Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types

PubMed Central

Ferreira, Lauren; Macaulay, Iain C.; Stubbington, Michael J.T.

2017-01-01

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell–specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates. PMID:28087841

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize[OA

PubMed Central

Gruis, Darren (Fred); Guo, Hena; Selinger, David; Tian, Qing; Olsen, Odd-Arne

2006-01-01

Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions. PMID:16698897

Single Insulin-Specific CD8+ T Cells Show Characteristic Gene Expression Profiles in Human Type 1 Diabetes

PubMed Central

Luce, Sandrine; Lemonnier, François; Briand, Jean-Paul; Coste, Joel; Lahlou, Najiba; Muller, Sylviane; Larger, Etienne; Rocha, Benedita; Mallone, Roberto; Boitard, Christian

2011-01-01

OBJECTIVE Both the early steps and the high recurrence of autoimmunity once the disease is established are unexplained in human type 1 diabetes. Because CD8+ T cells are central and insulin is a key autoantigen in the disease process, our objective was to characterize HLA class I–restricted autoreactive CD8+ T cells specific for preproinsulin (PPI) in recent-onset and long-standing type 1 diabetic patients and healthy control subjects. RESEARCH DESIGN AND METHODS We used HLA-A*02:01 tetramers complexed to PPI peptides to enumerate circulating PPI-specific CD8+ T cells in patients and characterize them using membrane markers and single-cell PCR. RESULTS Most autoreactive CD8+ T cells detected in recent-onset type 1 diabetic patients are specific for leader sequence peptides, notably PPI6–14, whereas CD8+ T cells in long-standing patients recognize the B-chain peptide PPI33–42 (B9–18). Both CD8+ T-cell specificities are predominantly naïve, central, and effector memory cells, and their gene expression profile differs from cytomegalovirus-specific CD8+ T cells. PPI6–14–specific CD8+ T cells detected in one healthy control displayed Il-10 mRNA expression, which was not observed in diabetic patients. CONCLUSIONS PPI-specific CD8+ T cells in type 1 diabetic patients include central memory and target different epitopes in new-onset versus long-standing disease. Our data support the hypothesis that insulin therapy may contribute to the expansion of autoreactive CD8+ T cells in the long term. PMID:21998398

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish

PubMed Central

Nagao, Yusuke; Takada, Hiroyuki; Miyadai, Motohiro; Adachi, Tomoko; Kamei, Yasuhiro; Hara, Ikuyo; Naruse, Kiyoshi; Hibi, Masahiko

2018-01-01

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type. PMID:29621239

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

PubMed

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 epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases. © 2017 American Heart Association, Inc.

Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

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

Londei, M.; Savill, C.M.; Verhoef, A.

Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovialmore » tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis.« less

Exploring the cellular basis of human disease through a large-scale mapping of deleterious genes to cell types.

PubMed

Cornish, Alex J; Filippis, Ioannis; David, Alessia; Sternberg, Michael J E

2015-09-01

Each cell type found within the human body performs a diverse and unique set of functions, the disruption of which can lead to disease. However, there currently exists no systematic mapping between cell types and the diseases they can cause. In this study, we integrate protein-protein interaction data with high-quality cell-type-specific gene expression data from the FANTOM5 project to build the largest collection of cell-type-specific interactomes created to date. We develop a novel method, called gene set compactness (GSC), that contrasts the relative positions of disease-associated genes across 73 cell-type-specific interactomes to map genes associated with 196 diseases to the cell types they affect. We conduct text-mining of the PubMed database to produce an independent resource of disease-associated cell types, which we use to validate our method. The GSC method successfully identifies known disease-cell-type associations, as well as highlighting associations that warrant further study. This includes mast cells and multiple sclerosis, a cell population currently being targeted in a multiple sclerosis phase 2 clinical trial. Furthermore, we build a cell-type-based diseasome using the cell types identified as manifesting each disease, offering insight into diseases linked through etiology. The data set produced in this study represents the first large-scale mapping of diseases to the cell types in which they are manifested and will therefore be useful in the study of disease systems. Overall, we demonstrate that our approach links disease-associated genes to the phenotypes they produce, a key goal within systems medicine.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

PubMed

Sandh, Gustaf; Ramström, Margareta; Stensjö, Karin

2014-12-04

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Androgen receptor (AR) pathophysiological roles in androgen-related diseases in skin, bone/muscle, metabolic syndrome and neuron/immune systems: lessons learned from mice lacking AR in specific cells

PubMed Central

Chang, Chawnshang; Yeh, Shuyuan; Lee, Soo Ok; Chang, Ta-min

2013-01-01

The androgen receptor (AR) is expressed ubiquitously and plays a variety of roles in a vast number of physiological and pathophysiological processes. Recent studies of AR knockout (ARKO) mouse models, particularly the cell type- or tissue-specific ARKO models, have uncovered many AR cell type- or tissue-specific pathophysiological roles in mice, which otherwise would not be delineated from conventional castration and androgen insensitivity syndrome studies. Thus, the AR in various specific cell types plays pivotal roles in production and maturation of immune cells, bone mineralization, and muscle growth. In metabolism, the ARs in brain, particularly in the hypothalamus, and the liver appear to participate in regulation of insulin sensitivity and glucose homeostasis. The AR also plays key roles in cutaneous wound healing and cardiovascular diseases, including atherosclerosis and abdominal aortic aneurysm. This article will discuss the results obtained from the total, cell type-, or tissue-specific ARKO models. The understanding of AR cell type- or tissue-specific physiological and pathophysiological roles using these in vivo mouse models will provide useful information in uncovering AR roles in humans and eventually help us to develop better therapies via targeting the AR or its downstream signaling molecules to combat androgen/AR-related diseases. PMID:24653668

Localization of migraine susceptibility genes in human brain by single-cell RNA sequencing.

PubMed

Renthal, William

2018-01-01

Background Migraine is a debilitating disorder characterized by severe headaches and associated neurological symptoms. A key challenge to understanding migraine has been the cellular complexity of the human brain and the multiple cell types implicated in its pathophysiology. The present study leverages recent advances in single-cell transcriptomics to localize the specific human brain cell types in which putative migraine susceptibility genes are expressed. Methods The cell-type specific expression of both familial and common migraine-associated genes was determined bioinformatically using data from 2,039 individual human brain cells across two published single-cell RNA sequencing datasets. Enrichment of migraine-associated genes was determined for each brain cell type. Results Analysis of single-brain cell RNA sequencing data from five major subtypes of cells in the human cortex (neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells) indicates that over 40% of known migraine-associated genes are enriched in the expression profiles of a specific brain cell type. Further analysis of neuronal migraine-associated genes demonstrated that approximately 70% were significantly enriched in inhibitory neurons and 30% in excitatory neurons. Conclusions This study takes the next step in understanding the human brain cell types in which putative migraine susceptibility genes are expressed. Both familial and common migraine may arise from dysfunction of discrete cell types within the neurovascular unit, and localization of the affected cell type(s) in an individual patient may provide insight into to their susceptibility to migraine.

Cross-Laboratory Analysis of Brain Cell Type Transcriptomes with Applications to Interpretation of Bulk Tissue Data

PubMed Central

Toker, Lilah; Rocco, Brad; Sibille, Etienne

2017-01-01

Establishing the molecular diversity of cell types is crucial for the study of the nervous system. We compiled a cross-laboratory database of mouse brain cell type-specific transcriptomes from 36 major cell types from across the mammalian brain using rigorously curated published data from pooled cell type microarray and single-cell RNA-sequencing (RNA-seq) studies. We used these data to identify cell type-specific marker genes, discovering a substantial number of novel markers, many of which we validated using computational and experimental approaches. We further demonstrate that summarized expression of marker gene sets (MGSs) in bulk tissue data can be used to estimate the relative cell type abundance across samples. To facilitate use of this expanding resource, we provide a user-friendly web interface at www.neuroexpresso.org. PMID:29204516

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

PubMed Central

Smith, Emily M.; Lajoie, Bryan R.; Jain, Gaurav; Dekker, Job

2016-01-01

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. PMID:26748519

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

PubMed Central

Huska, Matthew R.; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R.; Schwahn, Kevin; Cooper, Samantha B.; Yamamoto, Keith R.; Thomas-Chollier, Morgane; Vingron, Martin

2017-01-01

Abstract The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter–proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. PMID:27903902

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

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

PubMed

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

2007-01-01

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

Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

ERIC Educational Resources Information Center

Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

2013-01-01

The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

Cell-Specific Multifunctional Processing of Heterogeneous Cell Systems in a Single Laser Pulse Treatment

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Mutonga, Martin B. G.; Lapotko, Dmitri O.

2012-01-01

Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the two above cellular effects. PMID:23167546

Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell–Specific Genes

PubMed Central

Sunohara, Mitsuhiro; Pouldar, Tiffany M.; Wang, Hongjun; Liu, Yixin; Rieger, Megan E.; Tran, Evelyn; Flodby, Per; Siegmund, Kimberly D.; Crandall, Edward D.; Laird-Offringa, Ite A.

2017-01-01

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type–specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells’ roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription–polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell–specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases. PMID:27749084

Brain-specific enhancers for cell-based therapy

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

Visel, Axel; Rubenstein, John L.R.; Chen, Ying-Jiun

Herein are described a set of novel specific human enhancers for specific forebrain cell types used to study and select for human neural progenitor cells. This approach enables the ability to generate interneurons from human ES, iPS and iN cells, making them available for human transplantation and for molecular/cellular analyzes. These approaches are also directly applicable to generating other neuronal cell types, such as cortical and striatal projection neurons, which have implications for many human diseases.

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

PubMed

Kriegel, Alison J; Liu, Yong; Liu, Pengyuan; Baker, Maria Angeles; Hodges, Matthew R; Hua, Xing; Liang, Mingyu

2013-12-01

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Autoantigen-specific B-cell depletion overcomes failed immune tolerance in type 1 diabetes.

PubMed

Henry, Rachel A; Kendall, Peggy L; Thomas, James W

2012-08-01

Eliminating autoantigen-specific B cells is an attractive alternative to global B-cell depletion for autoimmune disease treatment. To identify the potential for targeting a key autoimmune B-cell specificity in type 1 diabetes, insulin-binding B cells were tracked within a polyclonal repertoire using heavy chain B-cell receptor (BCR) transgenic (VH125Tg) mice. Insulin-specific B cells are rare in the periphery of nonautoimmune VH125Tg/C57BL/6 mice and WT/NOD autoimmune mice, whereas they clearly populate 1% of mature B-cell subsets in VH125Tg/NOD mice. Autoantigen upregulates CD86 in anti-insulin B cells, suggesting they are competent to interact with T cells. Endogenous insulin occupies anti-insulin BCR beginning with antigen commitment in bone marrow parenchyma, as identified by a second anti-insulin monoclonal antibody. Administration of this monoclonal antibody selectively eliminates insulin-reactive B cells in vivo and prevents disease in WT/NOD mice. Unexpectedly, developing B cells are less amenable to depletion, despite increased BCR sensitivity. These findings exemplify how a critical type 1 diabetes B-cell specificity escapes immune tolerance checkpoints. Disease liability is corrected by eliminating this B-cell specificity, providing proof of concept for a novel therapeutic approach for autoimmune disease.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells

PubMed Central

Salopiata, Florian; Depner, Sofia; Wäsch, Marvin; Böhm, Martin E.; Mücke, Oliver; Plass, Christoph; Lehmann, Wolf D.; Kreutz, Clemens; Timmer, Jens; Klingmüller, Ursula

2016-01-01

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types. PMID:27494133

Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye.

PubMed

Johnston, Robert J; Otake, Yoshiaki; Sood, Pranidhi; Vogt, Nina; Behnia, Rudy; Vasiliauskas, Daniel; McDonald, Elizabeth; Xie, Baotong; Koenig, Sebastian; Wolf, Reinhard; Cook, Tiffany; Gebelein, Brian; Kussell, Edo; Nakagoshi, Hideki; Desplan, Claude

2011-06-10

How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification. Copyright © 2011 Elsevier Inc. All rights reserved.

Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy

PubMed Central

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

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

PubMed Central

Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

2017-01-01

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

Constellation Pharmacology: A new paradigm for drug discovery

PubMed Central

Schmidt, Eric W.; Olivera, Baldomero M.

2015-01-01

Constellation Pharmacology is a cell-based high-content phenotypic-screening platform that utilizes subtype-selective pharmacological agents to elucidate the cell-specific combinations (“constellations”) of key signaling proteins that define specific cell types. Heterogeneous populations of native cells, in which the different individual cell types have been identified and characterized, are the foundation for this screening platform. Constellation Pharmacology is useful for screening small molecules or for deconvoluting complex mixtures of biologically-active natural products. This platform has been used to purify natural products and discover their molecular mechanisms. In the on-going development of Constellation Pharmacology, there is a positive-feedback loop between the pharmacological characterization of cell types and screening for new drug candidates. As Constellation Pharmacology is used to discover compounds with novel targeting-selectivity profiles, those new compounds then further help to elucidate the constellations of specific cell types, thereby increasing the content of this high-content platform. PMID:25562646

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

PubMed

Smith, Emily M; Lajoie, Bryan R; Jain, Gaurav; Dekker, Job

2016-01-07

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Arabidopsis translatome cell-specific mRNA atlas: Mining suberin and cutin lipid monomer biosynthesis genes as an example for data application.

PubMed

Mustroph, Angelika; Bailey-Serres, Julia

2010-03-01

Plants consist of distinct cell types distinguished by position, morphological features and metabolic activities. We recently developed a method to extract cell-type specific mRNA populations by immunopurification of ribosome-associated mRNAs. Microarray profiles of 21 cell-specific mRNA populations from seedling roots and shoots comprise the Arabidopsis Translatome dataset. This gene expression atlas provides a new tool for the study of cell-specific processes. Here we provide an example of how genes involved in a pathway limited to one or few cell-types can be further characterized and new candidate genes can be predicted. Cells of the root endodermis produce suberin as an inner barrier between the cortex and stele, whereas the shoot epidermal cells form cutin as a barrier to the external environment. Both polymers consist of fatty acid derivates, and share biosynthetic origins. We use the Arabidopsis Translatome dataset to demonstrate the significant cell-specific expression patterns of genes involved in those biosynthetic processes and suggest new candidate genes in the biosynthesis of suberin and cutin.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

PubMed Central

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

2016-01-01

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. PMID:26976580

SOX2 regulates common and specific stem cell features in the CNS and endoderm derived organs.

PubMed

Hagey, Daniel W; Klum, Susanne; Kurtsdotter, Idha; Zaouter, Cecile; Topcic, Danijal; Andersson, Olov; Bergsland, Maria; Muhr, Jonas

2018-02-01

Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

PubMed

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Metabolic profiling of Arabidopsis thaliana epidermal cells

PubMed Central

Ebert, Berit; Zöller, Daniela; Erban, Alexander; Fehrle, Ines; Hartmann, Jürgen; Niehl, Annette; Kopka, Joachim; Fisahn, Joachim

2010-01-01

Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo. PMID:20150518

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation.

PubMed

Love, Michael I; Huska, Matthew R; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R; Schwahn, Kevin; Cooper, Samantha B; Yamamoto, Keith R; Thomas-Chollier, Morgane; Vingron, Martin; Meijsing, Sebastiaan H

2017-02-28

The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cell Type–Specific Three-Dimensional Structure of Thalamocortical Circuits in a Column of Rat Vibrissal Cortex

PubMed Central

de Kock, Christiaan P. J.; Bruno, Randy M.; Ramirez, Alejandro; Meyer, Hanno S.; Dercksen, Vincent J.; Helmstaedter, Moritz; Sakmann, Bert

2012-01-01

Soma location, dendrite morphology, and synaptic innervation may represent key determinants of functional responses of individual neurons, such as sensory-evoked spiking. Here, we reconstruct the 3D circuits formed by thalamocortical afferents from the lemniscal pathway and excitatory neurons of an anatomically defined cortical column in rat vibrissal cortex. We objectively classify 9 cortical cell types and estimate the number and distribution of their somata, dendrites, and thalamocortical synapses. Somata and dendrites of most cell types intermingle, while thalamocortical connectivity depends strongly upon the cell type and the 3D soma location of the postsynaptic neuron. Correlating dendrite morphology and thalamocortical connectivity to functional responses revealed that the lemniscal afferents can account for some of the cell type- and location-specific subthreshold and spiking responses after passive whisker touch (e.g., in layer 4, but not for other cell types, e.g., in layer 5). Our data provides a quantitative 3D prediction of the cell type–specific lemniscal synaptic wiring diagram and elucidates structure–function relationships of this physiologically relevant pathway at single-cell resolution. PMID:22089425

Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps.

PubMed

Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K; Ernst, Jason; Kellis, Manolis; Hardison, Ross C; Myers, Richard M; Wold, Barbara J

2013-12-01

We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity.

Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps

PubMed Central

Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K.; Ernst, Jason; Kellis, Manolis; Hardison, Ross C.; Myers, Richard M.; Wold, Barbara J.

2013-01-01

We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity. PMID:24170599

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

DOE PAGES

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

2016-01-01

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

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

Nourbakhsh-Rey, Mehrnoush; Libault, Marc

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hairmore » cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. Lastly, in this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses.« less

An atlas of active enhancers across human cell types and tissues

NASA Astrophysics Data System (ADS)

Andersson, Robin; Gebhard, Claudia; Miguel-Escalada, Irene; Hoof, Ilka; Bornholdt, Jette; Boyd, Mette; Chen, Yun; Zhao, Xiaobei; Schmidl, Christian; Suzuki, Takahiro; Ntini, Evgenia; Arner, Erik; Valen, Eivind; Li, Kang; Schwarzfischer, Lucia; Glatz, Dagmar; Raithel, Johanna; Lilje, Berit; Rapin, Nicolas; Bagger, Frederik Otzen; Jørgensen, Mette; Andersen, Peter Refsing; Bertin, Nicolas; Rackham, Owen; Burroughs, A. Maxwell; Baillie, J. Kenneth; Ishizu, Yuri; Shimizu, Yuri; Furuhata, Erina; Maeda, Shiori; Negishi, Yutaka; Mungall, Christopher J.; Meehan, Terrence F.; Lassmann, Timo; Itoh, Masayoshi; Kawaji, Hideya; Kondo, Naoto; Kawai, Jun; Lennartsson, Andreas; Daub, Carsten O.; Heutink, Peter; Hume, David A.; Jensen, Torben Heick; Suzuki, Harukazu; Hayashizaki, Yoshihide; Müller, Ferenc; Consortium, The Fantom; Forrest, Alistair R. R.; Carninci, Piero; Rehli, Michael; Sandelin, Albin

2014-03-01

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

PubMed Central

2009-01-01

Background Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution. Results In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells. Conclusion This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells. PMID:19712466

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

PubMed Central

Mayer, Robert; Brero, Alessandro; von Hase, Johann; Schroeder, Timm; Cremer, Thomas; Dietzel, Steffen

2005-01-01

Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation. PMID:16336643

Allelic exchange of pheromones and their receptors reprograms sexual identity in Cryptococcus neoformans.

