2005-01-01

    Dendritic cells play a pivotal role in the induction of antitumor immune responses. Immature dendritic cells are located intratumorally within colorectal cancer and intimately interact with tumor cells, whereas mature dendritic cells are present peripheral to the tumor. The majority of colorectal

  1. Intraobserver and Interobserver Variability of Fuhrman and Modified Fuhrman Grading Systems for Conventional Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Sibel Bektas

    2009-11-01

    Full Text Available The Fuhrman nuclear grade is the most widely used grading system for renal cell carcinoma. The aim of this study was to evaluate the intraobserver and interobserver variability of the Fuhrman and modified Fuhrman grading systems for conventional renal cell carcinoma. In this study, five pathologists independently classified 110 cases of conventional renal cell carcinoma according to the Fuhrman and modified (three- and two-tiered Fuhrman grading systems. The intraobserver and interobserver variability of these systems were assessed using κ statistics. The associations between the Fuhrman and modified Fuhrman grades, pathologic stage and tumor size were determined by correlation analysis. The intraobserver and interobserver combined mean κ values for four-tiered Fuhrman grading were 0.48 and 0.41, respectively. The highest agreement was detected in two-tiered modification (including grades 1 + 2 and 3 + 4; the intraobserver and inter-observer combined mean κ values were 0.67 and 0.62, respectively. Correlations between pathologic stage and tumor size with two-tiered modification (including grades 1 + 2 and 3 + 4 were greater than those in three- and four-tiered Fuhrman grading. Collapsing the Fuhrman grading into a two-tiered scheme improved the intraobserver and interobserver reproducibility.

  2. Highly Variable Plasma Concentrations of Voriconazole in Pediatric Hematopoietic Stem Cell Transplantation Patients

    Science.gov (United States)

    Wolfs, Tom; Jonker, Martine; de Waal, Marjolein; Egberts, Toine C. G.; Ververs, Tessa T.; Boelens, Jaap J.; Bierings, Marc

    2013-01-01

    Invasive fungal infections are of great concern in pediatric hematopoietic stem cell transplantation (HSCT) recipients. Voriconazole is usually the drug of first choice for treating or preventing invasive aspergillosis. Optimum trough levels (Ctroughs) are between 1 and 5 mg/liter. It is unclear whether these levels are reached with currently advised pediatric dosing schedules. Between 2007 and 2011, 11 patients voriconazole in the HSCT unit of UMC Utrecht. For children 12 years) (P = 0.034). The intrapatient variability in Ctrough ranged between 1 and 238%. Voriconazole was discontinued in six patients due to toxicity. These patients had a median Ctrough of 0.5 mg/liter at the initial dose (ranging from 0.5 to 2.6 mg/liter), and a medium maximal concentration of 4 mg/liter was reached. Inter- and intrapatient variability is a major concern in voriconazole treatment and necessitates therapeutic drug monitoring of dosing, especially in young children. PMID:23114771

  3. Antigen-Specific Monoclonal Antibodies Isolated from B Cells Expressing Constitutively Active STAT5

    NARCIS (Netherlands)

    Scheeren, F.A.; van Geelen, C.M.M.; Yasuda, E.; Spits, H.; Beaumont, T.

    2011-01-01

    Background: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. Methodology/Principal Findings: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer

  4. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer.

    Science.gov (United States)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders; Skov, Birgit G

    2014-12-01

    Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR PCR kit, Qiagen, UK; reference method). For immunohistochemistry, antibodies against exon19 deletions (clone 6B6), exon21 mutations (clone 43B2) from Cell Signaling Technology (Boston, USA) and EGFR variantIII (clone 218C9) from Dako (Copenhagen, DK) were applied. Protein expression was evaluated, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94.4-99.4%). Sensitivity of exon19 antibody was 63.2% (95% confidence interval=38.4-83.7%) and of exon21 antibody was 80.0% (95% confidence interval=44.4-97.5%). Seven exon19 and four exon21 mutations were false negatives (immunohistochemistry negative, RT-PCR positive). Two exon19 and three exon21 mutations were false positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC

  5. T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling

    Science.gov (United States)

    Germar, Kristine; Dose, Marei; Konstantinou, Tassos; Zhang, Jiangwen; Wang, Hongfang; Lobry, Camille; Arnett, Kelly L.; Blacklow, Stephen C.; Aifantis, Iannis; Aster, Jon C.; Gounari, Fotini

    2011-01-01

    Although transcriptional programs associated with T-cell specification and commitment have been described, the functional hierarchy and the roles of key regulators in structuring/orchestrating these programs remain unclear. Activation of Notch signaling in uncommitted precursors by the thymic stroma initiates the T-cell differentiation program. One regulator first induced in these precursors is the DNA-binding protein T-cell factor 1 (Tcf-1), a T-cell–specific mediator of Wnt signaling. However, the specific contribution of Tcf-1 to early T-cell development and the signals inducing it in these cells remain unclear. Here we assign functional significance to Tcf-1 as a gatekeeper of T-cell fate and show that Tcf-1 is directly activated by Notch signals. Tcf-1 is required at the earliest phase of T-cell determination for progression beyond the early thymic progenitor stage. The global expression profile of Tcf-1–deficient progenitors indicates that basic processes of DNA metabolism are down-regulated in its absence, and the blocked T-cell progenitors become abortive and die by apoptosis. Our data thus add an important functional relationship to the roadmap of T-cell development. PMID:22109558

  6. Cloning analysis of HBV-specific CD8 T cell receptor gene in patients with acute hepatitis B

    Directory of Open Access Journals (Sweden)