PubMed

Stanton, Brynne C; Giles, Steven S; Staudt, Mark W; Kruzel, Emilia K; Hull, Christina M

2010-02-26

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT-encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and alpha) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (alpha). We discovered that these "alpha(a)" cells effectively adopt a new mating type (that of a cells); they sense and respond to alpha factor, they elicit a mating response from alpha cells, and they fuse with alpha cells. In addition, alpha(a) cells lose the alpha cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between alpha and alpha(a) strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT-encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle.

PubMed

Kadi, Fawzi; Charifi, Nadia; Henriksson, Jan

2006-07-01

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6+/-8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4+/-8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.

Performance of mid infrared spectroscopy in skin cancer cell type identification

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2017-02-01

Marker free optical spectroscopy is a powerful tool for the rapid inspection of pathologically suspicious skin lesions and the non-invasive detection of early skin tumors. This goal can be reached by the combination of signal localization and the spectroscopical detection of chemical cell signatures. We here present the development and application of mid infrared spectroscopy (midIR) for the analysis of skin tumor cell types and three dimensional tissue phantoms towards the application of midIR spectroscopy for fast and reliable skin diagnostics. We developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional skin cancer phantoms. The cell systems were characterized with different systems in the midIR range up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially melanoma cells. Special attention and algorithm training was required for closely related mesenchymal cell types as dedifferentiated melanoma cells and fibroblasts. Proof of concept experiments with mixtures of in vivo fluorescence labelled skin cell types allowed the test of the new algorithms performance for the identification of specific cell types. The intense training of the software systems with various samples resulted in a increased sensitivity and specificity of the combined midIR and software system. These data highlight the potential of midIR spectroscopy as sensitive and specific future optical biopsy technology.

Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

PubMed Central

Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

2014-01-01

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

PubMed Central

2011-01-01

Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection. Results By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells. Conclusions These findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages. PMID:22151736

Linear viral load increase of a single HPV-type in women with multiple HPV infections predicts progression to cervical cancer.

PubMed

Depuydt, Christophe E; Thys, Sofie; Beert, Johan; Jonckheere, Jef; Salembier, Geert; Bogers, Johannes J

2016-11-01

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R(2) ) by linear regression. For each woman slopes and R(2) were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+, at least one of the HPV-types had a clonal progressive course (R(2)  ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type-specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R(2) and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types and elucidates HPV-genotype attribution. © 2016 UICC.

Chemical compound-based direct reprogramming for future clinical applications

PubMed Central

Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

PubMed Central

Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

The cell biology of lignification in higher plants

PubMed Central

Barros, Jaime; Serk, Henrik; Granlund, Irene; Pesquet, Edouard

2015-01-01

Background Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying. Scope Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level. Conclusions The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility. PMID:25878140

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.

PubMed

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.

Myelin-reactive “type B” T cells and T cells specific for low-affinity MHC-binding myelin peptides escape tolerance in HLA-DR transgenic mice

PubMed Central

Kawamura, Kazuyuki; McLaughlin, Katherine A.; Weissert, Robert; Forsthuber, Thomas G.

2009-01-01

Genes of the major histocompatibility complex (MHC) show the strongest genetic association with multiple sclerosis (MS) but the underlying mechanisms have remained unresolved. Here, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401 contribute to autoimmune central nervous system (CNS) demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon backcrossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific “type B” T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific “type B” T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and “type B” T cells can escape the induction of T cell tolerance and may promote MS. PMID:18713991

Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

PubMed

Gage, P J; Roller, M L; Saunders, T L; Scarlett, L M; Camper, S A

1996-01-01

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

PubMed

Ramberger, Melanie; Högl, Birgit; Stefani, Ambra; Mitterling, Thomas; Reindl, Markus; Lutterotti, Andreas

2017-03-01

Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients. © Sleep Research Society 2016. Published by Oxford University Press [on behalf of the Sleep Research Society].

Computational neuroanatomy: mapping cell-type densities in the mouse brain, simulations from the Allen Brain Atlas

NASA Astrophysics Data System (ADS)

Grange, Pascal

2015-09-01

The Allen Brain Atlas of the adult mouse (ABA) consists of digitized expression profiles of thousands of genes in the mouse brain, co-registered to a common three-dimensional template (the Allen Reference Atlas).This brain-wide, genome-wide data set has triggered a renaissance in neuroanatomy. Its voxelized version (with cubic voxels of side 200 microns) is available for desktop computation in MATLAB. On the other hand, brain cells exhibit a great phenotypic diversity (in terms of size, shape and electrophysiological activity), which has inspired the names of some well-studied cell types, such as granule cells and medium spiny neurons. However, no exhaustive taxonomy of brain cell is available. A genetic classification of brain cells is being undertaken, and some cell types have been chraracterized by their transcriptome profiles. However, given a cell type characterized by its transcriptome, it is not clear where else in the brain similar cells can be found. The ABA can been used to solve this region-specificity problem in a data-driven way: rewriting the brain-wide expression profiles of all genes in the atlas as a sum of cell-type-specific transcriptome profiles is equivalent to solving a quadratic optimization problem at each voxel in the brain. However, the estimated brain-wide densities of 64 cell types published recently were based on one series of co-registered coronal in situ hybridization (ISH) images per gene, whereas the online ABA contains several image series per gene, including sagittal ones. In the presented work, we simulate the variability of cell-type densities in a Monte Carlo way by repeatedly drawing a random image series for each gene and solving the optimization problem. This yields error bars on the region-specificity of cell types.

Immuno-Navigator, a batch-corrected coexpression database, reveals cell type-specific gene networks in the immune system

PubMed Central

Vandenbon, Alexis; Dinh, Viet H.; Mikami, Norihisa; Kitagawa, Yohko; Teraguchi, Shunsuke; Ohkura, Naganari; Sakaguchi, Shimon

2016-01-01

High-throughput gene expression data are one of the primary resources for exploring complex intracellular dynamics in modern biology. The integration of large amounts of public data may allow us to examine general dynamical relationships between regulators and target genes. However, obstacles for such analyses are study-specific biases or batch effects in the original data. Here we present Immuno-Navigator, a batch-corrected gene expression and coexpression database for 24 cell types of the mouse immune system. We systematically removed batch effects from the underlying gene expression data and showed that this removal considerably improved the consistency between inferred correlations and prior knowledge. The data revealed widespread cell type-specific correlation of expression. Integrated analysis tools allow users to use this correlation of expression for the generation of hypotheses about biological networks and candidate regulators in specific cell types. We show several applications of Immuno-Navigator as examples. In one application we successfully predicted known regulators of importance in naturally occurring Treg cells from their expression correlation with a set of Treg-specific genes. For one high-scoring gene, integrin β8 (Itgb8), we confirmed an association between Itgb8 expression in forkhead box P3 (Foxp3)-positive T cells and Treg-specific epigenetic remodeling. Our results also suggest that the regulation of Treg-specific genes within Treg cells is relatively independent of Foxp3 expression, supporting recent results pointing to a Foxp3-independent component in the development of Treg cells. PMID:27078110

Birthdating Studies Reshape Models for Pituitary Gland Cell Specification

PubMed Central

Davis, Shannon W.; Mortensen, Amanda H.; Camper, Sally A.

2011-01-01

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke’s pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the post-natal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke’s pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke’s pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process. PMID:21262217

Birthdating studies reshape models for pituitary gland cell specification.

PubMed

Davis, Shannon W; Mortensen, Amanda H; Camper, Sally A

2011-04-15

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process. Copyright © 2011 Elsevier Inc. All rights reserved.

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

PubMed Central

Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

2007-01-01

To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

Neuronal cell fate specification in Drosophila.

PubMed

Jan, Y N; Jan, L Y

1994-02-01

Recent work indicates that the Drosophila nervous system develops in a progressive process of cell fate specification. Expression of specific proneural genes in clusters of cells (the proneural clusters) in the cellular blastoderm endows these cells with the potential to form certain types of neural precursors. Intercellular interactions that involve both proneural genes and neurogenic genes then allow the neural precursors to be singled out from the proneural clusters. Expression of neural precursor genes in all neural precursors is likely to account for the universal aspects of neuronal differentiation, such as axonal outgrowth. Selective expression of certain neuronal-type selector genes further specifies the type of neuron(s) that a neural precursor will produce.

Microfluidic differential immunocapture biochip for specific leukocyte counting

PubMed Central

Hassan, Umer; Watkins, Nicholas N; Reddy, Bobby; Damhorst, Gregory; Bashir, Rashid

2016-01-01

Enumerating specific cell types from whole blood can be very useful for research and diagnostic purposes—e.g., for counting of cD4 and cD8 t cells in HIV/aIDs diagnostics. We have developed a biosensor based on a differential immunocapture technology to enumerate specific cells in 30 min using 10 µl of blood. this paper provides a comprehensive stepwise protocol to replicate our biosensor for cD4 and cD8 cell counts. the biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies. capture of other specific cells requires immobilization of their corresponding antibodies within the capture chamber. therefore, this protocol is useful for research into areas surrounding immunocapture-based biosensor development. the biosensor production requires 24 h, a one-time cell capture optimization takes 6–9 h, and the final cell counting experiment in a laboratory environment requires 30 min to complete. PMID:26963632

Two-Stage, In Silico Deconvolution of the Lymphocyte Compartment of the Peripheral Whole Blood Transcriptome in the Context of Acute Kidney Allograft Rejection

PubMed Central

Shannon, Casey P.; Balshaw, Robert; Ng, Raymond T.; Wilson-McManus, Janet E.; Keown, Paul; McMaster, Robert; McManus, Bruce M.; Landsberg, David; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage, in silico deconvolution approach that first predicts sample composition to biologically meaningful and homogeneous leukocyte sub-populations, and then performs cell type-specific differential expression analysis in these same sub-populations, from peripheral whole blood expression data. We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay. PMID:24733377

Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

PubMed

Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

2016-09-01

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

PubMed

Lascombe, I; Sallot, M; Vuillermoz, C; Weisz, A; Adessi, G L; Jouvenot, M

1998-04-30

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

PubMed Central

Pujol-Autonell, Irma; Serracant-Prat, Arnau; Cano-Sarabia, Mary; Ampudia, Rosa M.; Rodriguez-Fernandez, Silvia; Sanchez, Alex; Izquierdo, Cristina; Stratmann, Thomas; Puig-Domingo, Manuel; Maspoch, Daniel; Verdaguer, Joan; Vives-Pi, Marta

2015-01-01

Introduction The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. Objective To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. Methods A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. Results We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. Conclusions We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases. PMID:26039878

Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes.

PubMed

Pujol-Autonell, Irma; Serracant-Prat, Arnau; Cano-Sarabia, Mary; Ampudia, Rosa M; Rodriguez-Fernandez, Silvia; Sanchez, Alex; Izquierdo, Cristina; Stratmann, Thomas; Puig-Domingo, Manuel; Maspoch, Daniel; Verdaguer, Joan; Vives-Pi, Marta

2015-01-01

The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.

Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.

PubMed

Wile, Brian M; Ban, Kiwon; Yoon, Young-Sup; Bao, Gang

2014-10-01

Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

Targeted Deletion and Lipidomic Analysis Identify Epithelial Cell COX-2 as a Major Driver of Chemically-induced Skin Cancer

PubMed Central

Jiao, Jing; Ishikawa, Tomo-o; Dumlao, Darren S.; Norris, Paul C.; Magyar, Clara E.; Mikulec, Carol; Catapang, Art; Dennis, Edward A.; Fischer, Susan M.; Herschman, Harvey R.

2014-01-01

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX2) plays a critical role in DMBA/TPA-induced skin tumor induction. While many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell-type specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared to littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2 expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell-type specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biological responses. PMID:25063587

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

PubMed

Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

Bridging the Gap: Towards a Cell-Type Specific Understanding of Neural Circuits Underlying Fear Behaviors

PubMed Central

McCullough, KM; Morrison, FG; Ressler, KJ

2016-01-01

Fear and anxiety-related disorders are remarkably common and debilitating, and are often characterized by dysregulated fear responses. Rodent models of fear learning and memory have taken great strides towards elucidating the specific neuronal circuitries underlying the learning of fear responses. The present review addresses recent research utilizing optogenetic approaches to parse circuitries underlying fear behaviors. It also highlights the powerful advances made when optogenetic techniques are utilized in a genetically defined, cell-type specific, manner. The application of next-generation genetic and sequencing approaches in a cell-type specific context will be essential for a mechanistic understanding of the neural circuitry underlying fear behavior and for the rational design of targeted, circuit specific, pharmacologic interventions for the treatment and prevention of fear-related disorders. PMID:27470092

Evaluation of pharmacokinetic and pharmacodynamic profiles of liposomes for the cell type-specific delivery of small molecule drugs.

PubMed

Dasa, Siva Sai Krishna; Suzuki, Ryo; Mugler, Emily; Chen, Lanlin; Jansson-Löfmark, Rasmus; Michaëlsson, Erik; Lindfors, Lennart; Klibanov, Alexander L; French, Brent A; Kelly, Kimberly A

2017-11-01

Liposome-based drug formulations represent an exciting avenue of research as they increase efficacy to toxicity ratios. Current formulations rely on passive accumulation to the disease site where drug is taken up by the cells. Ligand mediated targeting increases the net accumulation of liposomes, however, an unexplored benefit is to potentially refine pharmacodynamics (PD) of a drug specifically to different cell types within diseased tissue. As a model system, we engineered cardiomyocyte- (I-1) and endothelial-targeted (B-40) liposomes to carry a VEGFR2 inhibitor (PTK787), and examined the effect of cell type-specific delivery on both pharmacokinetics (PK) and PD. Neovascularization in post-myocardial infarction was significantly reduced by B-40 liposomes loaded with PTK787 as compared to animals injected with I-1 liposomes, and profoundly more as compared to free PTK787. This study thus shows that the intraorgan targeting of drugs through cell type-specific delivery holds substantial promise towards lowering the minimal efficacious dose administered systemically. Published by Elsevier Inc.

Comparative photodynamic therapy cytotoxicity of mannose-conjugated chlorin and talaporfin sodium in cultured human and rat cells.

PubMed

Shinoda, Yo; Takahashi, Tsutomu; Akimoto, Jiro; Ichikawa, Megumi; Yamazaki, Hiromi; Narumi, Atsushi; Yano, Shigenobu; Fujiwara, Yasuyuki

2017-01-01

Photodynamic therapy (PDT) is a Food and Drug Administration authorized method for cancer treatment, which uses photosensitizer and laser photo-irradiation to generate reactive oxygen species to induce cell death in tumors. Photosensitizers have been progressively developed, from first to third generation, with improvements in cell specificity, reduced side effects and toxicity, increased sensitivity for irradiation and reduced persistence of photosensitizer in healthy cells. These improvements have been achieved by basic comparative experiments between current and novel photosensitizers using cell lines; however, photosensitizers should be carefully evaluated because they may have cell type specificity. In the present study, we compared a third-generation photosensitizer, β-mannose-conjugated chlorin (β-M-chlorin), with the second generation, talaporfin sodium (NPe6), using seven different rat and human cell lines and a neuronal/glial primary culture prepared from rat embryos. NPe6 was more effective than β-M-chlorin in human-derived cell lines, and β-M-chlorin was more effective than NPe6 in rat primary cultures and rat-derived cell lines, except for the rat pheochromocytoma cell line, PC12. These differences of phototoxicity in different cell types are not because of differences in photosensitivity between the photosensitizers, but rather are associated with different distribution and accumulation rates in the different cell types. These data suggest that evaluation of photosensitizers for PDT should be carried out using as large a variety of cell types as possible because each photosensitizer may have cell type specificity.

Toward the human cellular microRNAome.

PubMed

McCall, Matthew N; Kim, Min-Sik; Adil, Mohammed; Patil, Arun H; Lu, Yin; Mitchell, Christopher J; Leal-Rojas, Pamela; Xu, Jinchong; Kumar, Manoj; Dawson, Valina L; Dawson, Ted M; Baras, Alexander S; Rosenberg, Avi Z; Arking, Dan E; Burns, Kathleen H; Pandey, Akhilesh; Halushka, Marc K

2017-10-01

MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species. © 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.

Maternal T-Cell Engraftment Interferes With Human Leukocyte Antigen Typing in Severe Combined Immunodeficiency.

PubMed

Liu, Chang; Duffy, Brian; Bednarski, Jeffrey J; Calhoun, Cecelia; Lay, Lindsay; Rundblad, Barrett; Payton, Jacqueline E; Mohanakumar, Thalachallour

2016-02-01

To report the laboratory investigation of a case of severe combined immunodeficiency (SCID) with maternal T-cell engraftment, focusing on the interference of human leukocyte antigen (HLA) typing by blood chimerism. HLA typing was performed with three different methods, including sequence-specific primer (SSP), sequence-specific oligonucleotide, and Sanger sequencing on peripheral blood leukocytes and buccal cells, from a 3-month-old boy and peripheral blood leukocytes from his parents. Short tandem repeat (STR) testing was performed in parallel. HLA typing of the patient's peripheral blood leukocytes using the SSP method demonstrated three different alleles for each of the HLA-B and HLA-C loci, with both maternal alleles present at each locus. Typing results from the patient's buccal cells showed a normal pattern of inheritance for paternal and maternal haplotypes. STR enrichment testing of the patient's CD3+ T lymphocytes and CD15+ myeloid cells confirmed maternal T-cell engraftment, while the myeloid cell profile matched the patient's buccal cells. Maternal T-cell engraftment may interfere with HLA typing in patients with SCID. Selection of the appropriate typing methods and specimens is critical for accurate HLA typing and immunologic assessment before allogeneic hematopoietic stem cell transplantation. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

PubMed

Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z; Gittes, George K

2016-02-17

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas

PubMed Central

Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z.; Gittes, George K.

2016-01-01

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer. PMID:26884345

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

PubMed Central

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

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

PubMed Central

Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R.

2010-01-01

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste bud cells contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of rat circumvallate taste buds. In confocal images, we counted 21.5±0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7±1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP3R3),α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP3R3, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP3R3, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ~3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10-12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells. PMID:16841162

Epigenetic regulation of normal human mammary cell type-specific miRNAs

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

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

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 linkedmore » 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.« less

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Human stem cells and drug screening: opportunities and challenges.

PubMed

Ebert, Allison D; Svendsen, Clive N

2010-05-01

High-throughput screening technologies are widely used in the early stages of drug discovery to rapidly evaluate the properties of thousands of compounds. However, they generally rely on testing compound libraries on highly proliferative immortalized or cancerous cell lines, which do not necessarily provide an accurate indication of the effects of compounds in normal human cells or the specific cell type under study. Recent advances in stem cell technology have the potential to allow production of a virtually limitless supply of normal human cells that can be differentiated into any specific cell type. Moreover, using induced pluripotent stem cell technology, they can also be generated from patients with specific disease traits, enabling more relevant modelling and drug screens. This article discusses the opportunities and challenges for the use of stem cells in drug screening with a focus on induced pluripotent stem cells.

The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

Beyond generalized hair cells: Molecular cues for hair cell types

PubMed Central

Jahan, Israt; Pan, Ning; Kersigo, Jennifer; Fritzsch, Bernd

2012-01-01

Basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial for inner ear neurosensory development. The proneural TF Atoh1 regulates the differentiation of hair cells (HCs) whereas Neurog1 and Neurod1 regulate specification and differentiation of neurons, respectively, but also affect HC development. Expression of Delta and Jagged ligands in nascent HCs and Notch receptors in supporting cells induce supporting cell differentiation through the regulation of neurogenic bHLH TFs (such as Hes1, Hes5) and suppression of limited Atoh1 expression. In sensorineural hearing loss, HCs are lost followed by supporting cells and progressive degeneration of neurons, at least in rodents. Regaining complete hearing may require reconstituting the organ of Corti (OC) from scratch, including the two types of HCs, inner (IHC) and outer (OHC) hair cells with the precise sorting of two types of afferent (type I and II) and efferent (lateral, LOC and medial, MOC olivo-cochlear) innervation. We review effects of bHLH TF dosage and their cross-regulation to differentiate HC types in the OC. We categorize findings of specific gene expressions in HCs: 1. as markers without meaning for the regeneration task, 2. as stabilizers who are needed to maintain or complete differentiation, and 3. as decision making genes, expressed and acting early enough to be useful in this process. Only one TF has been characterized that fits the last aspect: Atoh1. We propose that temporal and intensity variations of Atoh1 are naturally modulated to differentiate specific types of HCs. Importantly, the molecular means to modify the Atoh1 expression are at least partially understood and can be readily implemented in the attempts to regenerate specific types of HCs. PMID:23201032

[Polyamines and cell growth: specific aspects in Amoeba proteus and in certain cancer cell lines].

PubMed

Dubois, J; Schenkel, E; Hanocq, M

1995-01-01

The differences between the metabolic schemes of polyamines can be the starting point to investigate the discovery of new antiparasitic or anticancer drugs which would be cell type specific. The studies, which were undertaken with the P388 cancer cells and Amoeba proteus, have shown that the pool of polyamine was very different in both cell types. Moreover, the cytotoxicity of putrescine, spermidine, spermine and 1-3 diaminopropane was found to be dependent of the activity of cell enzymes which could play a role to control cell proliferation by producing toxic metabolites.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

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

Han, Yanfu; Chai, Jiake, E-mail: cjk304@126.com; Sun, Tianjun

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. Inmore » this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.« less

Immunological metagene signatures derived from immunogenic cancer cell death associate with improved survival of patients with lung, breast or ovarian malignancies: A large-scale meta-analysis

PubMed Central

Garg, Abhishek D.; De Ruysscher, Dirk; Agostinis, Patrizia

2016-01-01

ABSTRACT The emerging role of the cancer cell-immune cell interface in shaping tumorigenesis/anticancer immunotherapy has increased the need to identify prognostic biomarkers. Henceforth, our primary aim was to identify the immunogenic cell death (ICD)-derived metagene signatures in breast, lung and ovarian cancer that associate with improved patient survival. To this end, we analyzed the prognostic impact of differential gene-expression of 33 pre-clinically-validated ICD-parameters through a large-scale meta-analysis involving 3,983 patients (‘discovery’ dataset) across lung (1,432), breast (1,115) and ovarian (1,436) malignancies. The main results were also substantiated in ‘validation’ datasets consisting of 818 patients of same cancer-types (i.e. 285 breast/274 lung/259 ovarian). The ICD-associated parameters exhibited a highly-clustered and largely cancer type-specific prognostic impact. Interestingly, we delineated ICD-derived consensus-metagene signatures that exhibited a positive prognostic impact that was either cancer type-independent or specific. Importantly, most of these ICD-derived consensus-metagenes (acted as attractor-metagenes and thereby) ‘attracted’ highly co-expressing sets of genes or convergent-metagenes. These convergent-metagenes also exhibited positive prognostic impact in respective cancer types. Remarkably, we found that the cancer type-independent consensus-metagene acted as an ‘attractor’ for cancer-specific convergent-metagenes. This reaffirms that the immunological prognostic landscape of cancer tends to segregate between cancer-independent and cancer-type specific gene signatures. Moreover, this prognostic landscape was largely dominated by the classical T cell activity/infiltration/function-related biomarkers. Interestingly, each cancer type tended to associate with biomarkers representing a specific T cell activity or function rather than pan-T cell biomarkers. Thus, our analysis confirms that ICD can serve as a platform for discovery of novel prognostic metagenes. PMID:27057433

IFNγ Signaling Endows DCs with the Capacity to Control Type I Inflammation during Parasitic Infection through Promoting T-bet+ Regulatory T Cells

PubMed Central

Lee, Hyang-Mi; Fleige, Anne; Forman, Ruth; Cho, Sunglim; Khan, Aly Azeem; Lin, Ling-Li; Nguyen, Duc T.; O'Hara-Hall, Aisling; Yin, Zhinan; Hunter, Christopher A.; Muller, Werner; Lu, Li-Fan

2015-01-01

IFNγ signaling drives dendritic cells (DCs) to promote type I T cell (Th1) immunity. Here, we show that activation of DCs by IFNγ is equally crucial for the differentiation of a population of T-bet+ regulatory T (Treg) cells specialized to inhibit Th1 immune responses. Conditional deletion of IFNγ receptor in DCs but not in Treg cells resulted in a severe defect in this specific Treg cell subset, leading to exacerbated immune pathology during parasitic infections. Mechanistically, IFNγ-unresponsive DCs failed to produce sufficient amount of IL-27, a cytokine required for optimal T-bet induction in Treg cells. Thus, IFNγ signalling endows DCs with the ability to efficiently control a specific type of T cell immunity through promoting a corresponding Treg cell population. PMID:25658840

Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype

PubMed Central

Bastow, K. F.; Darby, G.; Wildy, P.; Minson, A. C.

1980-01-01

We have isolated cells with a thymidine kinase-negative (tk−) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10−3 to 10−4, and resistance to either compound conferred simultaneous resistance to the other. tk− revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences. Images PMID:16789205

Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3.

PubMed

Fukushima, Keijo; Takahashi, Tadanobu; Ito, Seigo; Takaguchi, Masahiro; Takano, Maiko; Kurebayashi, Yuuki; Oishi, Kenta; Minami, Akira; Kato, Tatsuya; Park, Enoch Y; Nishimura, Hidekazu; Takimoto, Toru; Suzuki, Takashi

2014-09-01

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors. Copyright © 2014. Published by Elsevier Inc.

Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

PubMed

Coetzee, Simon G; Shen, Howard C; Hazelett, Dennis J; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J; Couch, Fergus J; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N A; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A; Pharoah, Paul D P; Noushmehr, Houtan; Gayther, Simon A

2015-07-01

Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

PubMed

Dorrell, Craig; Abraham, Stephanie L; Lanxon-Cookson, Kelsea M; Canaday, Pamela S; Streeter, Philip R; Grompe, Markus

2008-09-01

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

PubMed Central

Mossel, Eric C.; Wang, Jieru; Jeffers, Scott; Edeen, Karen E.; Wang, Shuanglin; Cosgrove, Gregory P.; Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Pearson, Leonard D.; Holmes, Kathryn V.; Mason, Robert J.

2008-01-01

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 hours and peaked at approximately 105 pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection. PMID:18022664

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

PubMed Central

Zechmann, Bernd; Liou, Liang-Chun; Koffler, Barbara E; Horvat, Lucija; Tomašić, Ana; Fulgosi, Hrvoje; Zhang, Zhaojie

2011-01-01

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress. PMID:22093747

Activated macrophages create lineage-specific microenvironments for pancreatic acinar- and β-cell regeneration in mice.

PubMed

Criscimanna, Angela; Coudriet, Gina M; Gittes, George K; Piganelli, Jon D; Esni, Farzad

2014-11-01

Although the cells that contribute to pancreatic regeneration have been widely studied, little is known about the mediators of this process. During tissue regeneration, infiltrating macrophages debride the site of injury and coordinate the repair response. We investigated the role of macrophages in pancreatic regeneration in mice. We used a saporin-conjugated antibody against CD11b to reduce the number of macrophages in mice following diphtheria toxin receptor-mediated cell ablation of pancreatic cells, and evaluated the effects on pancreatic regeneration. We analyzed expression patterns of infiltrating macrophages after cell-specific injury or from the pancreas of nonobese diabetic mice. We developed an in vitro culture system to study the ability of macrophages to induce cell-specific regeneration. Depletion of macrophages impaired pancreatic regeneration. Macrophage polarization, as assessed by expression of tumor necrosis factor-α, interleukin 6, interleukin 10, and CD206, depended on the type of injury. The signals provided by polarized macrophages promoted lineage-specific generation of acinar or endocrine cells. Macrophage from nonobese diabetic mice failed to provide signals necessary for β-cell generation. Macrophages produce cell type-specific signals required for pancreatic regeneration in mice. Additional study of these processes and signals might lead to new approaches for treating type 1 diabetes or pancreatitis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Changes in the Excitability of Neocortical Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by Advancing Disease.

PubMed

Kim, Juhyun; Hughes, Ethan G; Shetty, Ashwin S; Arlotta, Paola; Goff, Loyal A; Bergles, Dwight E; Brown, Solange P

2017-09-13

Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1 G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS. SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease. Copyright © 2017 the authors 0270-6474/17/379038-17$15.00/0.

Regulation of epidermal cell fate in Arabidopsis roots: the importance of multiple feedback loops

PubMed Central

Schiefelbein, John; Huang, Ling; Zheng, Xiaohua

2014-01-01

The specification of distinct cell types in multicellular organisms is accomplished via establishment of differential gene expression. A major question is the nature of the mechanisms that establish this differential expression in time and space. In plants, the formation of the hair and non-hair cell types in the root epidermis has been used as a model to understand regulation of cell specification. Recent findings show surprising complexity in the number and the types of regulatory interactions between the multiple transcription factor genes/proteins influencing root epidermis cell fate. Here, we describe this regulatory network and the importance of the multiple feedback loops for its establishment and maintenance. PMID:24596575

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

PubMed

Ulges, Alexander; Klein, Matthias; Reuter, Sebastian; Gerlitzki, Bastian; Hoffmann, Markus; Grebe, Nadine; Staudt, Valérie; Stergiou, Natascha; Bohn, Toszka; Brühl, Till-Julius; Muth, Sabine; Yurugi, Hajime; Rajalingam, Krishnaraj; Bellinghausen, Iris; Tuettenberg, Andrea; Hahn, Susanne; Reißig, Sonja; Haben, Irma; Zipp, Frauke; Waisman, Ari; Probst, Hans-Christian; Beilhack, Andreas; Buchou, Thierry; Filhol-Cochet, Odile; Boldyreff, Brigitte; Breloer, Minka; Jonuleit, Helmut; Schild, Hansjörg; Schmitt, Edgar; Bopp, Tobias

2015-03-01

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

Selective effect of irradiation on responses to thymus-independent antigen.

PubMed

Lee, S K; Woodland, R T

1985-02-01

Low doses of ionizing radiation have a selective immunosuppressive effect on in vivo B cell responses to thymus-independent (TI) antigens. The B cell response, assayed as direct anti-trinitrophenyl (TNP)-specific plaque-forming cells (PFC), induced by type 2, TI antigens (TNP-Ficoll or TNP-Dextran), was reduced, on the average, by 10-fold in animals exposed to 200 rad of ionizing radiation 24 hr before antigen challenge. In contrast, PFC responses to type 1, TI antigens (TNP-lipopolysaccharide or TNP-Brucella abortus) are unaffected in mice exposed to the same dose of radiation. Adoptive transfers showed that this selective immunosuppression is a result of the specific inactivation of the B cell subpopulation responding to type 2, TI antigens. These experiments suggest that physiologic differences exist in the B cell subpopulations of normal mice which respond to type 1, or type 2, TI antigens.

The Terminator mouse is a diphtheria toxin-receptor knock-in mouse strain for rapid and efficient enrichment of desired cell lineages.

PubMed

Guo, Jian-Kan; Shi, Hongmei; Koraishy, Farrukh; Marlier, Arnaud; Ding, Zhaowei; Shan, Alan; Cantley, Lloyd G

2013-11-01

Biomedical research often requires primary cultures of specific cell types, which are challenging to obtain at high purity in a reproducible manner. Here we engineered the murine Rosa26 locus by introducing the diphtheria toxin receptor flanked by loxP sites. The resultant strain was nicknamed the Terminator mouse. This approach results in diphtheria toxin-receptor expression in all non-Cre expressing cell types, making these cells susceptible to diphtheria toxin exposure. In primary cultures of kidney cells derived from the Terminator mouse, over 99.99% of cells were dead within 72 h of diphtheria toxin treatment. After crossing the Terminator with the podocin-Cre (podocyte specific) mouse or the Ggt-Cre (proximal tubule specific) mouse, diphtheria toxin treatment killed non-Cre expressing cells but spared podocytes and proximal tubule cells, respectively, enriching the primary cultures to over 99% purity, based on both western blotting and immunostaining of marker proteins. Thus, the Terminator mouse can be a useful tool to selectively and reproducibly obtain even low-abundant cell types at high quantity and purity.

Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

DOE PAGES

Botcheva, Krassimira; McCorkle, Sean R.

2014-11-21

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

Potential of mid IR spectroscopy in the rapid label free identification of skin malignancies

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2016-03-01

The rapid inspection of suspicious skin lesions for pathological cell types is the objective of optical point of care diagnostics technologies. A marker free fast diagnosis of skin malignancies would overcome the limitations of the current gold standard surgical biopsy. The time consuming and costly biopsy procedure requires the inspection of each sample by a trained pathologist, which limits the analysis of potentially malignant lesions. Optical technologies like RAMAN or infrared spectroscopy, which provide both, localization and chemical information, can be used to differentiate malignant from healthy tissue by the analysis of multi cell structures and cell type specific spectra. We here report the application of midIR spectroscopy towards fast and reliable skin diagnostics. Within the European research project MINERVA we developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional human skin equivalents. The standards were characterized in the established midIR range and also with newly developed systems for fast imaging up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially the tumor cells. The signals from single cell layers were sufficient for cell type differentiation. We have compared different midIR systems and found all of them suitable for specific cell type identification. Our data demonstrate the potential of midIR spectroscopy for fast image acquisition and an improved data processing as sensitive and specific optical biopsy technology.

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

PubMed Central

Nagao, Yusuke; Suzuki, Takao; Shimizu, Atsushi; Kimura, Tetsuaki; Seki, Ryoko; Adachi, Tomoko; Inoue, Chikako; Omae, Yoshihiro; Kamei, Yasuhiro; Hara, Ikuyo; Taniguchi, Yoshihito; Naruse, Kiyoshi; Wakamatsu, Yuko; Kelsh, Robert N.; Hibi, Masahiko; Hashimoto, Hisashi

2014-01-01

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor). PMID:24699463

Jacalin and peanut agglutinin (PNA) bindings in the taste bud cells of the rat: new reliable markers for type IV cells of the rat taste buds.

PubMed

Taniguchi, Ryo; Shi, Lei; Fujii, Masae; Ueda, Katsura; Honma, Shiho; Wakisaka, Satoshi

2005-12-01

Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (beta-1, 3) N-acetylgalactosamine (galactosyl (beta-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and alpha-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for alpha-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.

Human T-Cell Clones from Autoimmune Thyroid Glands: Specific Recognition of Autologous Thyroid Cells

NASA Astrophysics Data System (ADS)

Londei, Marco; Bottazzo, G. Franco; Feldmann, Marc

1985-04-01

The thyroid glands of patients with autoimmune diseases such as Graves' disease and certain forms of goiter contain infiltrating activated T lymphocytes and, unlike cells of normal glands, the epithelial follicular cells strongly express histocompatability antigens of the HLA-DR type. In a study of such autoimmune disorders, the infiltrating T cells from the thyroid glands of two patients with Graves' disease were cloned in mitogen-free interleukin-2 (T-cell growth factor). The clones were expanded and their specificity was tested. Three types of clones were found. One group, of T4 phenotype, specifically recognized autologous thyroid cells. Another, also of T4 phenotype, recognized autologous thyroid or blood cells and thus responded positively in the autologous mixed lymphocyte reaction. Other clones derived from cells that were activated in vivo were of no known specificity. These clones provide a model of a human autoimmune disease and their analysis should clarify mechanisms of pathogenesis and provide clues to abrogating these undesirable immune responses.

Improved specific energy Ni-H2 cell

NASA Astrophysics Data System (ADS)

Miller, L.

1985-07-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

Improved Specific Energy Ni-h2 Cell

NASA Technical Reports Server (NTRS)

Miller, L.

1985-01-01

Design optimization activities which have evolved and validated the necessary technology to produce Ni-H2 battery cells exhibiting a specific energy of 75-80 Whr/Kg (energy density approximately 73 Whr/L are summarized. Final design validation is currently underway with the production of battery cells for qualification and life testing. The INTELSAT type Ni-H2 battery cell design has been chosen for expository purposes. However, it should be recognized portions of the improved technology could be applied to the Air Force type Ni-H2 battery cell design with equal benefit.

The MYB23 Gene Provides a Positive Feedback Loop for Cell Fate Specification in the Arabidopsis Root Epidermis[C][W

PubMed Central

Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

2009-01-01

The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis. PMID:19395683

The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis.

PubMed

Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

2009-04-01

The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Cell-specific prediction and application of drug-induced gene expression profiles.