    Ning DING

    2011-05-01

    Full Text Available Objective To investigate the molecular mechanism of T cell receptor(TCR in CD8 T cell-mediated immune response to HBV in patients with acute hepatitis B(AHB.Methods Peripheral blood mononuclear cells(PBMCs were collected from HLA-A2-positive AHB patients.To determine HBsAg183-191 and HBsAg335-343-specific CD8 T cell frequencies,the PBMCs were stained by fluorescence-labeled anti-CD3,anti-CD8 and pentamers,and analyzed by flow cytometry.PBMCs from 6 patients were stimulated with epitopic peptide HBsAg335-343 in vitro for 3 to 4 weeks.HBV-specific CD8 T cells were isolated by magnetic activated cell sorting followed by flow florescence activated cell sorting.The mRNA of sorted cells was extracted after expanding by IL-2,anti-CD3 and anti-CD8.The full-length gene fragments of variable region of TCR α and β chains were gained by 5’-RACE,and then cloned and sequenced(≥50 clones for single chain of each sample.The gene families of TCR α and β chains were identified and the sequence characters of CDR3 were compared.Results Analysis of more than 600 cloned gene sequences of TCR α and β chains showed that the proliferated HBV-specific CD8 T cells from 6 AHB patients presented a predominant expression in TCR α and chains,with 2-4 α chain families and 1-4 chain families in each case.The α2,α14,α15,β3,β13 and 23 families were detected in more than one case.The chain genes were all 13 for all tested clones in one case.For the same α chain or-chain family,CDR3 sequences tended to be identical in one case but different among cases.Conclusions HBV-specific CD8 T cells with antigenic peptide-induced proliferation present predominance in the usage of TCR α and β chains.This property might be one of the important molecular factors influencing anti-HBV immunity.

  7. Rotavirus-specific helper T cell responses in newborns, infants, children, and adults.

    Science.gov (United States)

    Offit, P A; Hoffenberg, E J; Pia, E S; Panackal, P A; Hill, N L

    1992-06-01

    An obstacle to developing a successful rotavirus vaccine has been the inability to consistently correlate the humoral immune response with protection against disease. Transplacental transfer of maternal rotavirus-specific antibodies may obscure the capacity to discriminate an active from a passively acquired humoral immune response in infants. In an attempt to circumvent this problem, an assay was developed to detect rotavirus-specific helper T cells among circulating mononuclear cells. Rotavirus-specific lymphoproliferative responses and rotavirus-specific neutralizing antibody titers in blood were determined in 11 mother/newborn pairs at the time of delivery and in 54 infants, children, and adults ranging in age from 16 days to 40 years. Only 1 of 11 infants tested between 16 days and 6 months of age had detectable rotavirus-specific helper T cell activity whereas 8 of 11 had circulating rotavirus-specific neutralizing antibodies. Acquisition of rotavirus-specific helper T cell activity over the first few years of life correlated with the age at which infants and young children are known to be infected with rotavirus. These findings support the hypothesis that detection of rotavirus-specific lymphoproliferative activity in infants may more accurately determine previous exposure to rotavirus than detection of rotavirus-specific antibodies.

  8. Identification of germ cell-specific VASA and IFITM3 proteins in human ovarian endometriosis.

    Science.gov (United States)

    Fraunhoffer, Nicolas A; Meilerman Abuelafia, Analía; Stella, Inés; Galliano, Silvia; Barrios, Marcela; Vitullo, Alfredo D

    2015-10-07

    Endometriosis is a gynaecological disorder that affects 6-10 % of female population. It is characterized by the presence of endometrial tissue outside the uterus, most often in the pelvic peritoneum or ovaries. Recent studies have indicated that mesenchymal endometrial stem cells might get involved in endometriosis progression. Although germ line stem cells have been proved to exist in the ovary, their involvement in ovarian endometriosis has not been investigated. In this preliminary report we aimed to identify germinal stem cell markers in ovarian endometriosis. Ten paraffin-embedded ovarian endometriosis samples were screened for germ cell-specific proteins DDX4 (VASA) and IFITM3, and its relation with stem cell marker OCT4, proliferation marker PCNA and estrogen receptor alpha (ESR1), by immunohistochemistry, immunofluorescence and PCR. DDX4 and IFITM3 proteins were expressed in isolated cells and clusters of cells in the cortical region of ovarian endometriotic cysts. DDX4 and IFITM3 co-localized in cells from endometriotic stroma, and DDX4/IFITM3-expressing cells were positive for ESR1, OCT4 and PCNA. No cells expressing neither DDX4 nor IFITM3 were detected in normal endometrial tissue. The identification of germ cell-specific proteins DDX4 and IFITM3 provides the first evidence of ovarian-sourced cells in ovarian endometriotic lesions and opens up new directions towards understanding the still confusing pathogenesis of endometriosis.

  9. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    KAUST Repository

    Ghiotto, Fabio

    2011-01-01

    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

  10. Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs.

    Science.gov (United States)

    Niehoff, Julius; Matzkies, Matthias; Nguemo, Filomain; Hescheler, Jürgen; Reppel, Michael

    2016-01-01

    Heart rate variability (HRV) refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV) that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC)-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. The Microelectrode Array (MEA) technique was used to perform short-term recordings of extracellular field potentials (FPs) of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, αPig-44). Offline analysis was focused on time domain and nonlinear methods. The Poincaré-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 ± 8.7% of control at 10(-9) M, p < 0.05) and increased variability measures at higher concentrations (SDNN 258.8 ± 42.7% of control at 10(-5) M, p < 0.05). Amiodarone and Metoprolol did not alter BRV significantly. Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening. © 2016 The Author(s) Published by S. Karger AG, Basel.

  11. Concanavalin A-induced activation of lymphocytic choriomeningitis virus memory lymphocytes into specifically cytotoxic T cells

    DEFF Research Database (Denmark)

    Marker, O; Thomsen, Allan Randrup; Andersen, G T

    1977-01-01

    When spleen cells, which have been primed to Lymphocytic Choriomeningitis (LCM) virus during a primary infection several months previously, are stimulated in vitro with Con A. highly specific secondary cytotoxic effector cells are generated. The degree of cytotoxicity revealed by such Con A-stimu......-stimulated cells is higher than that of non-incubated spleen cells harvested nine days following the primary infection, and the effect is totally inhibited by anti-theta serum plus complement treatment of the effector cells immediately before the cytotoxic test....

  12. Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

    Science.gov (United States)

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

    2016-09-01

    Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction

    NARCIS (Netherlands)

    Mooijman, Dylan; Dey, Siddharth S; Boisset, Jean-Charles; Crosetto, Nicola; van Oudenaarden, Alexander

    2016-01-01

    The epigenetic DNA modification 5-hydroxymethylcytosine (5hmC) has crucial roles in development and gene regulation. Quantifying the abundance of this epigenetic mark at the single-cell level could enable us to understand its roles. We present a single-cell, genome-wide and strand-specific 5hmC

  14. Selection of scFvs specific for the HepG2 cell line using ribosome ...

    Indian Academy of Sciences (India)

    The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k ...