PubMed

Hodos, Rachel; Zhang, Ping; Lee, Hao-Chih; Duan, Qiaonan; Wang, Zichen; Clark, Neil R; Ma'ayan, Avi; Wang, Fei; Kidd, Brian; Hu, Jianying; Sontag, David; Dudley, Joel

2018-01-01

Gene expression profiling of in vitro drug perturbations is useful for many biomedical discovery applications including drug repurposing and elucidation of drug mechanisms. However, limited data availability across cell types has hindered our capacity to leverage or explore the cell-specificity of these perturbations. While recent efforts have generated a large number of drug perturbation profiles across a variety of human cell types, many gaps remain in this combinatorial drug-cell space. Hence, we asked whether it is possible to fill these gaps by predicting cell-specific drug perturbation profiles using available expression data from related conditions--i.e. from other drugs and cell types. We developed a computational framework that first arranges existing profiles into a three-dimensional array (or tensor) indexed by drugs, genes, and cell types, and then uses either local (nearest-neighbors) or global (tensor completion) information to predict unmeasured profiles. We evaluate prediction accuracy using a variety of metrics, and find that the two methods have complementary performance, each superior in different regions in the drug-cell space. Predictions achieve correlations of 0.68 with true values, and maintain accurate differentially expressed genes (AUC 0.81). Finally, we demonstrate that the predicted profiles add value for making downstream associations with drug targets and therapeutic classes.

Cell-specific prediction and application of drug-induced gene expression profiles

PubMed Central

Hodos, Rachel; Zhang, Ping; Lee, Hao-Chih; Duan, Qiaonan; Wang, Zichen; Clark, Neil R.; Ma'ayan, Avi; Wang, Fei; Kidd, Brian; Hu, Jianying; Sontag, David

2017-01-01

Gene expression profiling of in vitro drug perturbations is useful for many biomedical discovery applications including drug repurposing and elucidation of drug mechanisms. However, limited data availability across cell types has hindered our capacity to leverage or explore the cell-specificity of these perturbations. While recent efforts have generated a large number of drug perturbation profiles across a variety of human cell types, many gaps remain in this combinatorial drug-cell space. Hence, we asked whether it is possible to fill these gaps by predicting cell-specific drug perturbation profiles using available expression data from related conditions--i.e. from other drugs and cell types. We developed a computational framework that first arranges existing profiles into a three-dimensional array (or tensor) indexed by drugs, genes, and cell types, and then uses either local (nearest-neighbors) or global (tensor completion) information to predict unmeasured profiles. We evaluate prediction accuracy using a variety of metrics, and find that the two methods have complementary performance, each superior in different regions in the drug-cell space. Predictions achieve correlations of 0.68 with true values, and maintain accurate differentially expressed genes (AUC 0.81). Finally, we demonstrate that the predicted profiles add value for making downstream associations with drug targets and therapeutic classes. PMID:29218867

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

Clostridium perfringens Type E Virulence Traits Involved in Gut Colonization

PubMed Central

Redondo, Leandro M.; Carrasco, Juan M. Díaz; Redondo, Enzo A.; Delgado, Fernando; Miyakawa, Mariano E. Fernández

2015-01-01

Clostridium perfringens type E disease in ruminants has been characterized by hemorrhagic enteritis or sudden death. Although type E isolates are defined by the production of alpha and iota toxin, little is known about the pathogenesis of C. perfringens type E infections. Thus far, the role of iota toxin as a virulence factor is unknown. In this report, iota toxin showed positive effects on adherence and colonization of C. perfringens type E while having negative effect on the adherence of type A cells. In-vitro and in-vivo models suggest that toxinotype E would be particularly adapted to exploit the changes induced by iota toxin in the surface of epithelial cells. In addition, type E strains produce metabolites that affected the growth of potential intra-specific competitors. These results suggest that the alteration of the enterocyte morphology induced by iota toxin concomitantly with the specific increase of type E cell adhesion and the strong intra-specific growth inhibition of other strains could be competitive traits inherent to type E isolates that improve its fitness within the bovine gut environment. PMID:25799452

Immunity to herpes simplex virus type 2. Suppression of virus-induced immune responses in ultraviolet B-irradiated mice

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

Yasumoto, S.; Hayashi, Y.; Aurelian, L.

1987-10-15

Ultraviolet B irradiation (280 to 320 nm) of mice at the site of intradermal infection with herpes simplex virus type 2 increased the severity of the herpes simplex virus type 2 disease and decreased delayed-type hypersensitivity (DTH) responses to viral antigen. Decrease in DTH resulted from the induction of suppressor T cells, as evidenced by the ability of spleen cells from UV-irradiated mice to inhibit DTH and proliferative responses after adoptive transfer. Lymph node cells from UV-irradiated animals did not transfer suppression. DTH was suppressed at the induction but not the expression phase. Suppressor T cells were Lyt-1+, L3T4+, andmore » their activity was antigen-specific. However, after in vitro culture of spleen cells from UV-irradiated mice with herpes simplex virus type 2 antigen, suppressor activity was mediated by Lyt-2+ cells. Culture supernatants contained soluble nonantigen-specific suppressive factors.« less

Distinct roles of neuroepithelial-like and radial glia-like progenitor cells in cerebellar regeneration.

PubMed

Kaslin, Jan; Kroehne, Volker; Ganz, Julia; Hans, Stefan; Brand, Michael

2017-04-15

Zebrafish can regenerate after brain injury, and the regenerative process is driven by resident stem cells. Stem cells are heterogeneous in the vertebrate brain, but the significance of having heterogeneous stem cells in regeneration is not understood. Limited availability of specific stem cells might impair the regeneration of particular cell lineages. We studied regeneration of the adult zebrafish cerebellum, which contains two major stem and progenitor cell types: ventricular zone and neuroepithelial cells. Using conditional lineage tracing we demonstrate that cerebellar regeneration depends on the availability of specific stem cells. Radial glia-like cells are thought to be the predominant stem cell type in homeostasis and after injury. However, we find that radial glia-like cells play a minor role in adult cerebellar neurogenesis and in recovery after injury. Instead, we find that neuroepithelial cells are the predominant stem cell type supporting cerebellar regeneration after injury. Zebrafish are able to regenerate many, but not all, cell types in the cerebellum, which emphasizes the need to understand the contribution of different adult neural stem and progenitor cell subtypes in the vertebrate central nervous system. © 2017. Published by The Company of Biologists Ltd.

Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

PubMed Central

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Background Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. PMID:22879953

Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1.

PubMed

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as "replication domains", and recent findings indicate that replication timing is under developmental and cell type-specific regulation. We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome.

Genetic identification of brain cell types underlying schizophrenia.

PubMed

Skene, Nathan G; Bryois, Julien; Bakken, Trygve E; Breen, Gerome; Crowley, James J; Gaspar, Héléna A; Giusti-Rodriguez, Paola; Hodge, Rebecca D; Miller, Jeremy A; Muñoz-Manchado, Ana B; O'Donovan, Michael C; Owen, Michael J; Pardiñas, Antonio F; Ryge, Jesper; Walters, James T R; Linnarsson, Sten; Lein, Ed S; Sullivan, Patrick F; Hjerling-Leffler, Jens

2018-06-01

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.

The mathematical cell model reconstructed from interference microscopy data

NASA Astrophysics Data System (ADS)

Rogotnev, A. A.; Nikitiuk, A. S.; Naimark, O. B.; Nebogatikov, V. O.; Grishko, V. V.

2017-09-01

The mathematical model of cell dynamics is developed to link the dynamics of the phase cell thickness with the signs of the oncological pathology. The measurements of irregular oscillations of cancer cells phase thickness were made with laser interference microscope MIM-340 in order to substantiate this model. These data related to the dynamics of phase thickness for different cross-sections of cells (nuclei, nucleolus, and cytoplasm) allow the reconstruction of the attractor of dynamic system. The attractor can be associated with specific types of collective modes of phase thickness responsible for the normal and cancerous cell dynamics. Specific type of evolution operator was determined using an algorithm of designing of the mathematical cell model and temporal phase thickness data for cancerous and normal cells. Qualitative correspondence of attractor types to the cell states was analyzed in terms of morphological signs associated with maximum value of mean square irregular oscillations of phase thickness dynamics.

Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

PubMed Central

Krönig, Malte; Walter, Max; Drendel, Vanessa; Werner, Martin; Jilg, Cordula A.; Richter, Andreas S.; Backofen, Rolf; McGarry, David; Follo, Marie; Schultze-Seemann, Wolfgang; Schüle, Roland

2015-01-01

A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. PMID:25514598

Adoptive Transfer of Tumor-Specific Th2 Cells Eradicates Tumors by Triggering an In Situ Inflammatory Immune Response.

PubMed

Lorvik, Kristina Berg; Hammarström, Clara; Fauskanger, Marte; Haabeth, Ole Audun Werner; Zangani, Michael; Haraldsen, Guttorm; Bogen, Bjarne; Corthay, Alexandre

2016-12-01

Adoptive cell therapy (ACT) trials to date have focused on transfer of autologous tumor-specific cytotoxic CD8 + T cells; however, the potential of CD4 + T helper (Th) cells for ACT is gaining interest. While encouraging results have been reported with IFNγ-producing Th1 cells, tumor-specific Th2 cells have been largely neglected for ACT due to their reported tumor-promoting properties. In this study, we tested the efficacy of idiotype-specific Th2 cells for the treatment of mice with MHC class II-negative myeloma. Th2 ACT efficiently eradicated subcutaneous myeloma in an antigen-specific fashion. Transferred Th2 cells persisted in vivo and conferred long-lasting immunity. Cancer eradication mediated by tumor-specific Th2 cells did not require B cells, natural killer T cells, CD8 + T cells, or IFNγ. Th2 ACT was also curative against B-cell lymphoma. Upon transfer, Th2 cells induced a type II inflammation at the tumor site with massive infiltration of M2-type macrophages producing arginase. In vivo blockade of arginase strongly inhibited Th2 ACT, consistent with a key role of arginase and M2 macrophages in myeloma elimination by Th2 cells. These results illustrate that cancer eradication may be achieved by induction of a tumor-specific Th2 inflammatory immune response at the tumor site. Thus, ACT with tumor-specific Th2 cells may represent a highly efficient immunotherapy protocol against cancer. Cancer Res; 76(23); 6864-76. ©2016 AACR. ©2016 American Association for Cancer Research.

GAD-specific T cells are induced by GAD-alum treatment in Type-1 diabetes patients.

PubMed

Pihl, Mikael; Barcenilla, Hugo; Axelsson, Stina; Chéramy, Mikael; Åkerman, Linda; Johansson, Ingela; Ludvigsson, Johnny; Casas, Rosaura

2017-03-01

Administration of Glutamic Acid Decarboxylase (GAD) 65 formulated in aluminium hydroxide preserved insulin secretion in a phase II trial in recent onset Type 1 Diabetes. A subsequent European phase III trial was closed at 15months after failing to reach primary endpoint, but the majority of the Swedish patients completed the 21months follow-up. We studied the frequencies and phenotype of T cells, suppressive capacity of Tregs, GAD 65 -induced proliferation, and frequencies of T cells with a GAD 65 -specific TCR in Swedes participating in the trial. Stimulation with GAD 65 induced activated T cells and also cells with a suppressive phenotype. Activated GAD 65 -specific effector T cells were detected by tetramer staining while the frequency of GAD 65 -specific Treg was not affected by the treatment. Additional doses of GAD-alum increased frequencies of CD25 + CD127 + , but had no effect on CD25 hi CD127 lo . Our findings indicate that GAD-alum treatment primarily induced activated T cells. GAD 65 -specific cells were mainly of activated phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic tracing of the gustatory neural pathway originating from Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae

PubMed Central

Yamamoto, Kurumi; Ishimaru, Yoshiro; Ohmoto, Makoto; Matsumoto, Ichiro; Asakura, Tomiko; Abe, Keiko

2011-01-01

Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate and foliate papillae located in the posterior region of the tongue, though not in fungiform papillae or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in circumvallate and foliate papillae, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal nerve bundles in the nodose/petrosal ganglion. WGA signals were also observed in a small population of neurons in the geniculate ganglion. This result demonstrates the anatomical connection between taste receptor cells in the foliate papillae and the chorda tympani nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract. These findings demonstrate that the approximately 10 kb 5’-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from taste receptor cells in the posterior region of the tongue. PMID:21883212

Satellite cell response to concurrent resistance exercise and high-intensity interval training in sedentary, overweight/obese, middle-aged individuals.

PubMed

Pugh, Jamie K; Faulkner, Steve H; Turner, Mark C; Nimmo, Myra A

2018-02-01

Sarcopenia can begin from the 4-5th decade of life and is exacerbated by obesity and inactivity. A combination of resistance exercise (RE) and endurance exercise is recommended to combat rising obesity and inactivity levels. However, work continues to elucidate whether interference in adaptive outcomes occur when RE and endurance exercise are performed concurrently. This study examined whether a single bout of concurrent RE and high-intensity interval training (HIIT) alters the satellite cell response following exercise compared to RE alone. Eight sedentary, overweight/obese, middle-aged individuals performed RE only (8 × 8 leg extensions at 70% 1RM), or RE + HIIT (10 × 1 min at 90% HR max on a cycle ergometer). Muscle biopsies were collected from the vastus lateralis before and 96 h after the RE component to determine muscle fiber type-specific total (Pax7 + cells) and active (MyoD + cells) satellite cell number using immunofluorescence microscopy. Type-I-specific Pax7 + (P = 0.001) cell number increased after both exercise trials. Type-I-specific MyoD + (P = 0.001) cell number increased after RE only. However, an elevated baseline value in RE + HIIT compared to RE (P = 0.046) was observed, with no differences between exercise trials at 96 h (P = 0.21). Type-II-specific Pax7 + and MyoD + cell number remained unchanged after both exercise trials (all P ≥ 0.13). Combining a HIIT session after a single bout of RE does not interfere with the increase in type-I-specific total, and possibly active, satellite cell number, compared to RE only. Concurrent RE + HIIT may offer a time-efficient way to maximise the physiological benefits from a single bout of exercise in sedentary, overweight/obese, middle-aged individuals.

Single-cell-type Proteomics: Toward a Holistic Understanding of Plant Function*

PubMed Central

Dai, Shaojun; Chen, Sixue

2012-01-01

Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the protein characteristics of each type of cell will not only reveal specific cell functions, but also enhance understanding of how an organism works. Most plant proteomics studies have focused on using tissues and organs containing a mixture of different cells. Recent single-cell-type proteomics efforts on pollen grains, guard cells, mesophyll cells, root hairs, and trichomes have shown utility. We expect that high resolution proteomic analyses will reveal novel functions in single cells. This review provides an overview of recent developments in plant single-cell-type proteomics. We discuss application of the approach for understanding important cell functions, and we consider the technical challenges of extending the approach to all plant cell types. Finally, we consider the integration of single-cell-type proteomics with transcriptomics and metabolomics with the goal of providing a holistic understanding of plant function. PMID:22982375

Computational deconvolution of genome wide expression data from Parkinson's and Huntington's disease brain tissues using population-specific expression analysis

PubMed Central

Capurro, Alberto; Bodea, Liviu-Gabriel; Schaefer, Patrick; Luthi-Carter, Ruth; Perreau, Victoria M.

2015-01-01

The characterization of molecular changes in diseased tissues gives insight into pathophysiological mechanisms and is important for therapeutic development. Genome-wide gene expression analysis has proven valuable for identifying biological processes in neurodegenerative diseases using post mortem human brain tissue and numerous datasets are publically available. However, many studies utilize heterogeneous tissue samples consisting of multiple cell types, all of which contribute to global gene expression values, confounding biological interpretation of the data. In particular, changes in numbers of neuronal and glial cells occurring in neurodegeneration confound transcriptomic analyses, particularly in human brain tissues where sample availability and controls are limited. To identify cell specific gene expression changes in neurodegenerative disease, we have applied our recently published computational deconvolution method, population specific expression analysis (PSEA). PSEA estimates cell-type-specific expression values using reference expression measures, which in the case of brain tissue comprises mRNAs with cell-type-specific expression in neurons, astrocytes, oligodendrocytes and microglia. As an exercise in PSEA implementation and hypothesis development regarding neurodegenerative diseases, we applied PSEA to Parkinson's and Huntington's disease (PD, HD) datasets. Genes identified as differentially expressed in substantia nigra pars compacta neurons by PSEA were validated using external laser capture microdissection data. Network analysis and Annotation Clustering (DAVID) identified molecular processes implicated by differential gene expression in specific cell types. The results of these analyses provided new insights into the implementation of PSEA in brain tissues and additional refinement of molecular signatures in human HD and PD. PMID:25620908

Targeted deletion and lipidomic analysis identify epithelial cell COX-2 as a major driver of chemically induced skin cancer.

PubMed

Jiao, Jing; Ishikawa, Tomo-O; Dumlao, Darren S; Norris, Paul C; Magyar, Clara E; Mikulec, Carol; Catapang, Art; Dennis, Edward A; Fischer, Susan M; Herschman, Harvey R

2014-11-01

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses. Cox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors. ©2014 American Association for Cancer Research.

Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.

PubMed

Zaret, K S; Watts, J; Xu, J; Wandzioch, E; Smale, S T; Sekiya, T

2008-01-01

The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Cell fate control in the developing central nervous system

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

Guérout, Nicolas; Li, Xiaofei; Barnabé-Heider, Fanie, E-mail: Fanie.Barnabe-Heider@ki.se

The principal neural cell types forming the mature central nervous system (CNS) are now understood to be diverse. This cellular subtype diversity originates to a large extent from the specification of the earlier proliferating progenitor populations during development. Here, we review the processes governing the differentiation of a common neuroepithelial cell progenitor pool into mature neurons, astrocytes, oligodendrocytes, ependymal cells and adult stem cells. We focus on studies performed in mice and involving two distinct CNS structures: the spinal cord and the cerebral cortex. Understanding the origin, specification and developmental regulators of neural cells will ultimately impact comprehension and treatmentsmore » of neurological disorders and diseases. - Highlights: • Similar mechanisms regulate cell fate in different CNS cell types and structures. • Cell fate regulators operate in a spatial–temporal manner. • Different neural cell types rely on the generation of a diversity of progenitor cells. • Cell fate decision is dictated by the integration of intrinsic and extrinsic signals.« less

Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye

PubMed Central

Morrison, Carolyn A.; Chen, Hao; Cook, Tiffany; Brown, Stuart

2018-01-01

Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye. PMID:29324767

A Learning Model for L/M Specificity in Ganglion Cells

NASA Technical Reports Server (NTRS)

Ahumada, Albert J.

2016-01-01

An unsupervised learning model for developing LM specific wiring at the ganglion cell level would support the research indicating LM specific wiring at the ganglion cell level (Reid and Shapley, 2002). Removing the contributions to the surround from cells of the same cone type improves the signal-to-noise ratio of the chromatic signals. The unsupervised learning model used is Hebbian associative learning, which strengthens the surround input connections according to the correlation of the output with the input. Since the surround units of the same cone type as the center are redundant with the center, their weights end up disappearing. This process can be thought of as a general mechanism for eliminating unnecessary cells in the nervous system.

GiniClust: detecting rare cell types from single-cell gene expression data with Gini index.

PubMed

Jiang, Lan; Chen, Huidong; Pinello, Luca; Yuan, Guo-Cheng

2016-07-01

High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

PubMed

Van Leene, Jelle; Hollunder, Jens; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Stals, Hilde; Van Isterdael, Gert; Verkest, Aurine; Neirynck, Sandy; Buffel, Yelle; De Bodt, Stefanie; Maere, Steven; Laukens, Kris; Pharazyn, Anne; Ferreira, Paulo C G; Eloy, Nubia; Renne, Charlotte; Meyer, Christian; Faure, Jean-Denis; Steinbrenner, Jens; Beynon, Jim; Larkin, John C; Van de Peer, Yves; Hilson, Pierre; Kuiper, Martin; De Veylder, Lieven; Van Onckelen, Harry; Inzé, Dirk; Witters, Erwin; De Jaeger, Geert

2010-08-10

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

PubMed Central

Klink, Vincent P.; Overall, Christopher C.; Alkharouf, Nadim W.; MacDonald, Margaret H.; Matthews, Benjamin F.

2010-01-01

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process. PMID:20508855

Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

PubMed Central

GM, Cooper; EL, Lensie; JJ, Cray; MR, Bykowski; GE, DeCesare; MA, Smalley; MP, Mooney; PG, Campbell; JE, Losee

2010-01-01

Background Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell-types. Also, few have investigated the potential connection between a tissue-specific pathology and cells derived from seemingly unrelated tissues. Here, we compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods Cells were derived from bone marrow, adipose, and muscle of 10 day-old wild-type rabbits (WT; n=17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (CS; n=18). Cells were stimulated with bone morphogenetic protein 4 (BMP4) and alkaline phosphatase expression and cell proliferation were assessed. Results In WT rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from CS rabbit bone marrow and muscle were significantly more osteogenic than WT. Adipose-derived cells demonstrated no significant differences. While muscle-derived cells were most osteogenic in WT rabbits, bone marrow-derived cells were most osteogenic in CS rabbits. Conclusions Results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from wild-type derived cells. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis. PMID:20871482

TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

NASA Astrophysics Data System (ADS)

Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-02-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Root Type-Specific Reprogramming of Maize Pericycle Transcriptomes by Local High Nitrate Results in Disparate Lateral Root Branching Patterns1[OPEN

PubMed Central

Lithio, Andrew

2016-01-01

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching patterns of lateral roots upon local high nitrate stimulation. In particular, brace roots displayed an exceptional architectural plasticity compared to other root types. Transcriptome profiling revealed root type-specific transcriptomic reprogramming of pericycle cells upon local high nitrate stimulation. The alteration of the transcriptomic landscape of brace root pericycle cells in response to local high nitrate stimulation was most significant. Root type-specific transcriptome diversity in response to local high nitrate highlighted differences in the functional adaptability and systemic shoot nitrogen starvation response during development. Integration of morphological, anatomical, and transcriptomic data resulted in a framework underscoring similarity and diversity among root types grown in heterogeneous nitrate environments. PMID:26811190

Yersinia pestis targets neutrophils via complement receptor 3

PubMed Central

Merritt, Peter M.; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S.; Marketon, Melanie M.

2015-01-01

Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins due to reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria toward neutrophils during plague infection. PMID:25359083

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

PubMed

Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

2013-03-06

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Establishing an unusual cell type: How to make a dikaryon

PubMed Central

Kruzel, Emilia K.; Hull, Christina M.

2010-01-01

Summary The dikaryons of basidiomycete fungi represent an unusual cell type required for complete sexual development. Dikaryon formation occurs via the activities of cell type-specific homeodomain transcription factors, which form regulatory complexes to establish the dikaryotic state. Decades of classical genetic and cell biological studies in mushrooms have provided a foundation for more recent molecular studies in the pathogenic species Ustilago maydis and Cryptococcus neoformans. Studies in these systems have revealed novel mechanisms of regulation that function downstream of classic homeodomain complexes to ensure that dikaryons are established and propagated. Comparisons of these dikaryon-specific networks promise to reveal the nature of regulatory network evolution and the adaptations responsible for driving complex eukaryotic development. PMID:21036099

Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits.

PubMed

Buchanan, Katherine A; Blackman, Arne V; Moreau, Alexandre W; Elgar, Dale; Costa, Rui P; Lalanne, Txomin; Tudor Jones, Adam A; Oyrer, Julia; Sjöström, P Jesper

2012-08-09

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns. Copyright © 2012 Elsevier Inc. All rights reserved.

Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

PubMed Central

Mazzoni, Esteban O; Mahony, Shaun; Closser, Michael; Morrison, Carolyn A; Nedelec, Stephane; Williams, Damian J; An, Disi; Gifford, David K; Wichterle, Hynek

2013-01-01

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types. PMID:23872598

Systems Biology Analysis of Heterocellular Signaling.

PubMed

Tape, Christopher J

2016-08-01

Tissues comprise multiple heterotypic cell types (e.g., epithelial, mesenchymal, and immune cells). Communication between heterotypic cell types is essential for biological cohesion and is frequently dysregulated in disease. Despite the importance of heterocellular communication, most systems biology techniques do not report cell-specific signaling data from mixtures of cells. As a result, our existing perspective of cellular behavior under-represents the influence of heterocellular signaling. Recent technical advances now permit the resolution of systems-level cell-specific signaling data. This review discusses how new physical, spatial, and isotopic resolving methods are facilitating unique systems biology studies of heterocellular communication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cuticular Waxes of Arabidopsis thaliana Shoots: Cell-Type-Specific Composition and Biosynthesis

PubMed Central

Hegebarth, Daniela; Jetter, Reinhard

2017-01-01

It is generally assumed that all plant epidermis cells are covered with cuticles, and the distinct surface geometries of pavement cells, guard cells, and trichomes imply functional differences and possibly different wax compositions. However, experiments probing cell-type-specific wax compositions and biosynthesis have been lacking until recently. This review summarizes new evidence showing that Arabidopsis trichomes have fewer wax compound classes than pavement cells, and higher amounts of especially long-chain hydrocarbons. The biosynthesis machinery generating this characteristic surface coating is discussed. Interestingly, wax compounds with similar, long hydrocarbon chains had been identified previously in some unrelated species, not all of them bearing trichomes. PMID:28686187

Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

PubMed Central

Li, Wenyan; Shen, Jun

2016-01-01

Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

PubMed

Shi, Rui; Wang, Jack P; Lin, Ying-Chung; Li, Quanzi; Sun, Ying-Hsuan; Chen, Hao; Sederoff, Ronald R; Chiang, Vincent L

2017-05-01

Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

Single-Cell Tracking Reveals a Role for Pre-Existing CCR5+ Memory Th1 Cells in the Control of Rhinovirus-A39 After Experimental Challenge in Humans.

PubMed

Muehling, Lyndsey M; Turner, Ronald B; Brown, Kenneth B; Wright, Paul W; Patrie, James T; Lahtinen, Sampo J; Lehtinen, Markus J; Kwok, William W; Woodfolk, Judith A

2018-01-17

Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4+ T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4+ subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5+ Th1 effectors, and included a minor interleukin-21+ T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5+ Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5+ effector memory types were comodulated. In the nose, CXCR3-CCR5+ T cells present during acute infection were activated effector memory type, whereas CXCR3+ cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. We conclude that virus-specific CCR5+ effector memory CD4+ T cells primed by previous exposure to related viruses contribute to the control of rhinovirus. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

The epidermis coordinates auxin-induced stem growth in response to shade

PubMed Central

Procko, Carl; Burko, Yogev; Long, Jeff A.; Chory, Joanne

2016-01-01

Growth of a complex multicellular organism requires coordinated changes in diverse cell types. These cellular changes generate organs of the correct size, shape, and functionality. In plants, the growth hormone auxin induces stem elongation in response to shade; however, which cell types of the stem perceive the auxin signal and contribute to organ growth is poorly understood. Here, we blocked the transcriptional response to auxin within specific tissues to show that auxin signaling is required in many cell types for correct hypocotyl growth in shade, with a key role for the epidermis. Combining genetic manipulations in Arabidopsis thaliana with transcriptional profiling of the hypocotyl epidermis from Brassica rapa, we show that auxin acts in the epidermis in part by inducing activity of the locally acting, growth-promoting brassinosteroid pathway. Our findings clarify cell-specific auxin function in the hypocotyl and highlight the complexity of cell type interactions within a growing organ. PMID:27401556

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

PubMed

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

PubMed

Goon, Peter K C; Hanon, Emmanuel; Igakura, Tadahiko; Tanaka, Yuetsu; Weber, Jonathan N; Taylor, Graham P; Bangham, Charles R M

2002-05-01

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

Leukemia mortality by cell type in petroleum workers with potential exposure to benzene.

PubMed Central

Raabe, G K; Wong, O

1996-01-01

Workers in the petroleum industry are potentially exposed to a variety of petrochemicals, including benzene or benzene-containing liquids. Although a large number of studies of petroleum workers have been conducted to examine leukemia and other cancer risks, few existing studies have investigated cell-type-specific leukemias. One of the major reasons for the lack of cell-type-specific analysis was the small number of deaths by cell type in individual studies. In the present investigation, all cohort studies of petroleum workers in the United States and the United Kingdom were combined into a single database for cell-type-specific leukemia analysis. The majority of these workers were petroleum refinery employees, but production, pipeline, and distribution workers in the petroleum industry were also included. The combined cohort consisted of more than 208,000 petroleum workers, who contributed more than 4.6 million person-years of observation. Based on a meta-analysis of the combined data, cell-type-specific leukemia risks were expressed in terms of standardized mortality ratios (meta-SMRs). The meta-SMR for acute myeloid leukemia was 0.96. The lack of an increase of acute myeloid leukemia was attributed to the low levels of benzene exposure in the petroleum industry, particularly in comparison to benzene exposure levels in some previous studies of workers in other industries, who had been found to experience an increased risk of acute myeloid leukemia. Similarly, no increase in chronic myeloid, acute lymphocytic, or chronic lymphocytic leukemias was found in petroleum workers (meta-SMRs of 0.89, 1.16, and 0.84, respectively). Stratified meta-analyses restricted to refinery studies or to studies with at least 15 years of follow-up yielded similar results. The findings of the present investigation are consistent with those from several recent case-control studies of cell-type-specific leukemia. Patterns and levels of benzene exposure in the petroleum industry are reviewed. The results of the present epidemiologic investigation are discussed in conjunction with recent advances in leukemogenesis from other scientific disciplines. PMID:9118924

A rapid, automated approach for quantitation of rotavirus and reovirus infectivity.

PubMed

Iskarpatyoti, Jason A; Willis, Janet Z; Guan, John; Morse, E Ashley; Ikizler, Miné; Wetzel, J Denise; Dermody, Terence S; Contractor, Nikhat

2012-09-01

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types. Copyright © 2012 Elsevier B.V. All rights reserved.

B cell function in the immune response to helminths

PubMed Central

Harris, Nicola

2010-01-01

Similar T helper (Th)2-type immune responses are generated against different helminths parasites, but the mechanisms that initiate Th2 immunity, and the specific immune components that mediate protection against these parasites, can vary greatly. B cells are increasingly recognized as important during the Th2-type immune response to helminths, and B cell activation might be a target for effective vaccine development. Antibody production is a function of B cells during helminth infection and understanding how polyclonal and antigen-specific antibodies contribute should provide important insights into how protective immunity develops. In addition, B cells might also contribute to the host response against helminths through antibody-independent functions including, antigen-presentation, as well as regulatory and effector activity. In this review, we examine the role of B cells during Th2-type immune response to these multicellular parasites. PMID:21159556

Comparison of cell-type-specific vs transmural aortic gene expression in experimental aneurysms.

PubMed

Sho, Eiketsu; Sho, Mien; Nanjo, Hiroshi; Kawamura, Koichi; Masuda, Hirotake; Dalman, Ronald L

2005-05-01

Abdominal aortic aneurysm (AAA) progression and disease resistance are related to mural cellularity; adventitial macrophages and neocapillaries predominate in larger, advanced aneurysms, whereas smaller AAAs have fewer macrophages and retain more medial smooth muscle cells (SMCs). Expression analysis of mRNA derived from the entire aorta may mask the role that specific cell types play in modulating disease progression. We used laser capture microdissection (LCM) to isolate SMC and macrophage-predominant mural cell populations for gene expression analysis in variable-flow AAA. Rat AAAs were created via porcine pancreatic elastase (PPE) infusion. Aortic flow was increased via femoral arteriovenous fistula creation (HF-AAA) or reduced via unilateral iliac ligation (LF-AAA) in selected cohorts. SMC and macrophage-predominant cell populations were isolated via LCM and analyzed for expression of pro-inflammatory transcription factors and chemokines, cytokines, and proteolytic enzymes via real-time polymerase chain reaction. Aortic PPE infusion precipitated endothelial cell (EC) denudation, SMC apoptosis, and elastic lamellar degeneration. Increased aortic flow (HF > NF > LF) stimulated restorative EC and SMC proliferation (45.8 +/- 6.6 > 30.5 +/- 2.1 > 21 +/- 3.6 and 212.2 +/- 9.8 > 136.5 +/- 8.9 > 110 +/- 13.5, respectively, for both cell types; P < .05) at 5 days after PPE infusion, while simultaneously reducing medial SMC apoptosis and transmural macrophage infiltration. Expression of nuclear factor kappa B (NF-kappab), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage migration inhibitory (MIF), heparin-binding EGF-like factor (HB-EGF) and inducible nitric oxide synthase (iNOS) varied between cell types and flow conditions at all time points examined. Gelatinolytic protease expression varied by cell type in response to flow loading (eg, increased in SMCs, decreased in macrophages), consistent with observed patterns of elastolysis and SMC proliferation reported in prior experiments. Flow differentially regulates cell-specific AAA gene expression. Whole-organ analysis of AAA tissue lysates obscures important cellular responses to inflammation and flow, and may explain previous seemingly contradictory observations regarding proteolysis and cell proliferation. Cell-type specific expression and functional analyses may substantially clarify the pathophysiology of AAA disease. Understanding aneurysmal aortic degeneration at the most fundamental level is a critical precursor to the development of next-generation therapies such as drug-eluting endografts and/or medical therapies to limit expansion of preclinical AAA in high-risk or elderly patients. Although animal modeling is necessary to gain insight into the early initiating events of AAA disease, the methods used in such analyses have critical bearing on the conclusions drawn regarding pathogenesis and potential therapeutic derivations. By analyzing cell-type-specific gene expression rather than whole-organ tissue lysates, the precise roles of important mediators such as metalloproteinases can be placed in the appropriate context. Further refinement of these techniques may allow cell-specific therapies to be applied at defined time points in disease progression with improved patient outcome and reduced procedural morbidity.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

PubMed

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-05-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

PubMed Central

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-01-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. PMID:26957309

The Evolution of Human Cells in Terms of Protein Innovation

PubMed Central

Sardar, Adam J.; Oates, Matt E.; Fang, Hai; Forrest, Alistair R.R.; Kawaji, Hideya; Gough, Julian; Rackham, Owen J.L.

2014-01-01

Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. PMID:24692656

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

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

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Genetic Induction of Cytolytic Susceptibility in Breast Cancer Cells

DTIC Science & Technology

2001-07-01

human tumor cells have been contrasted with breast cancer cells for this same reason. The original two tasks continue to provide the focus for this...tissue type but may be generally applicable to many or all types of human tumors. The complementary question to that of cell type specificity was...transfection with either the adenoviral ElA oncogene or the E7 oncogene from human papilloma virus type 18. The question was whether both ElA and E7

HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

PubMed Central

Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

2013-01-01

Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

Different role of zinc transporter 8 between type 1 diabetes mellitus and type 2 diabetes mellitus.

PubMed

Yi, Bo; Huang, Gan; Zhou, Zhiguang

2016-07-01

Diabetes can be simply classified into type 1 diabetes mellitus and type 2 diabetes mellitus. Zinc transporter 8 (ZnT8), a novel islet autoantigen, is specifically expressed in insulin-containing secretory granules of β-cells. Genetic studies show that the genotypes of SLC30A8 can determine either protective or diabetogenic response depending on environmental and lifestyle factors. The ZnT8 protein expression, as well as zinc content in β-cells, was decreased in diabetic mice. Thus, ZnT8 might participate in insulin biosynthesis and release, and subsequently involved deteriorated β-cell function through direct or indirect mechanisms in type 1 diabetes mellitus and type 2 diabetes mellitus. From a clinical feature standpoint, the prevalence of ZnT8A is gradiently increased in type 2 diabetes mellitus, latent autoimmune diabetes in adults and type 1 diabetes mellitus. The frequency and epitopes of ZnT8-specific T cells and cytokine release by ZnT8-specific T cells are also different in diabetic patients and healthy controls. Additionally, the response to ZnT8 administration is also different in type 1 diabetes mellitus and type 2 diabetes mellitus. In the present review, we summarize the literature about clinical aspects of ZnT8 in the pathogenesis of diabetes, and suggest that ZnT8 might play a different role between type 1 diabetes mellitus and type 2 diabetes mellitus. © 2015 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

PubMed

Neiman, Daniel; Moss, Joshua; Hecht, Merav; Magenheim, Judith; Piyanzin, Sheina; Shapiro, A M James; de Koning, Eelco J P; Razin, Aharon; Cedar, Howard; Shemer, Ruth; Dor, Yuval

2017-12-19

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic β-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3 + embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and β-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and β-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for β-cell reprogramming in diabetes and diagnosis of β-cell death using methylation patterns of circulating DNA. Copyright © 2017 the Author(s). Published by PNAS.

Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

PubMed

Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D; Lichterfeld, Mathias; Yu, Xu G

2015-06-01

The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

PubMed

Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

2010-07-01

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

Spatial distribution of the messenger ribonucleic acid and protein of the nuclear receptor coactivator, amplified in breast cancer-3, in mice.