  15. A new marker set that identifies fetal cells in maternal circulation with high specificity

    DEFF Research Database (Denmark)

    Hatt, Lotte; Brinch, Marie; Singh, Ripudaman

    2014-01-01

    OBJECTIVE: Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. METHOD: Blood samples from 78 normal pregnancies...... were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. RESULTS: Fetal...... or more fetal cells with FISH signals could be found in a sample. CONCLUSION: The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic. © 2014 John Wiley & Sons, Ltd....

  16. Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance.

    Science.gov (United States)

    Leborgne, N; Teulieres, C; Travert, S; Rols, M P; Teissie, J; Boudet, A M

    1995-05-01

    The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long-term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

  17. Bordetella pertussis attachment to respiratory epithelial cells can be impaired by fimbriae-specific antibodies

    NARCIS (Netherlands)

    Rodriguez, ME; Hellwig, SMM; Vidakovics, MLAP; Berbers, GAM; van de Winkel, JGJ

    Bordetella pertussis attachment to host cells is a crucial step in colonization. In this study, we investigated the specificity of antibodies, induced either by vaccination or infection, capable of reducing bacterial adherence to respiratory epithelial cells. Both sera and purified anti-B. pertussis

  18. Concanavalin A-induced activation of lymphocytic choriomeningitis virus memory lymphocytes into specifically cytotoxic T cells

    DEFF Research Database (Denmark)

    Marker, O; Thomsen, Allan Randrup; Andersen, G T

    1977-01-01

    When spleen cells, which have been primed to Lymphocytic Choriomeningitis (LCM) virus during a primary infection several months previously, are stimulated in vitro with Con A. highly specific secondary cytotoxic effector cells are generated. The degree of cytotoxicity revealed by such Con A...

  19. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    WINTEC

    Abstract. The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepa- titis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology model- ling and ...

  20. DC-ATLAS : a systems biology resource to dissect receptor specific signal transduction in dendritic cells

    NARCIS (Netherlands)

    Cavalieri, D.; Rivero, D.; Beltrame, L.; Buschow, S.I.; Calura, E.; Rizzetto, L.; Gessani, S.; Gauzzi, M.C.; Reith, W.; Baur, A.; Bonaiuti, R.; Brandizi, M.; Filippo, C. De; D'Oro, U.; Draghici, S.; Dunand-Sauthier, I.; Gatti, E.; Granucci, F.; Gundel, M.; Kramer, M.; Kuka, M.; Lanyi, A.; Melief, C.J.; Montfoort, N. van; Ostuni, R.; Pierre, P.; Popovici, R.; Rajnavolgyi, E.; Schierer, S.; Schuler, G.; Soumelis, V.; Splendiani, A.; Stefanini, I.; Torcia, M.G.; Zanoni, I.; Zollinger, R.; Figdor, C.G.; Austyn, J.M.

    2010-01-01

    BACKGROUND: The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research,

  1. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...

  2. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

  3. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    NARCIS (Netherlands)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated

  4. A novel conditional Aire allele enables cell-specific ablation of the immune tolerance regulator Aire.

    Science.gov (United States)

    Dobeš, Jan; Edenhofer, Frank; Vobořil, Matouš; Brabec, Tomáš; Dobešová, Martina; Čepková, Adéla; Klein, Ludger; Rajewsky, Klaus; Filipp, Dominik

    2017-11-30

    Medullary thymic epithelial cell (mTEC)-restricted expression of autoimmune regulator (Aire) is essential for establishment of immune tolerance. Recently, Aire was also shown to be expressed in cells of hematopietic and reproductive lineages. Thus, the generation of Aire fl/fl mouse strain enables the investigation of the cell-specific function of Aire. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. HaloTag Protein-Mediated Specific Labeling of Living Cells with Quantum Dots

    OpenAIRE

    So, Min-kyung; Yao, Hequan; Rao, Jianghong

    2008-01-01

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand...

  6. Endometrial natural killer (NK) cells reveal a tissue-specific receptor repertoire.

    Science.gov (United States)

    Feyaerts, D; Kuret, T; van Cranenbroek, B; van der Zeeuw-Hingrez, S; van der Heijden, O W H; van der Meer, A; Joosten, I; van der Molen, R G

    2018-02-13

    Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR

  7. Specific photoimmuno-theranostics for detection and elimination of skin cancer cells

    OpenAIRE

    Barth, Stefan; Fischer, Rainer; Hussain, Ahmad

    2016-01-01

    The technology provided herein generally relates to novel specific photoimmuno-theranostics for the use in detection and elimination of skin cancer cells. The technology also relates to novel methods which generate homogeneous and specific photoimmuno-theranostics reagents in a simple, controlled and efficient way. This method combines molecular optical imaging, photodynamic therapy and immunotherapy using SNAP-tag technology.

  8. Associations between pathogen-specific clinical mastitis and somatic cell count patterns

    NARCIS (Netherlands)

    Haas, de Y.; Veerkamp, R.F.; Barkema, H.W.; Gröhn, Y.T.; Schukken, Y.H.