PubMed

Zhang, Hao; Liao, Lan; Kuang, Shao-Qing; Xu, Jianming

2003-04-01

Transcriptional activities of nuclear receptors are modulated by coactivators and corepressors. The amplified in breast cancer-3 protein (AIB3, also known as ASC-2, RAP250, PRIP, TRBP, and NCR) is a newly identified nuclear receptor coactivator that is amplified and overexpressed in breast cancers. This study aims to investigate the spatial expression of AIB3 mRNA and protein in various murine tissues. Quantitative measurements revealed that the concentrations of AIB3 mRNA differ substantially in different tissues in a descending order from the following: testis, brain, thymus, white fat, pituitary, ovary, adrenal gland, lung, uterus, kidney, heart, skeletal muscle, liver, and virgin mammary gland. The AIB3 mRNA level in the testis is 165-fold higher than that in the virgin mammary gland. Specific antiserum was generated and used to map the distribution of AIB3 protein by immunohistochemistry. Although AIB3 protein was detected in many tissues, the AIB3 immunoreactivities varied significantly from cell type to cell type. High levels of AIB3 immunoreactivity were observed in hormone target cells including the testicular Sertoli cells, follicular granulosa cells, and epithelial cells of the prostate, uterus, mammary gland, and kidney tubules. Medium and low levels of AIB3 immunoreactivities were also detected in a variety of other cell types. These results demonstrate that AIB3 mRNA and protein are preferentially expressed in specific cell types, suggesting that AIB3 may support the function of nuclear receptors in a cell type-specific manner.

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

PubMed

Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Adult Non-Hodgkin Lymphoma Treatment (PDQ®)—Health Professional Version

Cancer.gov

Non-Hodgkin lymphomas (NHL) include indolent types (follicular lymphoma, Waldenstrom macroglobulinemia, and MALT) and aggressive types (diffuse large cell, Burkitt, and mantle cell). Treatment and prognosis depend on the specific type. Get comprehensive information on NHL classification and treatment in this clinician summary.

Parsing Stem Cell Lineage Development Using High Content Image Analysis of Epigenetic Spatial Markers.

PubMed

Kim, Joseph J; Moghe, Prabhas V

2018-06-14

This unit describes a protocol for acquiring and analyzing high-content super-resolution images of human stem cell nuclei for the characterization and classification of the cell differentiation paths based on distinct patterns of epigenetic mark organization. Here, we describe the cell culture, immunocytochemical labeling, super-resolution imaging parameters, and MATLAB-based quantitative image analysis approaches for monitoring human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs) as the cells differentiate towards various lineages. Although this protocol uses specific cell types as examples, this approach could be easily extended to a variety of cell types and nuclear epigenetic and mechanosensitive biomarkers that are relevant to specific cell developmental scenarios. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation.

PubMed

Cho, Josalyn L; Ling, Morris F; Adams, David C; Faustino, Lucas; Islam, Sabina A; Afshar, Roshi; Griffith, Jason W; Harris, Robert S; Ng, Aylwin; Radicioni, Giorgia; Ford, Amina A; Han, Andre K; Xavier, Ramnik; Kwok, William W; Boucher, Richard; Moon, James J; Hamilos, Daniel L; Kesimer, Mehmet; Suter, Melissa J; Medoff, Benjamin D; Luster, Andrew D

2016-10-05

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4 + T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4 + T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation. Copyright © 2016, American Association for the Advancement of Science.

Cell-type Specific Optogenetic Mice for Dissecting Neural Circuitry Function

PubMed Central

Zhao, Shengli; Ting, Jonathan T.; Atallah, Hisham E.; Qiu, Li; Tan, Jie; Gloss, Bernd; Augustine, George J.; Deisseroth, Karl; Luo, Minmin; Graybiel, Ann M.; Feng, Guoping

2011-01-01

Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function, and dysfunction. We used a Bacterial Artificial Chromosome (BAC) transgenic strategy to express Channelrhodopsin2 (ChR2) under the control of cell-type specific promoter elements. We provide a detailed functional characterization of the newly established VGAT-ChR2-EYFP, ChAT-ChR2-EYFP, TPH2-ChR2-EYFP and Pvalb-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action potential firing of GABAergic, cholinergic, serotonergic, and parvalbumin+ neuron subsets using blue light. This resource of cell type-specific ChR2 mouse lines will facilitate the precise mapping of neuronal connectivity and the dissection of the neural basis of behavior. PMID:21985008

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

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

Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook

2007-06-29

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types,more » including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.« less

Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

PubMed

Koch, P J; Goldschmidt, M D; Walsh, M J; Zimbelmann, R; Schmelz, M; Franke, W W

1991-05-01

Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

Regulation of CAPRICE transcription by MYB proteins for root epidermis differentiation in Arabidopsis.

PubMed

Koshino-Kimura, Yoshihiro; Wada, Takuji; Tachibana, Tatsuhiko; Tsugeki, Ryuji; Ishiguro, Sumie; Okada, Kiyotaka

2005-06-01

Epidermal cell differentiation in Arabidopsis root is studied as a model system for understanding cell fate specification. Two types of MYB-related transcription factors are involved in this cell differentiation. One of these, CAPRICE (CPC), encoding an R3-type MYB protein, is a positive regulator of hair cell differentiation and is preferentially transcribed in hairless cells. We analyzed the regulatory mechanism of CPC transcription. Deletion analyses of the CPC promoter revealed that hairless cell-specific transcription of the CPC gene required a 69 bp sequence, and a tandem repeat of this region was sufficient for its expression in epidermis. This region includes two MYB-binding sites, and the epidermis-specific transcription of CPC was abolished when base substitutions were introduced in these sites. We showed by gel mobility shift experiments and by yeast one-hybrid assay that WEREWOLF (WER), which is an R2R3-type MYB protein, directly binds to this region. We showed that WER also binds to the GL2 promoter region, indicating that WER directly regulates CPC and GL2 transcription by binding to their promoter regions.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

PubMed

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-08

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Endothelial cells are not required for specification of respiratory progenitors

PubMed Central

Havrilak, Jamie A.; Melton, Kristin R.; Shannon, John M.

2017-01-01

Crosstalk between mesenchymal and epithelial cells influences organogenesis in multiple tissues, such as lung, pancreas, liver, and the nervous system. Lung mesenchyme comprises multiple cell types, however, and precise identification of the mesenchymal cell type(s) that drives early events in lung development remains unknown. Endothelial cells have been shown to be required for some aspects of lung epithelial patterning, lung stem cell differentiation, and regeneration after injury. Furthermore, endothelial cells are involved in early liver and pancreas development. From these observations we hypothesized that endothelial cells might also be required for early specification of the respiratory field and subsequent lung bud initiation. We first blocked VEGF signaling in E8.5 cultured foreguts with small molecule VEGFR inhibitors and found that lung specification and bud formation were unaltered. However, when we examined E9.5 mouse embryos carrying a mutation in the VEGFR Flk-1, which do not develop endothelial cells, we found that respiratory progenitor specification was impeded. Because the E9.5 embryos were substantially smaller than control littermates, suggesting the possibility of developmental delay, we isolated and cultured foreguts from mutant and control embryos on E8.5, when no size differences were apparent. We found that both specification of the respiratory field and lung bud formation occurred in mutant and control explants. These observations were unaffected by the presence or absence of serum. We also observed that hepatic specification and initiation occurred in the absence of endothelial cells, and that expansion of the liver epithelium in culture did not differ between mutant and control explants. Consistent with previously published results, we also found that pancreatic buds were not maintained in cultured foreguts when endothelial cells were absent. Our observations support the conclusion that endothelial cells are not required for early specification of lung progenitors and bud initiation, and that the diminished lung specification seen in E9.5 Flk−/− embryos is likely due to developmental delay resulting from the insufficient delivery of oxygen, nutrients, and other factors in the absence of a vasculature. PMID:28501476

Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination.

PubMed

Dong, Xiaomin; Chen, Kenian; Cuevas-Diaz Duran, Raquel; You, Yanan; Sloan, Steven A; Zhang, Ye; Zong, Shan; Cao, Qilin; Barres, Ben A; Wu, Jia Qian

2015-12-01

Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.

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

PubMed

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.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana

PubMed Central

Van Leene, Jelle; Hollunder, Jens; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Stals, Hilde; Van Isterdael, Gert; Verkest, Aurine; Neirynck, Sandy; Buffel, Yelle; De Bodt, Stefanie; Maere, Steven; Laukens, Kris; Pharazyn, Anne; Ferreira, Paulo C G; Eloy, Nubia; Renne, Charlotte; Meyer, Christian; Faure, Jean-Denis; Steinbrenner, Jens; Beynon, Jim; Larkin, John C; Van de Peer, Yves; Hilson, Pierre; Kuiper, Martin; De Veylder, Lieven; Van Onckelen, Harry; Inzé, Dirk; Witters, Erwin; De Jaeger, Geert

2010-01-01

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)–cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK–cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants. PMID:20706207

New lessons learned from disease modeling with induced Pluripotent Stem Cells

PubMed Central

Onder, Tamer T.; Daley, George Q.

2012-01-01

Cellular reprogramming and generation of induced pluripotent stem cells (iPSCs) from adult cell types has enabled the creation of patient-specific stem cells for use in disease modeling. To date, many iPSC lines have been generated from a variety of disorders, which have then been differentiated into disease-relevant cell types. When a disease-specific phenotype is detectable in such differentiated cells, the reprogramming technology provides a new opportunity to identify aberrant disease-associated pathways and drugs that can block them. Here, we highlight recent progress as well as limitations in the use of iPSCs to recapitulate disease phenotypes and to screen for therapeutics in vitro. PMID:22749051

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

Homeostasis or channelopathy? Acquired cell type-specific ion channel changes in temporal lobe epilepsy and their antiepileptic potential

PubMed Central

Wolfart, Jakob; Laker, Debora

2015-01-01

Neurons continuously adapt the expression and functionality of their ion channels. For example, exposed to chronic excitotoxicity, neurons homeostatically downscale their intrinsic excitability. In contrast, the “acquired channelopathy” hypothesis suggests that proepileptic channel characteristics develop during epilepsy. We review cell type-specific channel alterations under different epileptic conditions and discuss the potential of channels that undergo homeostatic adaptations, as targets for antiepileptic drugs (AEDs). Most of the relevant studies have been performed on temporal lobe epilepsy (TLE), a widespread AED-refractory, focal epilepsy. The TLE patients, who undergo epilepsy surgery, frequently display hippocampal sclerosis (HS), which is associated with degeneration of cornu ammonis subfield 1 pyramidal cells (CA1 PCs). Although the resected human tissue offers insights, controlled data largely stem from animal models simulating different aspects of TLE and other epilepsies. Most of the cell type-specific information is available for CA1 PCs and dentate gyrus granule cells (DG GCs). Between these two cell types, a dichotomy can be observed: while DG GCs acquire properties decreasing the intrinsic excitability (in TLE models and patients with HS), CA1 PCs develop channel characteristics increasing intrinsic excitability (in TLE models without HS only). However, thorough examination of data on these and other cell types reveals the coexistence of protective and permissive intrinsic plasticity within neurons. These mechanisms appear differentially regulated, depending on the cell type and seizure condition. Interestingly, the same channel molecules that are upregulated in DG GCs during HS-related TLE, appear as promising targets for future AEDs and gene therapies. Hence, GCs provide an example of homeostatic ion channel adaptation which can serve as a primer when designing novel anti-epileptic strategies. PMID:26124723

Jointly characterizing epigenetic dynamics across multiple human cell types

PubMed Central

An, Lin; Yue, Feng; Hardison, Ross C

2016-01-01

Advanced sequencing technologies have generated a plethora of data for many chromatin marks in multiple tissues and cell types, yet there is lack of a generalized tool for optimal utility of those data. A major challenge is to quantitatively model the epigenetic dynamics across both the genome and many cell types for understanding their impacts on differential gene regulation and disease. We introduce IDEAS, an integrative and discriminative epigenome annotation system, for jointly characterizing epigenetic landscapes in many cell types and detecting differential regulatory regions. A key distinction between our method and existing state-of-the-art algorithms is that IDEAS integrates epigenomes of many cell types simultaneously in a way that preserves the position-dependent and cell type-specific information at fine scales, thereby greatly improving segmentation accuracy and producing comparable annotations across cell types. PMID:27095202

Multicolor flow-cytometric analysis of milk allergen-specific T-helper type 2 cells revealed coexpression of interleukin-4 with Foxp3.

PubMed

Yamawaki, Kazuo; Inuo, Chisato; Nomura, Takayasu; Tanaka, Kenichi; Nakajima, Yoichi; Kondo, Yasuto; Yoshikawa, Tetsushi; Urisu, Atsuo; Tsuge, Ikuya

2015-12-01

Allergen-specific T-helper type 2 (TH2) cells play an important role in the development of allergic inflammation; however, investigations of the properties of allergen-specific T cells have been challenging in humans. Despite clear evidence that forkhead box p3 (Foxp3) is expressed in conventional effector T cells, its function has remained unknown. To characterize allergen-specific TH2 cells in milk allergy, with particular focus on the expression of Foxp3. Twenty-one children with milk allergy and 11 children without milk allergy were studied. Peripheral blood mononuclear cells from subjects were stimulated with milk allergen for 6 hours and analyzed using multicolor flow cytometry to identify CD154(+) allergen-specific T-helper cells. Simultaneously, the expression of intracellular cytokines and Foxp3 was analyzed. The milk allergy group had significantly larger numbers of milk allergen-specific interleukin (IL)-4- and IL-5-producing CD4(+) T cells than the control group. Subjects in the milk allergy group had significantly more CD154(+)CD4(+) IL-10-producing cells and CD154(+)Foxp3(+)CD4(+) cells than those in the control group. In addition, the number of milk allergen-specific CD154(+)Foxp3(+)CD4(+) cells strongly correlated with that of CD154(+)IL4(+)CD4(+) cells. Bcl-2 expression in CD154(+)IL-4(+)Foxp3(+) T-helper cells was significantly lower compared with that in total CD4 cells. Increased numbers of IL-4-producing allergen-specific T-helper cells were found in patients with milk allergy. In addition, Foxp3 was coexpressed with IL-4 in allergen-specific TH2 cells from patients. This coexpression was associated with lower Bcl-2 levels and could contribute to the phenotype and function of TH2 cells. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

groHMM: a computational tool for identifying unannotated and cell type-specific transcription units from global run-on sequencing data.

PubMed

Chae, Minho; Danko, Charles G; Kraus, W Lee

2015-07-16

Global run-on coupled with deep sequencing (GRO-seq) provides extensive information on the location and function of coding and non-coding transcripts, including primary microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and enhancer RNAs (eRNAs), as well as yet undiscovered classes of transcripts. However, few computational tools tailored toward this new type of sequencing data are available, limiting the applicability of GRO-seq data for identifying novel transcription units. Here, we present groHMM, a computational tool in R, which defines the boundaries of transcription units de novo using a two state hidden-Markov model (HMM). A systematic comparison of the performance between groHMM and two existing peak-calling methods tuned to identify broad regions (SICER and HOMER) favorably supports our approach on existing GRO-seq data from MCF-7 breast cancer cells. To demonstrate the broader utility of our approach, we have used groHMM to annotate a diverse array of transcription units (i.e., primary transcripts) from four GRO-seq data sets derived from cells representing a variety of different human tissue types, including non-transformed cells (cardiomyocytes and lung fibroblasts) and transformed cells (LNCaP and MCF-7 cancer cells), as well as non-mammalian cells (from flies and worms). As an example of the utility of groHMM and its application to questions about the transcriptome, we show how groHMM can be used to analyze cell type-specific enhancers as defined by newly annotated enhancer transcripts. Our results show that groHMM can reveal new insights into cell type-specific transcription by identifying novel transcription units, and serve as a complete and useful tool for evaluating functional genomic elements in cells.

Aptamer delivery of siRNA, radiopharmaceutics and chemotherapy agents in cancer.

PubMed

de Almeida, Carlos E B; Alves, Lais Nascimento; Rocha, Henrique F; Cabral-Neto, Januário Bispo; Missailidis, Sotiris

2017-06-20

Aptamers are oligonucleotide reagents with high affinity and specificity, which among other therapeutic and diagnostic applications have the capability of acting as delivery agents. Thus, aptamers are capable of carrying small molecules, nanoparticles, radiopharmaceuticals or fluorescent agents as well as nucleic acid therapeutics specifically to their target cells. In most cases, the molecules may possess interesting therapeutic properties, but their lack of specificity for a particular cell type, or ability to internalise in such a cell, hinders their clinical development, or cause unwanted side effects. Thus, chemotherapy or radiotherapy agents, famous for their side effects, can be coupled to aptamers for specific delivery. Equally, siRNA have great therapeutic potential and specificity, but one of their shortcomings remain the delivery and internalisation into cells. Various methodologies have been proposed to date, including aptamers, to resolve this problem. Therapeutic or imaging reagents benefit from the adaptability and ease of chemical manipulation of aptamers, their high affinity for the specific marker of a cell type, and their internalisation ability via cell mediated endocytosis. In this review paper, we explore the potential of the aptamers as delivery agents and offer an update on current status and latest advancements. Copyright © 2017 Elsevier B.V. All rights reserved.

Culture Models for Studying Thyroid Biology and Disorders

PubMed Central

Toda, Shuji; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Matsunobu, Aki; Yamamoto, Mihoko; Ootani, Akifumi; Yamasaki, Fumio; Koike, Eisuke; Sugihara, Hajime

2011-01-01

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. PMID:22363871

Hormones that Stimulate the Growth of Blood Cells.

ERIC Educational Resources Information Center

Golde, David W.; Gasson, Judith C.

1988-01-01

Describes the nature and action of hematopoietic proteins which regulate the production of specific sets of blood cells. Discusses the production of these hematopoietins by recombinant-DNA methods in an effort to enable physicians to treat patients by eliciting production of specific types of blood cells. (CW)

The impact of polyunsaturated fatty acids on DNA methylation and expression of DNMTs in human colorectal cancer cells.