    2004-01-01

    Associations were estimated between pathogen-specific cases of clinical mastitis (CM) and somatic cell count (SCC) patterns based on deviations from the typical curve for SCC during lactation and compared with associations between pathogen-specific CM and lactation average SCC. Data from 274 Dutch

  9. Functional activation of myelin-specific T cells by virus-induced molecular mimicry.

    Science.gov (United States)

    Olson, Julie K; Eagar, Todd N; Miller, Stephen D

    2002-09-01

    Molecular mimicry is the process by which T cells activated in response to determinants on an infecting microorganism cross-react with self epitopes, leading to an autoimmune disease. Normally, infection of SJL/J mice with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent CNS infection, leading to a chronic progressive, CD4(+) T cell-mediated demyelinating disease. Myelin damage is initiated by T cell responses to virus persisting in CNS APCs, and progressive demyelinating disease (50 days postinfection) is perpetuated by myelin epitope-specific CD4(+) T cells activated by epitope spreading. We developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant myelin epitope, proteolipid protein (PLP) 139-151, into the coding region of a nonpathogenic TMEV variant. PLP139-TMEV-infected mice developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLP139-151-specific CD4(+) Th1 responses within 10-14 days postinfection. The current studies demonstrate that the early onset demyelinating disease induced by PLP139-TMEV is the direct result of autoreactive PLP139-151-specific CD4(+) T cell responses. PLP139-151-specific CD4(+) T cells from PLP139-TMEV-infected mice transferred demyelinating disease to naive recipients and PLP139-151-specific tolerance before infection prevented clinical disease. Finally, infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139-151-specific CD4(+) T cells could be activated in the periphery and traffic to the CNS. Collectively, infection with PLP139-151 mimic encoding TMEV serves as an excellent model for molecular mimicry by inducing pathologic myelin-specific CD4(+) T cells via a natural virus infection.

  10. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

    2011-08-26

    Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

  11. Modeling retinal degeneration using patient-specific induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Zi-Bing Jin

    Full Text Available Retinitis pigmentosa (RP is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

  12. Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

    Science.gov (United States)

    Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, Helge

    2013-01-01

    Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies. PMID:23734198

  13. cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules.

    Directory of Open Access Journals (Sweden)

    Erik Limpens

    Full Text Available Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur and proximal region (where symbiosomes are mainly differentiating, as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ". This digital "in situ" offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

  14. Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

    Science.gov (United States)

    Cohly, H. H.; Morrison, D. R.; Zouhair Atassi, M. Z.

    1989-01-01

    Non-immune SJL (H-2s) spleen cells were fused with non-secreting, non-antigen presenting (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B-cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones presented both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

  15. Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.

    Science.gov (United States)

    Gao, Hong; Prasad, Gaddamanugu L; Zacharias, Wolfgang

    2013-02-01

    To examine the effects of standardized (reference) tobacco preparations on human oral cavity cells, two oral squamous cell carcinoma cell lines (101A, 101B) and normal human gingival epithelial cells (HGEC) were treated with cigarette smoke total particulate matter (TPM), smokeless tobacco extracted with complete artificial saliva (ST/CAS), or whole-smoke conditioned media (WS-CM). EC-50 values, as determined by sulforhodamine B assays, varied among the cell types and agents. When normalized to nicotine content, cytotoxicity for WS-CM and TPM was higher compared to that observed with ST/CAS. Nicotine alone had no or only minimal cytotoxicity for all cell types in the applied range. Activation of pro-apoptotic caspase-3 was examined in all cell types at their respective EC-50 doses for the three agents. TPM, but not ST/CAS or WS-CM significantly activated caspase-3 in all three cell types. Fluorescence-activated cell sorting (FACS) for expression of the early apoptosis marker Annexin V and for nuclear staining by 7-aminoactinomycin (7-AAD) revealed different extents of apoptosis versus non-apoptotic cell death for the three agents. These data characterize differential responses of normal and malignant oral cells after exposure to TPM, ST/CAS, or WS-CM. They assist in understanding differential effects of combustible versus non-combustible tobacco products, and in identifying novel biomarkers for tobacco smoke exposure and effect in the oral cavity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation.

    Directory of Open Access Journals (Sweden)

    Alexander R Mendenhall

    Full Text Available In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to

  17. Trojan horse lymphocytes: a vesicular stomatitis virus-specific T-cell clone lyses target cells by carrying virus.

    OpenAIRE

    Hom, R C; Soman, G; Finberg, R

    1989-01-01

    We have isolated a vesicular stomatitis virus (VSV)-specific CD4+ CD8- murine T-cell clone. This clone proliferates only in response to VSV and lyses infected tumor cells bearing class II major histocompatibility antigens in short-term chromium release assays. In addition, the cell has VSV antigens on its surface and is capable of killing uninfected tumor cells without major histocompatibility antigen restriction in a 2-day assay. This latter cytolytic activity is eliminated by anti-VSV antib...

  18. RORγt-specific transcriptional interactomic inhibition suppresses autoimmunity associated with TH17 cells

    Science.gov (United States)

    Park, Tae-Yoon; Park, Sung-Dong; Cho, Jen-Young; Moon, Jae-Seung; Kim, Na-Yeon; Park, Kyungsoo; Seong, Rho Hyun; Lee, Sang-Won; Morio, Tomohiro; Bothwell, Alfred L. M.; Lee, Sang-Kyou

    2014-01-01

    The nuclear hormone receptor retinoic acid-related orphan receptor gamma t (RORγt) is a transcription factor (TF) specific to TH17 cells that produce interleukin (IL)-17 and have been implicated in a wide range of autoimmunity. Here, we developed a novel therapeutic strategy to modulate the functions of RORγt using cell-transducible form of transcription modulation domain of RORγt (tRORγt-TMD), which can be delivered effectively into the nucleus of cells and into the central nerve system (CNS). tRORγt-TMD specifically inhibited TH17-related cytokines induced by RORγt, thereby suppressing the differentiation of naïve T cells into TH17, but not into TH1, TH2, or Treg cells. tRORγt-TMD injected into experimental autoimmune encephalomyelitis (EAE) animal model can be delivered effectively in the splenic CD4+ T cells and spinal cord-infiltrating CD4+ T cells, and suppress the functions of TH17 cells. The clinical severity and incidence of EAE were ameliorated by tRORγt-TMD in preventive and therapeutic manner, and significant reduction of both infiltrating CD4+ IL-17+ T cells and inflammatory cells into the CNS was observed. As a result, the number of spinal cord demyelination was also reduced after tRORγt-TMD treatment. With the same proof of concept, tTbet-TMD specifically blocking TH1 differentiation improved the clinical incidence of rheumatoid arthritis (RA). Therefore, tRORγt-TMD and tTbet-TMD can be novel therapeutic reagents with the natural specificity for the treatment of inflammatory diseases associated with TH17 or TH1. This strategy can be applied to treat various diseases where a specific transcription factor has a key role in pathogenesis. PMID:25527718