PubMed

Sarabi, Mostafa Moradi; Naghibalhossaini, Fakhraddin

2018-05-01

Growing evidence suggests a role of polyunsaturated fatty acids (PUFA) in the prevention of various types of malignancy, including colorectal cancer (CRC). No published studies have yet examined the direct effect of PUFA treatment on DNA methylation in CRC cells. In this study, 5 human CRC cells were treated with 100 μM DHA, EPA, and LA for 6 days and changes in their global- and gene-specific DNA methylation status as well as expression of DNA methyl transferases (DNMT) were investigated. Cell-type specific differences in DNA methylation and expression of DNMTs were observed in PUFA-treated cells. DHA and EPA treatment induced global hypermethylation in HT29/219 and HCT116 cells, but reduced methylation in Caco2 cells (p < 0.05). Among 10 tumor related genes tested in 5 CRC cell lines, DHA and EPA induced promoter demethylation of Cox2 in HT29/219, p14 and PPARγ in HCT116, and ECAD in SW742 cells. Cell-type specific differences in expression of DNMT1, DNMT3a, and 3b genes were also observed between PUFA-treated and control cells (p < 0.05). Overall, treatment of PUFAs coordinately induced the expression of DNMTs in HT29/219, but suppressed in other 4 cell lines investigated in this study. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Transcriptional and electrophysiological maturation of neocortical fastspiking GABAergic interneurons

PubMed Central

Okaty, Benjamin W; Miller, Mark N; Sugino, Ken; Hempel, Chris M; Nelson, Sacha B

2009-01-01

Fast-spiking (FS) interneurons are important elements of neocortical circuitry that constitute the primary source of synaptic inhibition in adult cortex and impart temporal organization on ongoing cortical activity. The highly specialized intrinsic membrane and firing properties that allow cortical FS interneurons to perform these functions are due to equally specialized gene expression, which is ultimately coordinated by cell-type-specific transcriptional regulation. While embryonic transcriptional events govern the initial steps of cell-type specification in most cortical interneurons, including FS cells, the electrophysiological properties that distinguish adult cortical cell types emerge relatively late in postnatal development, and the transcriptional events that drive this maturational process are not known. To address this, we used mouse whole-genome microarrays and whole-cell patch clamp to characterize the transcriptional and electrophysiological maturation of cortical FS interneurons between postnatal day 7 (P7) and P40. We found that the intrinsic and synaptic physiology of FS cells undergoes profound regulation over the first four postnatal weeks, and that these changes are correlated with largely monotonic but bidirectional transcriptional regulation of thousands of genes belonging to multiple functional classes. Using our microarray screen as a guide, we discovered that upregulation of 2-pore K+ leak channels between P10 and P25 contributes to one of the major differences between the intrinsic membrane properties of immature and adult FS cells, and found a number of other candidate genes that likely confer cell-type specificity on mature FS cells. PMID:19474331

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

PubMed

Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

2014-03-01

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

PubMed

Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.

Cancer treatment by photodynamic therapy combined with NK-cell-line-based adoptive immunotherapy

NASA Astrophysics Data System (ADS)

Korbelik, Mladen; Sun, Jinghai

1998-05-01

Treatment of solid cancers by photodynamic therapy (PDT) triggers a strong acute inflammatory reaction localized to the illuminated malignant tissue. This event is regulated by a massive release of various potent mediators which have a profound effect not only on local host cell populations, but also attract different types of immune cells to the treated tumor. Phagocytosis of PDT-damaged cancerous cells by antigen presenting cells, such as activated tumor associated macrophages, enables the recognition of even poorly immunogenic tumors by specific immune effector cells and the generation of immune memory populations. Because of its inflammatory/immune character, PDT is exceptionally responsive to adjuvant treatments with various types of immunotherapy. Combining PDT with immuneactivators, such as cytokines or other specific or non-specific immune agents, rendered marked improvements in tumor cures with various cancer models. Another clinically attractive strategy is adoptive immunotherapy, and the prospects of its use in conjunction with PDT are outlined.

An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

PubMed Central

Clevers, H; Lonberg, N; Dunlap, S; Lacy, E; Terhorst, C

1989-01-01

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive. Images PMID:2583122

STUDIES ON NON-HEMOLYTIC STREPTOCOCCI ISOLATED FROM THE RESPIRATORY TRACT OF MAN

PubMed Central

Horsfall, Frank L.

1951-01-01

The type specific immunological properties of certain non-hemolytic streptococci, including Str. salivarius type I and type II, present in the respiratory tract of human beings appear to be dependent upon the presence of capsular polysaccharides. The levans formed from sucrose by Str. salivarius (encapsulated S cells or non-encapsulated R variants), or by cell-free enzymes derived from these microorganisms, are indistinguishable immunologically and show no evidence of type specificity. Such levans appear to be immunologically distinct from and unrelated to the capsular polysaccharides of the microorganisms which produce them. PMID:14824398

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

Reconstruction of genome-scale human metabolic models using omics data.

PubMed

Ryu, Jae Yong; Kim, Hyun Uk; Lee, Sang Yup

2015-08-01

The impact of genome-scale human metabolic models on human systems biology and medical sciences is becoming greater, thanks to increasing volumes of model building platforms and publicly available omics data. The genome-scale human metabolic models started with Recon 1 in 2007, and have since been used to describe metabolic phenotypes of healthy and diseased human tissues and cells, and to predict therapeutic targets. Here we review recent trends in genome-scale human metabolic modeling, including various generic and tissue/cell type-specific human metabolic models developed to date, and methods, databases and platforms used to construct them. For generic human metabolic models, we pay attention to Recon 2 and HMR 2.0 with emphasis on data sources used to construct them. Draft and high-quality tissue/cell type-specific human metabolic models have been generated using these generic human metabolic models. Integration of tissue/cell type-specific omics data with the generic human metabolic models is the key step, and we discuss omics data and their integration methods to achieve this task. The initial version of the tissue/cell type-specific human metabolic models can further be computationally refined through gap filling, reaction directionality assignment and the subcellular localization of metabolic reactions. We review relevant tools for this model refinement procedure as well. Finally, we suggest the direction of further studies on reconstructing an improved human metabolic model.

Nephron segment-specific gene expression using AAV vectors.

PubMed

Asico, Laureano D; Cuevas, Santiago; Ma, Xiaobo; Jose, Pedro A; Armando, Ines; Konkalmatt, Prasad R

2018-02-26

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na + /glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Selective targeting of alveolar type II respiratory epithelial cells by anti-surfactant protein-C antibody-conjugated lipoplexes

PubMed Central

Wu, Yun; Ma, Junyu; Woods, Parker S.; Chesarino, Nicholas M.; Liu, Chang; Lee, L. James; Nana-Sinkam, Serge P.; Davis, Ian C.

2015-01-01

Alveolar type II (ATII) respiratory epithelial cells are essential to normal lung function. They may be also central to the pathogenesis of diseases such as acute lung injury, pulmonary fibrosis, and pulmonary adenocarcinoma. Hence, ATII cells are important therapeutic targets. However, effective ATII cell-specific drug delivery in vivo requires carriers of an appropriate size, which can cross the hydrophobic alveolar surfactant film and polar aqueous layer overlying ATII cells, and be taken up without inducing ATII cell dysfunction, pulmonary inflammation, lung damage, or excessive systemic spread and side-effects. We have developed lipoplexes as a versatile nanoparticle carrier system for drug/RNA delivery. To optimize their pulmonary localization and ATII cell specificity, lipoplexes were conjugated to an antibody directed against the ATII cell-specific antigen surfactant protein-C (SP-C) then administered to C57BL/6 mice via the nares. Intranasally-administered, anti-SP-C-conjugated lipoplexes targeted mouse ATII cells with >70% specificity in vivo, were retained within ATII cells for at least 48 hours, and did not accumulate at significant levels in other lung cell types or viscera. 48 hours after treatment with anti-SP-C-conjugated lipoplexes containing the test microRNA miR-486, expression of mature miR-486 was approximately 4-fold higher in ATII cells than whole lung by qRT-PCR, and was undetectable in other viscera. Lipoplexes induced no weight loss, hypoxemia, lung dysfunction, pulmonary edema, or pulmonary inflammation over a 6-day period. These findings indicate that ATII cell-targeted lipoplexes exhibit all the desired characteristics of an effective drug delivery system for treatment of pulmonary diseases that result primarily from ATII cell dysfunction. PMID:25687308

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function*

PubMed Central

Lowery, Jason; Kuczmarski, Edward R.; Herrmann, Harald; Goldman, Robert D.

2015-01-01

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks. PMID:25957409

T Cell Proliferation and Colitis Are Initiated by Defined Intestinal Microbes.

PubMed

Chiaranunt, Pailin; Tometich, Justin T; Ji, Junyi; Hand, Timothy W

2018-07-01

Inflammatory bowel disease has been associated with the dysregulation of T cells specific to Ags derived from the intestinal microbiota. How microbiota-specific T cells are regulated is not completely clear but is believed to be mediated by a combination of IgA, regulatory T cells, and type 3 innate lymphoid cells. To test the role of these regulatory components on microbiota-specific T cells, we bred CBir1 TCR transgenic (CBir1Tg) mice (specific to flagellin from common intestinal bacteria) onto a lymphopenic Rag1 -/- background. Surprisingly, T cells from CBir1Tg mice bred onto a Rag1 -/- background could not induce colitis and did not differentiate to become effectors under lymphopenic conditions, despite deficits in immunoregulatory factors, such as IgA, regulatory T cells, and type 3 innate lymphoid cells. In fact, upon transfer of conventional CBir1Tg T cells into lymphopenic mice, the vast majority of proliferating T cells responded to Ags other than CBir1 flagellin, including those found on other bacteria, such as Helicobacter spp. Thus, we discovered a caveat in the CBir1Tg model within our animal facility that illustrates the limitations of using TCR transgenics at mucosal surfaces, where multiple TCR specificities can respond to the plethora of foreign Ags. Our findings also indicate that T cell specificity to the microbiota alone is not sufficient to induce T cell activation and colitis. Instead, other interrelated factors, such as the composition and ecology of the intestinal microbiota and host access to Ag, are paramount in controlling the activation of microbiota-specific T cell clones. Copyright © 2018 by The American Association of Immunologists, Inc.

Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

PubMed

Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

2016-10-01

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

Simultaneous neuron- and astrocyte-specific fluorescent marking

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

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko

2015-03-27

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignmentmore » of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.« less

The neuron identity problem: form meets function.

PubMed

Fishell, Gord; Heintz, Nathaniel

2013-10-30

A complete understanding of nervous system function cannot be achieved without the identification of its component cell types. In this Perspective, we explore a series of related issues surrounding cell identity and how revolutionary methods for labeling and probing specific neuronal types have clarified this question. Specifically, we ask the following questions: what is the purpose of such diversity, how is it generated, how is it maintained, and, ultimately, how can one unambiguously identity one cell type from another? We suggest that each cell type can be defined by a unique and conserved molecular ground state that determines its capabilities. We believe that gaining an understanding of these molecular barcodes will advance our ability to explore brain function, enhance our understanding of the biochemical basis of CNS disorders, and aid in the development of novel therapeutic strategies. Copyright © 2013 Elsevier Inc. All rights reserved.

Innate lymphoid cells and their stromal microenvironments.

PubMed

Kellermayer, Zoltán; Vojkovics, Dóra; Balogh, Péter

2017-09-01

In addition to the interaction between antigen presenting cells, T and B lymphocytes, recent studies have revealed important roles for a diverse set of auxiliary cells that profoundly influence the induction and regulation of immune responses against pathogens. Of these the stromal cells composed of various non-hematopoietic constituents are crucial for the creation and maintenance of specialized semi-static three-dimensional lymphoid tissue microenvironment, whereas the more recently described innate lymphoid cells are generated by the diversification of committed lymphoid precursor cells independently from clonally rearranged antigen receptor genes. Recent findings have revealed important contributions by innate lymphoid cells in inflammation and protection against pathogens in a tissue-specific manner. Importantly, lymphoid stromal cells also influence the onset of immune responses in tissue-specific fashion, raising the possibility of tissue-specific stromal - innate lymphoid cell collaboration. In this review we summarize the main features and interactions between these two cells types, with particular emphasis on ILC type 3 cells and their microenvironmental partners. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Split T Cell Tolerance against a Self/Tumor Antigen: Spontaneous CD4+ but Not CD8+ T Cell Responses against p53 in Cancer Patients and Healthy Donors

PubMed Central

Tsuji, Takemasa; Matsuzaki, Junko; Ritter, Erika; Miliotto, Anthony; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J.; Gnjatic, Sacha

2011-01-01

Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4+ and CD8+ T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8+ T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4+ T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4+ T cells but not CD8+ T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4+ T cells in healthy donors originated from a CD45RA− antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4+ T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events. PMID:21858191

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

PubMed

Knittel, T; Aurisch, S; Neubauer, K; Eichhorst, S; Ramadori, G

1996-08-01

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

Chick cerebellar Purkinje cells express omega-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels.

PubMed

Angulo, M C; Parra, P; Dieudonné, S

1998-03-01

Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds and later in mammals. A specific calcium channel, the P-type channel, has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. P-type channels play an essential role in synaptic transmission of mammalian cerebellum. It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. We found that omega-conotoxin GVIA (omega-CgTx GVIA), a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

Blocking Virus Replication during Acute Murine Cytomegalovirus Infection Paradoxically Prolongs Antigen Presentation and Increases the CD8+ T Cell Response by Preventing Type I IFN-Dependent Depletion of Dendritic Cells.

PubMed

Loo, Christopher P; Snyder, Christopher M; Hill, Ann B

2017-01-01

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8 + T cell response, which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication, we found that increased virus replication drove increased effector CD8 + T cell differentiation, as expected. Paradoxically, however, increased virus replication dramatically decreased the size of the CD8 + T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs, but they did not inhibit the response to "inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8 + T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells. Copyright © 2016 by The American Association of Immunologists, Inc.

The role of myostatin and activin receptor IIB in the regulation of unloading-induced myofiber type-specific skeletal muscle atrophy.

PubMed

Babcock, Lyle W; Knoblauch, Mark; Clarke, Mark S F

2015-09-15

Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB. Wistar rats underwent HLS for 10 days, after which the tibialis anterior was harvested for frozen cross sectioning. Simultaneous multichannel immunofluorescent staining combined with differential interference contrast imaging was employed to analyze myofiber type-specific expression of myostatin and actRIIB and myofiber type cross-sectional area (CSA) across fiber types, myonuclei, and satellite cells. Hindlimb suspension (HLS) induced significant myofiber type-specific atrophy in myosin heavy chain (MHC) IIx (P < 0.05) and MHC IIb myofibers (P < 0.05). Myostatin staining associated with myonuclei was less in HLS rats compared with controls, while satellite cell staining for myostatin remained unchanged. In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared with ambulatory control rats (P < 0.01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS. Our data suggest that differential expression of actRIIB may be responsible for myostatin-induced myofiber type-selective atrophy observed during chronic unloading. Copyright © 2015 the American Physiological Society.

Purification and immunochemical characterization of type e polysaccharide antigen of Streptococcus mutans.

PubMed

Hamada, S; Slade, H D

1976-07-01

The type-specific antigen of Streptococcus mutans strain MT703, serotype e, has been chromatographically purified and characterized. Two chromatographic fractions were obtained from saline extracts which reacted with both anti-MT703 whole-cell serum and Lancefield group E serum. The major fraction (eI) was identified as a polysaccharide composed of 37% glucose, 56% rhamnose, 5% protein, and 0.3% phosphorus, whereas the minor fraction (eII) contained 66% protein in addition to 10% glucose and 17% rhamnose. The immunological specificity of these antigens was found to be the same by immunodiffusion in agar gel. Another fraction with a negative charge (eIII) reacted with polyglycerophosphate antisera from Streptococcus mutans and Streptococcus pyogenes. For comparison, the MT703 antigen in a hot trichloroacetic acid extract (eA) and the group E antigen from a saline extract of cells of strain K129 (EI) were similarly purified by anionic ion-exchange chromatography. Although the ratio of glucose and rhamnose in eA was 1:0.9 and in eI and eII approximately 1:1.5, reactions of identity were obtained in gel diffusion against specific anti-e serum. This difference in ratio is probably a result of the extraction procedures. Both the type e and group E antisera were reactive with both eI and EI antigens. The adsorption of group E antiserum with MT703 cells removed all E antibody, whereas type e-specific antibody remained after adsorption with K129 cells. These results suggest that eI antigen possesses both e and E specificities, whereas EI possesses E only. These findings were supported by the quantitative precipitin test and immunodiffusion and/or immunoelectrophoretic patterns in agar gel. Methyl-beta-D-glucopyranoside markedly inhibited the precipitin reaction in both type e and group E sera. However, a significantly stronger inhibition by cellobiose of type e serum than of group E serum indicates that a beta-linked glucose-glucose dimer is the predominant antigenic determinant of the e specificity. The presence of both e and E specificities on a single polysaccharide molecule was demonstrated by the use of purified e antigen released from a specific e-anti-e complex. This antigen reacted with a group E-specific serum as well as a type e-specific serum. An examination of five S. mutans type e strains showed the presence of group E specificity also, whereas the I, II, and IV serotypes of group E streptococci only possessed the group E specificity.

Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

PubMed Central

Ding, Siyuan; Khoury-Hanold, William; Iwasaki, Akiko; Robek, Michael D.

2014-01-01

Type III interferon (IFN-λ) exhibits potent antiviral activity similar to IFN-α/β, but in contrast to the ubiquitous expression of the IFN-α/β receptor, the IFN-λ receptor is restricted to cells of epithelial origin. Despite the importance of IFN-λ in tissue-specific antiviral immunity, the molecular mechanisms responsible for this confined receptor expression remain elusive. Here, we demonstrate that the histone deacetylase (HDAC) repression machinery mediates transcriptional silencing of the unique IFN-λ receptor subunit (IFNLR1) in a cell-type-specific manner. Importantly, HDAC inhibitors elevate receptor expression and restore sensitivity to IFN-λ in previously nonresponsive cells, thereby enhancing protection against viral pathogens. In addition, blocking HDAC activity renders nonresponsive cell types susceptible to the pro-apoptotic activity of IFN-λ, revealing the combination of HDAC inhibitors and IFN-λ to be a potential antitumor strategy. These results demonstrate that the type III IFN response may be therapeutically harnessed by epigenetic rewiring of the IFN-λ receptor expression program. PMID:24409098

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

PubMed

Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

2011-06-06

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection

PubMed Central

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

Background 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. Methodology/Principal Findings 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. Conclusions and Significance 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. PMID:22962591

Regulation of cytokine polarization and T cell recruitment to inflamed paws in mouse collagen-induced arthritis by the chemokine receptor CXCR6.