  19. HCV genotype-3a T cell immunity: specificity, function and impact of therapy

    Science.gov (United States)

    Humphreys, Isla S; von Delft, Annette; Brown, Anthony; Hibbert, Linda; Collier, Jane D; Foster, Graham R; Rahman, Monira; Christian, Annabel; Klenerman, Paul; Barnes, Eleanor

    2012-01-01

    Background Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. Objectives The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-α/ribavirin on T cell immunity. Design T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-γ ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. Results CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. Conclusion HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness. PMID:22337948

  20. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    Science.gov (United States)

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

  1. Sarcoidosis Th17 Cells are ESAT-6 Antigen Specific but Demonstrate Reduced IFN-γ Expression

    Science.gov (United States)

    Richmond, Bradley W.; Ploetze, Kristen; Isom, Joan; Chambers-Harris, Isfahan; Braun, Nicole A.; Taylor, Thyneice; Abraham, Susamma; Mageto, Yolanda; Culver, Dan A.; Oswald-Richter, Kyra A.; Drake, Wonder P.

    2013-01-01

    Rationale Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. Methods Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. Results Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p=0.03 and p=0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (psarcoidosis patients produced less IFN-γ than healthy controls. Conclusions Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis. PMID:23073617

  2. Characterization of eight novel proteins with male germ cell-specific expression in mouse

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    Eddy Edward M

    2008-07-01

    Full Text Available Abstract Background Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library. Methods Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea. Results The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1, Sfap1 (Sperm flagellum associated protein 1 and Sfap2 (Sperm flagellum associated protein 2. Conclusion We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in

  3. Cell specific radiation dosimetry in skeleton from life-span carcinogenesis studies

    Energy Technology Data Exchange (ETDEWEB)

    Webster, S.S.J.

    1993-04-05

    The osteogenic sarcoma is the dominant life-threatening pathology in lifespan studies of beagles exposed to alpha-emitting bone-seeking radionuclides. It was deduced from these studies that certain skeletal sites are more prone to develop tumors. This project sought to determine the bone cells at risk and their cell-specific radiation dose. The cell-specific radiation dose values are related to loss and high Ra-226 and Pu-239 induced osteogenic sarcoma sites, to test different dose response hypothesis and predict the extent of effects in humans.

  4. Socio-Ecological Variables Associated with Context-Specific Sitting Time in Belgian Older Adults: A One-Year Follow-Up Study

    Science.gov (United States)

    Busschaert, Cedric; Scherrens, Anne-Lore; De Bourdeaudhuij, Ilse; Cardon, Greet; Van Cauwenberg, Jelle; De Cocker, Katrien

    2016-01-01

    Introduction Knowledge about variables associated with context-specific sitting time in older adults is limited. Therefore, this study explored cross-sectional and longitudinal associations of socio-demographic, social-cognitive, physical-environmental and health-related variables with sitting during TV viewing, computer use and motorized transport in older adults. Methods A sample of Belgian older adults completed structured interviews on context-specific sitting time and associated variables using a longitudinal study design. Objective measurements of grip strength and physical performance were also completed. Complete baseline data were available of 258 participants (73.98±6.16 years) of which 229 participants remained in the study at one year follow-up (retention rate: 91.60%). Cross-sectional correlates (baseline data) and longitudinal predictors (change-scores in relation with change in sitting time) were explored through multiple linear regression analyses. Results Per context-specific sitting time, most of the cross-sectional correlates differed from the longitudinal predictors. Increases over time in enjoyment of watching TV (+one unit), encouragement of partner to watch less TV (+one unit) and TV time of partner (+30.0 min/day) were associated with respectively 9.1 min/day (pvariables predicted changes in context-specific sitting time. Variables at the social-cognitive level were most frequently related to context-specific sitting. PMID:27997603

  5. Common Variable Immunodeficiency Associated with Hepatosplenic T-Cell Lymphoma Mimicking Juvenile Systemic Lupus Erythematosus

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    A. A. Jesus

    2011-01-01

    Full Text Available Common variable immunodeficiency (CVID is a heterogeneous disorder with susceptibility to infections, autoimmune manifestations, and cancer. To our knowledge, CIVD with T-cell lymphoma mimicking juvenile systemic lupus erythematosus (JSLE was not described in the literature, and one case was reported herein. An 8-year-old female was admitted in our Pediatric Immunology Unit with a clinical history of hypogammaglobulinemia, recurrent upper respiratory infections, and pneumonias. She had a marked decrease of three serum immunoglobulin isotypes, and the diagnosis of CVID was established. At the age of 17 years, she presented with oral ulceration, nonerosive arthritis, nephritis, serositis, cytopenia, positive antiphospholipid antibodies, and positive antinuclear antibody fulfilling the American College of Rheumatology (ACR criteria for SLE. She was treated with intravenous methylprednisolone for three consecutive days, and intravenous immunoglobulin, and maintenance therapy of chloroquine, azathioprine and prednisone 40 mg/day. Two months later, she died of septic shock secondary to acute pneumonia. The necropsy showed hepatosplenic T-cell lymphoma with diffuse involvement of bone marrow, spleen, liver, and lungs. The lymphoma cells were positive for CD3 immunostaining and negative for CD20 and lysozyme. In conclusion, the association of CVID and hepatosplenic T-cell lymphoma may simulate JSLE diagnosis.

  6. Common variable immunodeficiency associated with hepatosplenic T-cell lymphoma mimicking juvenile systemic lupus erythematosus.

    Science.gov (United States)

    Jesus, A A; Jacob, C M A; Silva, C A; Dorna, M; Pastorino, A C; Carneiro-Sampaio, M

    2011-01-01

    Common variable immunodeficiency (CVID) is a heterogeneous disorder with susceptibility to infections, autoimmune manifestations, and cancer. To our knowledge, CIVD with T-cell lymphoma mimicking juvenile systemic lupus erythematosus (JSLE) was not described in the literature, and one case was reported herein. An 8-year-old female was admitted in our Pediatric Immunology Unit with a clinical history of hypogammaglobulinemia, recurrent upper respiratory infections, and pneumonias. She had a marked decrease of three serum immunoglobulin isotypes, and the diagnosis of CVID was established. At the age of 17 years, she presented with oral ulceration, nonerosive arthritis, nephritis, serositis, cytopenia, positive antiphospholipid antibodies, and positive antinuclear antibody fulfilling the American College of Rheumatology (ACR) criteria for SLE. She was treated with intravenous methylprednisolone for three consecutive days, and intravenous immunoglobulin, and maintenance therapy of chloroquine, azathioprine and prednisone 40  mg/day. Two months later, she died of septic shock secondary to acute pneumonia. The necropsy showed hepatosplenic T-cell lymphoma with diffuse involvement of bone marrow, spleen, liver, and lungs. The lymphoma cells were positive for CD3 immunostaining and negative for CD20 and lysozyme. In conclusion, the association of CVID and hepatosplenic T-cell lymphoma may simulate JSLE diagnosis.

  7. Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification.

    Science.gov (United States)

    Angibaud, Julie; Baudouin, Stéphane J; Louveau, Antoine; Nerrière-Daguin, Véronique; Bonnamain, Virginie; Csaba, Zsolt; Dournaud, Pascal; Naveilhan, Philippe; Noraz, Nelly; Pellier-Monnin, Véronique; Boudin, Hélène

    2012-02-01

    Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.

  8. Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor beta-chain gene.

    Science.gov (United States)

    Epplen, J T; Bartels, F; Becker, A; Nerz, G; Prester, M; Rinaldy, A; Simon, M M

    1986-06-01

    Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor beta-chain rearrangements and respective mRNAs expressed. beta-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the beta-chain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the beta-chain diversity-joining region (D beta--J beta) junction cannot be explained by known germ-line D beta and J beta elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive beta-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations.

  9. Diagnostic delay in oral squamous cell carcinoma: the role of cognitive and psychological variables.

    Science.gov (United States)

    Panzarella, Vera; Pizzo, Giuseppe; Calvino, Francesco; Compilato, Domenico; Colella, Giuseppe; Campisi, Giuseppina

    2014-03-01

    This retrospective study investigated, in two cohorts of subjects living in Southern Italy and awaiting treatment for oral squamous cell carcinoma (OSCC), the variables related to diagnostic delay ascribable to the patient, with particular reference to the cognitive and psychological ones. A total of 156 patients with OSCC (mean age: 62 years, M/F: 2.39∶1) were recruited at the Universities of Palermo and Naples. Risk factors related to patient delay included: sociodemographic, health-related, cognitive and psychological variables. The analysis was conducted by considering two different delay ranges: dichotomous (≤1 month vs. >1 month) and polytomous (3 months) delay. Data were investigated by univariate and multivariate analyses and a P value ≤0.05 was considered statistically significant. For both delay measurements, the most relevant variables were: 'Personal experience of cancer' (dichotomous delay: P=0.05, odds ratio (OR)=0.33, 95% confidence interval (CI)=0.11-0.99; polytomous delay: P=0.006, Chi-square=10.224) and 'Unawareness' (dichotomous delay: Pdichotomous and for polytomous categorization of delay, respectively. The findings of this study indicated that, in the investigated cohorts, the knowledge about cancer issues is strongly linked to the patient delay. Educational interventions on the Mediterranean population are necessary in order to increase the patient awareness and to emphasize his/her key role in early diagnosis of OSCC.

  10. Variability in cell response of Cronobacter sakazakii after mild-heat treatments and its impact on food safety

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    Julio eParra-Flores

    2016-04-01

    Full Text Available Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula (PIF and follow-up formulae (FUF. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell were below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness were, in most of the cases, was above 0.7.

  11. Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity.

    Science.gov (United States)

    Richardson, T B; Kaspers, J; Porter, C D

    2004-05-01

    Transcriptional targeting is an important aspect of developing gene therapy vectors in order to restrict transgene expression to selected target cells. One approach, when using retroviral vectors, is to replace viral transcriptional control elements within the long terminal repeat (LTR) with sequences imparting the desired specificity. We have developed such hybrid LTR retroviruses, incorporating sequences from each of the human promoters for flt-1, ICAM-2 and KDR, as part of our antivascular cancer gene therapy strategy targeting tumour endothelial cells. The chosen fragments were used to replace the enhancer or combined enhancer and proximal promoter regions of the viral LTR. All showed activity in primary human breast microvascular endothelial cells, with viruses incorporating ICAM-2 sequences exhibiting the greatest specificity versus nonendothelial cells in vitro and a marked alteration of specificity towards endothelial cells in a subcutaneous xenograft model in vivo. Moreover, our study documents the effect of enhancer and/or proximal promoter deletion on LTR activity and reports that differential dependence in different cell lines can give the false impression of specificity if experiments are not adequately controlled. This finding also has implications for other retroviral vector designs seeking to provide transcriptional specificity and for their safety with respect to prevention of gene activation at sites of proviral integration.

  12. Targeting specific cells in the brain with nanomedicines for CNS therapies.

    Science.gov (United States)

    Zhang, Fan; Lin, Yi-An; Kannan, Sujatha; Kannan, Rangaramanujam M

    2016-10-28

    Treatment of Central Nervous System (CNS) disorders still remains a major clinical challenge. The Blood-Brain Barrier (BBB), known as the major hindrance, greatly limits therapeutics penetration into the brain. Moreover, even though some therapeutics can cross BBB based on their intrinsic properties or via the use of proper nanoscale delivery vehicles, their therapeutic efficacy is still often limited without the specific uptake of drugs by the cancer or disease-associated cells. As more studies have started to elucidate the pathological roles of major cells in the CNS (for example, microglia, neurons, and astrocytes) for different disorders, nanomedicines that can enable targeting of specific cells in these diseases may provide great potential to boost efficacy. In this review, we aim to briefly cover the pathological roles of endothelial cells, microglia, tumor-associated microglia/macrophage, neurons, astrocytes, and glioma in CNS disorders and to highlight the recent advances in nanomedicines that can target specific disease-associated cells. Furthermore, we summarized some strategies employed in nanomedicine to achieve specific cell targeting or to enhance the drug neuroprotective effects in the CNS. The specific targeting at the cellular level by nanotherapy can be a more precise and effective means not only to enhance the drug availability but also to reduce side effects. Published by Elsevier B.V.

  13. Generation and Application of Male Mice with Specific Expression of Green Fluorescent Protein in Germ Cells.

    Science.gov (United States)

    Wang, Zhiru; Li, Jun; Cao, Dong; Liu, Xiaomei; Zhu, Desheng

    2016-10-01

    The study aimed to generate a mouse line with green fluorescent protein (GFP) specifically expressed in male germ cells to assess testicular toxicity. The mouse line with GFP specifically expressed in male germ cells was generated by mating a germ cell-specific transgenic Cre male mouse with a double-fluorescent reporter female mouse using Cre/loxP. The mouse line was administered ethylene glycol monomethyl ether (EGME) by oral gavage. Then, the green fluorescence intensity in the testes was used as an indicator to examine the potential for testicular toxicity testing by molecular biology, histopathology, and in vivo imaging techniques. Specific testicular GFP expression was observed in mice. GFP was mainly expressed in the germ cell lineage and concentrated in secondary spermatocytes/spermatocytes and spermatozoa. After administration of EGME, at the organ level, the green fluorescent intensity of the testes was decreased by 11 days and had disappeared by 34 days. Frozen testicular sections stained with DAPI showed significantly decreased green fluorescence in secondary spermatocytes and sperm cells. These observations were consistent with the testis weight and results of testicular histopathology. With the application of in vivo imaging becoming popular, this mouse line with GFP specifically expressed in the male germ cells may have some advantages for the study of reproductive toxicity.

  14. Can Variability of Pattern ERG Signal Help to Detect Retinal Ganglion Cells Dysfunction in Glaucomatous Eyes?

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    Alberto Mavilio

    2015-01-01

    Full Text Available Objective. To evaluate variability of steady-state pattern electroretinogram (SS-PERG signal in normal, suspected, and glaucomatous eyes. Methods. Twenty-one subjects with suspected glaucoma due to disc abnormalities (GS, 37 patients with early glaucoma (EG, and 24 normal control (NC were tested with spectral-domain optical coherence tomography (SD-OCT, standard automated perimetry (SAP, and SS-PERG. Mean deviation (MD, pattern standard deviation (PSD, retinal nerve fiber layer (RNFL, and ganglionar complex cells (GCC were evaluated. The SS-PERG was recorded five consecutive times and the amplitude and phase of second harmonic were measured. PERG amplitude and coefficient of variation of phase (CVphase were recorded, and correlation with structural and functional parameters of disease, by means of one-way ANOVA and Pearson’s correlation, was analysed. Results. PERG amplitude was reduced, as expression of retinal ganglion cells (RGCs dysfunction, in EG patients and GS subjects compared to NC patients (P<0.0001. CVphase was significantly increased in EG patients and GS subjects, compared to healthy (P<0.0001, and it was also correlated with PSD (P=0.0009, GCC (P=0.028, and RNFL (P=0.0078 only in EG patients. Conclusions. Increased intrasession variability of phase in suspected glaucomatous eyes may be a sign of RGCs dysfunction.

  15. Association between morphometric variables and nocturnal desaturation in sickle-cell anemia

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    Cristina Salles

    2014-07-01

    Full Text Available OBJECTIVE: to evaluate associations between morphometric variables, cervical circumference (CC, and abdominal circumference (AC with the presence of nocturnal desaturation in children and adolescents with sickle-cell anemia. METHODS: all patients were submitted to baseline polysomnography, oral cavity measurements (maxillary intermolar distance, mandibular intermolar distance, and overjet, and CC and AC measurements. RESULTS: a total of 85 patients were evaluated. A positive correlation was observed between the height/age Z-score and CC measurement (r = 0.233, p = 0.031. The presence of nocturnal desaturation was associated with CC (59.2± 9.3 vs. 67.5 ± 10.7, p = 0.006 and AC measurements (27.0 ± 2.0 vs. 29.0± 2.1, p = 0.028. There was a negative correlation between desaturation and maxillary intermolar distance (r = -0.365, p = 0.001 and mandibular intermolar distance (r = -0.233, p = 0.037. CONCLUSIONS: the morphometric variables of CC and AC may contribute to raise suspicion of nocturnal desaturation in children and adolescents with sickle-cell anemia.

  16. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

    Science.gov (United States)

    García, Javier; Puentes, Alvaro; Rodríguez, Luis; Ocampo, Marisol; Curtidor, Hernando; Vera, Ricardo; Lopez, Ramses; Valbuena, John; Cortes, Jimena; Vanegas, Magnolia; Barrero, Carlos; Patarroyo, Manuel A.; Urquiza, Mauricio; Patarroyo, Manuel E.

    2005-01-01

    The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins. PMID:16131654

  17. The contribution of specific cell subpopulations to submandibular salivary gland branching morphogenesis.

    Science.gov (United States)

    Kwon, Hae Ryong; Larsen, Melinda

    2015-06-01

    Branching morphogenesis is the developmental program responsible for generating a large surface to volume ratio in many secretory and absorptive organs. To accomplish branching morphogenesis, spatiotemporal regulation of specific cell subpopulations is required. Here, we review recent studies that define the contributions of distinct cell subpopulations to specific cellular processes during branching morphogenesis in the mammalian submandibular salivary gland, including the initiation of the gland, the coordination of cleft formation, and the contribution of stem/progenitor cells to morphogenesis. In conclusion, we provide an overview of technological advances that have opened opportunities to further probe the contributions of specific cell subpopulations and to define the integration of events required for branching morphogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines.

    Science.gov (United States)

    García, Javier; Puentes, Alvaro; Rodríguez, Luis; Ocampo, Marisol; Curtidor, Hernando; Vera, Ricardo; Lopez, Ramses; Valbuena, John; Cortes, Jimena; Vanegas, Magnolia; Barrero, Carlos; Patarroyo, Manuel A; Urquiza, Mauricio; Patarroyo, Manuel E

    2005-09-01

    The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.

  19. Virus-specific cytotoxic T cells in chronic active Epstein-Barr virus infection.

    Science.gov (United States)

    Shibayama, Haruna; Imadome, Ken-Ichi; Onozawa, Erika; Tsuzura, Akiho; Miura, Osamu; Koyama, Takatoshi; Arai, Ayako

    2017-01-01

    Chronic active Epstein-Barr virus infection (CAEBV) is a disease characterized by clonally proliferating and activated EBV-infected T or NK cells accompanied by chronic inflammation and T- or NK-cell neoplasms. However, the mechanism for developing CAEBV has not been clarified to date. Because the decreased number or inactivation of EBV-specific cytotoxic T lymphocytes (CTLs) resulted in the development of EBV-positive B-cell neoplasms, we investigated the number of CTLs in CAEBV patients using the tetrameric complexes of HLA-restricted EBV-specific peptides. Among the seven patients examined, EBV-specific CTLs were detected in the peripheral blood mononuclear cells (PBMCs) of four cases but were not detected in three cases. The ratio of EBV-specific CTLs in PBMCs tended to be higher in the patients with active disease than in those with inactive disease. In two patients in whom EBV-specific CTLs had not been detected, CTLs appeared after the eradication of EBV-infected T cells by allogeneic bone marrow transplantation. These results suggested that the failure of CTLs had a role in developing CAEBV, although the induction number and function of EBV-specific CTLs might vary in each patient.

  20. Antiproliferative and Apoptotic Effects of a Specific Antiprostate Stem Cell Single Chain Antibody on Human Prostate Cancer Cells

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    Foroogh Nejatollahi

    2013-01-01

    Full Text Available Prostate stem cell antigen (PSCA is a highly glycosylated cell surface protein which is overexpressed in several malignancies including prostate, pancreas, and urinary bladder cancers. Tumor suppression has been reported by anti-PSCA antibody. Small and high affinity single chain antibodies (scFv have been introduced as effective agents for cancer immunotargeting approaches. In the present study, we used a phage antibody display library of scFv and selected two antibodies against two immunodominant epitopes of PSCA by panning process. The reactivity of the scFvs for the corresponding epitopes was determined by phage ELISA. The binding specificity of antibodies to PSCA-expressing prostate cancer cell line, DU-145, was analyzed by flow cytometry. The antiproliferative and apoptotic induction effects were evaluated by MTT and Annexin-V assays, respectively. Results represented functional scFv C5-II which could bind specifically to DU-145 cells and significantly inhibited the proliferation of these cells (61% with no effect on PSCA-negative cells. The antibody also induced apoptosis in the PSCA expressing cells. The percentage of the apoptotic cells after 24 hrs of exposure to 500 scFv/cell was 33.80%. These results demonstrate that the functional anti-PSCA scFv C5-II has the potential to be considered as a new agent for targeted therapy of prostate cancer.

  1. lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis

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    DiNardo, Stephen; Okegbe, Tishina; Wingert, Lindsey; Freilich, Sarah; Terry, Natalie

    2011-01-01

    To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (CySC). Here, we show that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led us to examine the developmental relationship between CySCs and hub cells. In contrast to a previous report, we did not observe significant conversion of steady-state CySC progeny to hub fate. However, we found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, lines mutant embryos exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and we found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche. PMID:21486923

  2. A nanocomplex that is both tumor cell-selective and cancer gene-specific for anaplastic large cell lymphoma

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    Zu Youli

    2011-01-01

    Full Text Available Abstract Background Many in vitro studies have demonstrated that silencing of cancerous genes by siRNAs is a potential therapeutic approach for blocking tumor growth. However, siRNAs are not cell type-selective, cannot specifically target tumor cells, and therefore have limited in vivo application for siRNA-mediated gene therapy. Results In this study, we tested a functional RNA nanocomplex which exclusively targets and affects human anaplastic large cell lymphoma (ALCL by taking advantage of the abnormal expression of CD30, a unique surface biomarker, and the anaplastic lymphoma kinase (ALK gene in lymphoma cells. The nanocomplexes were formulated by incorporating both ALK siRNA and a RNA-based CD30 aptamer probe onto nano-sized polyethyleneimine-citrate carriers. To minimize potential cytotoxicity, the individual components of the nanocomplexes were used at sub-cytotoxic concentrations. Dynamic light scattering showed that formed nanocomplexes were ~140 nm in diameter and remained stable for more than 24 hours in culture medium. Cell binding assays revealed that CD30 aptamer probes selectively targeted nanocomplexes to ALCL cells, and confocal fluorescence microscopy confirmed intracellular delivery of the nanocomplex. Cell transfection analysis showed that nanocomplexes silenced genes in an ALCL cell type-selective fashion. Moreover, exposure of ALCL cells to nanocomplexes carrying both ALK siRNAs and CD30 RNA aptamers specifically silenced ALK gene expression, leading to growth arrest and apoptosis. Conclusions Taken together, our findings indicate that this functional RNA nanocomplex is both tumor cell type-selective and cancer gene-specific for ALCL cells.

  3. Risk stratification of plasma cell neoplasm: insights from plasma cell-specific cytoplasmic immunoglobulin fluorescence in situ hybridization (cIg FISH) vs. conventional FISH.

    Science.gov (United States)

    Dong, Henry; Yang, Hai-Su; Jagannath, Sundar; Stephenson, Christine F; Brenholz, Pauline; Mazumder, Amitabha; Chari, Ajai

    2012-10-01

    We directly compared the results of routine fluorescence in situ hybridization (FISH) and plasma cell-specific cytoplasmic immunoglobulin (cIg) FISH from 75 paired samples for myeloma risk stratification. CIg FISH improves test specificity and sensitivity and tends to eliminate borderline results. It proves that most plasma cells (PCs) consistently carry the abnormality in myelomas with an IGH translocation, whereas routine FISH detects these cells only at variably low levels. Routine cytogenetic analysis of plasma cell neoplasms (PCNs) has a low sensitivity. Conventional fluorescence in situ hybridization (FISH) is not plasma cell (PC) specific and results are diluted by other cells in the sample. Although PC-specific FISH testing has been recommended for multiple myeloma (MM) risk stratification, eg, by combining cytoplasmic immunoglobulin (cIg) staining with FISH, the benefits of cIg FISH have never been directly demonstrated in a controlled study. Seventy-five samples from patients with PCNs were analyzed by concomitant conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13, 17p-, and +3. The results were compared for their reliability, specific