PubMed

Slauenwhite, Drew; Gebremeskel, Simon; Doucette, Carolyn D; Hoskin, David W; Johnston, Brent

2014-11-01

The chemokine receptor CXCR6 is highly expressed on lymphocytes isolated from the synovium of patients with rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis, suggesting that CXCR6 regulates immune cell activation or infiltration into arthritic joints. This study was undertaken to examine the role of CXCR6 in T cell activation and arthritis development. A collagen-induced arthritis model was used to examine arthritis development in wild-type and CXCR6(-/-) mice. CXCR6 expression, lymphocyte accumulation, and intracellular cytokine production were examined by flow cytometry. Collagen-specific antibodies were measured in the serum. Collagen-specific recall responses were examined in vitro via proliferation and cytokine release assays. T cell homing to inflamed joints was examined using competitive adoptive transfer of dye-labeled lymphocytes from wild-type and CXCR6(-/-) mice. The numbers of CXCR6+ T cells were increased in the paws and draining lymph nodes of arthritic mice. The incidence of arthritis, disease severity, extent of T cell accumulation, and levels of collagen-specific IgG2a antibodies were significantly reduced in CXCR6(-/-) mice compared to wild-type mice. T cells from wild-type mice exhibited Th1 (interferon-γ [IFNγ]) polarization in the inguinal lymph nodes following immunization. At disease peak, this shifted to a Th17 (interleukin-17A [IL-17A]) response in the popliteal lymph nodes. T cells in CXCR6(-/-) mice exhibited impaired cytokine polarization, resulting in a decreased frequency and number of IL-17A- and IFNγ-producing cells. Recruitment of activated CXCR6(-/-) mouse T cells to the inflamed paws was impaired compared to recruitment of wild-type mouse T cells. These experiments demonstrate that CXCR6 plays important roles in the pathogenesis of arthritis through its effects on both T cell cytokine polarization and homing of T cells to inflamed joints. Copyright © 2014 by the American College of Rheumatology.

Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression

PubMed Central

Libbrecht, Maxwell W.; Ay, Ferhat; Hoffman, Michael M.; Gilbert, David M.; Bilmes, Jeffrey A.; Noble, William Stafford

2015-01-01

The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term “specific expression domains.” We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. PMID:25677182

Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression.

PubMed

Libbrecht, Maxwell W; Ay, Ferhat; Hoffman, Michael M; Gilbert, David M; Bilmes, Jeffrey A; Noble, William Stafford

2015-04-01

The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term "specific expression domains." We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. © 2015 Libbrecht et al.; Published by Cold Spring Harbor Laboratory Press.

Intermolecular Interactions of Homologs of Germ Plasm Components in Mammalian Germ Cells

PubMed Central

Fox, Mark S.; Clark, Amander T.; El Majdoubi, Mohammed; Vigne, Jean-Louis; Urano, Jun; Hostetler, Chris E.; Griswold, Michael D.; Weiner, Richard I.; Pera, Renee A. Reijo

2007-01-01

In some species such as flies, worms, frogs, and fish the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically-distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration, that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells. PMID:16996493

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

PubMed

Rivière, Guillaume; Lienhard, Daniel; Andrieu, Thomas; Vieau, Didier; Frey, Brigitte M; Frey, Felix J

2011-04-01

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Ligand-independent TLR signals generated by ectopic overexpression of MyD88 generate local and systemic anti-tumor immunity

PubMed Central

Hartman, Zachary C.; Osada, Takuya; Glass, Oliver; Yang, Xiao Y.; Lei, Gang-jun; Lyerly, H. Kim; Clay, Timothy M.

2010-01-01

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anti-cancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll Like-Receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and demonstrate that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T-cells. Thus, Ad-MyD88 initiated robust anti-tumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy. PMID:20823152

Klebsiella pneumoniae type 3 fimbriae agglutinate yeast in a mannose-resistant manner.

PubMed

Stahlhut, Steen G; Struve, Carsten; Krogfelt, Karen A

2012-03-01

The ability of bacterial pathogens to express different fimbrial adhesins plays a significant role in virulence. Thus, specific detection of fimbrial expression is an important task in virulence characterization and epidemiological studies. Most clinical Klebsiella pneumoniae isolates express type 1 and type 3 fimbriae, which are characterized by mediation of mannose-sensitive agglutination of yeast cells and agglutination of tannic acid-treated ox red blood cells (RBCs), respectively. It has been observed that K. pneumoniae isolates agglutinate yeast cells and commercially available sheep RBCs in a mannose-resistant manner. Thus, this study was initiated to identify the adhesin involved. Screening of a mutant library surprisingly revealed that the mannose-resistant agglutination of yeast and sheep RBCs was mediated by type 3 fimbriae. Specific detection of type 1 fimbriae expression in K. pneumoniae was feasible only by the use of guinea pig RBCs. This was further verified by the use of isogenic fimbriae mutants and by cloning and expressing K. pneumoniae fimbrial gene clusters in Escherichia coli. Yeast agglutination assays are commonly used to detect type 1 fimbriae expression but should not be used for bacterial species able to express type 3 fimbriae. For these species, the use of guinea pig blood for specific type 1 fimbriae detection is essential. The use of commercially available sheep RBCs or yeast is an easy alternative to traditional methods to detect type 3 fimbriae expression. Easy and specific detection of expression of type 1 and type 3 fimbriae is essential in the continuous characterization of these important adhesive virulence factors present in members of the Enterobacteriaceae.

Competent for commitment: you've got to have heart!

PubMed

Jain, Rajan; Epstein, Jonathan A

2018-01-01

The mature heart is composed primarily of four different cell types: cardiac myocytes, endothelium, smooth muscle, and fibroblasts. These cell types derive from pluripotent progenitors that become progressively restricted with regard to lineage potential, giving rise to multipotent cardiac progenitor cells and, ultimately, the differentiated cell types of the heart. Recent studies have begun to shed light on the defining characteristics of the intermediary cell types that exist transiently during this developmental process and the extrinsic and cell-autonomous factors that influence cardiac lineage decisions and cellular competence. This information will shape our understanding of congenital and adult cardiac disease and guide regenerative therapeutic approaches. In addition, cardiac progenitor specification can serve as a model for understanding basic mechanisms regulating the acquisition of cellular identity. In this review, we present the concept of "chromatin competence" that describes the potential for three-dimensional chromatin organization to function as the molecular underpinning of the ability of a progenitor cell to respond to inductive lineage cues and summarize recent studies advancing our understanding of cardiac cell specification, gene regulation, and chromatin organization and how they impact cardiac development. © 2018 Jain and Epstein; Published by Cold Spring Harbor Laboratory Press.

Characteristics of allelic gene expression in human brain cells from single-cell RNA-seq data analysis.

PubMed

Zhao, Dejian; Lin, Mingyan; Pedrosa, Erika; Lachman, Herbert M; Zheng, Deyou

2017-11-10

Monoallelic expression of autosomal genes has been implicated in human psychiatric disorders. However, there is a paucity of allelic expression studies in human brain cells at the single cell and genome wide levels. In this report, we reanalyzed a previously published single-cell RNA-seq dataset from several postmortem human brains and observed pervasive monoallelic expression in individual cells, largely in a random manner. Examining single nucleotide variants with a predicted functional disruption, we found that the "damaged" alleles were overall expressed in fewer brain cells than their counterparts, and at a lower level in cells where their expression was detected. We also identified many brain cell type-specific monoallelically expressed genes. Interestingly, many of these cell type-specific monoallelically expressed genes were enriched for functions important for those brain cell types. In addition, function analysis showed that genes displaying monoallelic expression and correlated expression across neuronal cells from different individual brains were implicated in the regulation of synaptic function. Our findings suggest that monoallelic gene expression is prevalent in human brain cells, which may play a role in generating cellular identity and neuronal diversity and thus increasing the complexity and diversity of brain cell functions.

Human induced pluripotent stem cells and their use in drug discovery for toxicity testing.

PubMed

Scott, Clay W; Peters, Matthew F; Dragan, Yvonne P

2013-05-10

Predicting human safety risks of novel xenobiotics remains a major challenge, partly due to the limited availability of human cells to evaluate tissue-specific toxicity. Recent progress in the production of human induced pluripotent stem cells (hiPSCs) may fill this gap. hiPSCs can be continuously expanded in culture in an undifferentiated state and then differentiated to form most cell types. Thus, it is becoming technically feasible to generate large quantities of human cell types and, in combination with relatively new detection methods, to develop higher-throughput in vitro assays that quantify tissue-specific biological properties. Indeed, the first wave of large scale hiSC-differentiated cell types including patient-derived hiPSCS are now commercially available. However, significant improvements in hiPSC production and differentiation processes are required before cell-based toxicity assays that accurately reflect mature tissue phenotypes can be delivered and implemented in a cost-effective manner. In this review, we discuss the promising alignment of hiPSCs and recently emerging technologies to quantify tissue-specific functions. We emphasize liver, cardiovascular, and CNS safety risks and highlight limitations that must be overcome before routine screening for toxicity pathways in hiSC-derived cells can be established. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Ethical challenges for using human cells in clinical cell therapy.

PubMed

Hermerén, Göran

2012-01-01

In this chapter, different challenges for using human cells in clinical cell therapy are identified and discussed. Several types of challenges are defined and described, with particular attention to the relation between ethical and scientific challenges. Some challenges are cell and disease specific: they are raised by research on special types of cells with certain methods in order to pave the way for cell therapies of particular diseases. But since scientific work is carried out in a societal and value-loaded context, the relations between scientific, ethical, and regulatory challenges are complex. Three theses are first discussed: there is not one fixed list of ethical challenges, some challenges are disease and cell type specific; there are challenges at all stages of the translation from bench to bedside, and the challenges are related to the various stages of translation. Moreover, experimental and ethical research needs to be integrated. Finally, a fourth thesis is suggested: if a constructive and well-argued position is desired, it is necessary to be specific not only about the scientific details but also about the value premises. Everybody is for justice, integrity, and respect for persons. But what precisely does this mean when it is applied to the choices scientists and regulators have to face in their daily work? Copyright © 2012 Elsevier B.V. All rights reserved.

Cell-type-specific role for nucleus accumbens neuroligin-2 in depression and stress susceptibility.

PubMed

Heshmati, Mitra; Aleyasin, Hossein; Menard, Caroline; Christoffel, Daniel J; Flanigan, Meghan E; Pfau, Madeline L; Hodes, Georgia E; Lepack, Ashley E; Bicks, Lucy K; Takahashi, Aki; Chandra, Ramesh; Turecki, Gustavo; Lobo, Mary Kay; Maze, Ian; Golden, Sam A; Russo, Scott J

2018-01-30

Behavioral coping strategies are critical for active resilience to stress and depression; here we describe a role for neuroligin-2 (NLGN-2) in the nucleus accumbens (NAc). Neuroligins (NLGN) are a family of neuronal postsynaptic cell adhesion proteins that are constituents of the excitatory and inhibitory synapse. Importantly, NLGN-3 and NLGN-4 mutations are strongly implicated as candidates underlying the development of neuropsychiatric disorders with social disturbances such as autism, but the role of NLGN-2 in neuropsychiatric disease states is unclear. Here we show a reduction in NLGN-2 gene expression in the NAc of patients with major depressive disorder. Chronic social defeat stress in mice also decreases NLGN-2 selectively in dopamine D1-positive cells, but not dopamine D2-positive cells, within the NAc of stress-susceptible mice. Functional NLGN-2 knockdown produces bidirectional, cell-type-specific effects: knockdown in dopamine D1-positive cells promotes subordination and stress susceptibility, whereas knockdown in dopamine D2-positive cells mediates active defensive behavior. These findings establish a behavioral role for NAc NLGN-2 in stress and depression; provide a basis for targeted, cell-type specific therapy; and highlight the role of active behavioral coping mechanisms in stress susceptibility.

FlpStop, a tool for conditional gene control in Drosophila

PubMed Central

Fisher, Yvette E; Yang, Helen H; Isaacman-Beck, Jesse; Xie, Marjorie; Gohl, Daryl M; Clandinin, Thomas R

2017-01-01

Manipulating gene function cell type-specifically is a common experimental goal in Drosophila research and has been central to studies of neural development, circuit computation, and behavior. However, current cell type-specific gene disruption techniques in flies often reduce gene activity incompletely or rely on cell division. Here we describe FlpStop, a generalizable tool for conditional gene disruption and rescue in post-mitotic cells. In proof-of-principle experiments, we manipulated apterous, a regulator of wing development. Next, we produced conditional null alleles of Glutamic acid decarboxylase 1 (Gad1) and Resistant to dieldrin (Rdl), genes vital for GABAergic neurotransmission, as well as cacophony (cac) and paralytic (para), voltage-gated ion channels central to neuronal excitability. To demonstrate the utility of this approach, we manipulated cac in a specific visual interneuron type and discovered differential regulation of calcium signals across subcellular compartments. Thus, FlpStop will facilitate investigations into the interactions between genes, circuits, and computation. DOI: http://dx.doi.org/10.7554/eLife.22279.001 PMID:28211790

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

A Self-Directed Method for Cell-Type Identification and Separation of Gene Expression Microarrays

PubMed Central

Zuckerman, Neta S.; Noam, Yair; Goldsmith, Andrea J.; Lee, Peter P.

2013-01-01

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures - these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets. PMID:23990767

Selective Vulnerability of Specific Retinal Ganglion Cell Types and Synapses after Transient Ocular Hypertension.

PubMed

Ou, Yvonne; Jo, Rebecca E; Ullian, Erik M; Wong, Rachel O L; Della Santina, Luca

2016-08-31

Key issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs. Among the α-like RGCs studied, αOFF-transient RGCs exhibited higher rates of cell death, with corresponding reductions in dendritic area, dendritic complexity, and synapse density. Functionally, OFF-transient RGCs displayed decreases in spontaneous activity and receptive field size. In contrast, neither αOFF-sustained nor αON-sustained RGCs displayed decreases in light responses, although they did exhibit a decrease in excitatory postsynaptic sites, suggesting that synapse loss may be one of the earliest signs of degeneration. Interestingly, presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer, corroborating the hypothesis that RGCs with dendrites stratifying in the OFF sublamina may be damaged early. Indeed, OFF arbors of ON-OFF RGCs lose complexity more rapidly than ON arbors. Our results reveal type-specific differences in RGC responses to injury with a selective vulnerability of αOFF-transient RGCs, and furthermore, an increased susceptibility of synapses in the OFF sublamina. The selective vulnerability of specific RGC types offers new avenues for the design of more sensitive functional tests and targeted neuroprotection. Conflicting reports regarding the selective vulnerability of specific retinal ganglion cell (RGC) types in glaucoma exist. We examine, for the first time, the effects of transient intraocular pressure elevation on the structure and function of various RGC types. Among the α-like RGCs studied, αOFF-transient RGCs are the most vulnerable to transient transient intraocular pressure elevation as measured by rates of cell death, morphologic alterations in dendrites and synapses, and physiological dysfunction. Specifically, we found that presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer. Our results suggest selective vulnerability both of specific types of RGCs and of specific inner plexiform layer sublaminae, opening new avenues for identifying novel diagnostic and treatment targets in glaucoma. Copyright © 2016 the authors 0270-6474/16/369240-13$15.00/0.

Attentional modulation of cell-class specific gamma-band synchronization in awake monkey area V4

PubMed Central

Vinck, Martin; Womelsdorf, Thilo; Buffalo, Elizabeth A.; Desimone, Robert; Fries, Pascal

2013-01-01

Summary Selective visual attention is subserved by selective neuronal synchronization, entailing precise orchestration among excitatory and inhibitory cells. We tentatively identified these as broad (BS) and narrow spiking (NS) cells and analyzed their synchronization to the local field potential in two macaque monkeys performing a selective visual attention task. Across cells, gamma phases scattered widely but were unaffected by stimulation or attention. During stimulation, NS cells lagged BS cells on average by ~60° and gamma synchronized twice as strongly. Attention enhanced and reduced the gamma locking of strongly and weakly activated cells, respectively. During a pre-stimulus attentional cue period, BS cells showed weak gamma synchronization, while NS cells gamma synchronized as strongly as with visual stimulation. These analyses reveal the cell-type specific dynamics of the gamma cycle in macaque visual cortex and suggest that attention affects neurons differentially depending on cell type and activation level. PMID:24267656

Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

DTIC Science & Technology

2017-09-01

1) define functional roles for individual genes and cell types in development of obesity and insulin resistance and 2) examine novel targets against...which we can design therapies to target specific pathogenic or or health-promoting cell types. 15. SUBJECT TERMS Obesity , Type 2 Diabetes Mellitus...compromised with chronic overnutrition ( obesity ). 4 KEYWORDS: Obesity , Diabetes, Insulin Resistance, Adipose, Adipocytes, Stromal Vascular Fraction, Single

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis.

PubMed

Wang, Y T; Liu, W; Seah, J N; Lam, C S; Xiang, J H; Korzh, V; Kwang, J

2002-12-10

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Dectin-1 diversifies Aspergillus fumigatus–specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

PubMed Central

Hohl, Tobias M.; Collins, Nichole; Leiner, Ingrid; Gallegos, Alena; Saijo, Shinobu; Coward, Jesse W.; Iwakura, Yoichiro

2011-01-01

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus–infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus–infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus–specific CD4 T cells. Our findings indicate that Dectin-1–mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus–specific CD4 T cells and enabling Th17 differentiation. PMID:21242294

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

PubMed

Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera for studies of chromosome structure and function, gene mapping, embryogenesis, characterization and biosynthesis of developmental and differentiation antigens. These antigens are those that are specific for various cell types and tissues, species specific antigen, antigens involved in chemotaxis, immunogenetics and clinical genetics including genetically inherited disorders, chromosome aberrations and transplantation antigens. Besides these monoclonal antibodies, their complexes have recently been investigated as exquisitely sensitive probes to be guided to target cells or organs. They have been used to deliver cytotoxic drugs to malignant cells or enzymes to specific cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

A Cre Mouse Line for Probing Irradiance- and Direction-Encoding Retinal Networks

PubMed Central

Sabbah, Shai

2017-01-01

Abstract Cell type-specific Cre driver lines have revolutionized the analysis of retinal cell types and circuits. We show that the transgenic mouse Rbp4-Cre selectively labels several retinal neuronal types relevant to the encoding of absolute light intensity (irradiance) and visual motion. In the ganglion cell layer (GCL), most marked cells are wide-field spiking polyaxonal amacrine cells (ACs) with sustained irradiance-encoding ON responses that persist during chemical synaptic blockade. Their arbors spread about 1 mm across the retina and are restricted to the inner half of the ON sublamina of the inner plexiform layer (IPL). There, they costratify with dendrites of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs), to which they are tracer coupled. We propose that synaptically driven and intrinsic photocurrents of M2 cells pass through gap junctions to drive AC light responses. Also marked in this mouse are two types of RGCs. R-cells have a bistratified dendritic arbor, weak directional tuning, and irradiance-encoding ON responses. However, they also receive excitatory OFF input, revealed during ON-channel blockade. Serial blockface electron microscopic (SBEM) reconstruction confirms OFF bipolar input, and reveals that some OFF input derives from a novel type of OFF bipolar cell (BC). R-cells innervate specific layers of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC). The other marked RGC type (RDS) is bistratified, transient, and ON-OFF direction selective (DS). It apparently innervates the nucleus of the optic tract (NOT). The Rbp4-Cre mouse will be valuable for targeting these cell types for further study and for selectively manipulating them for circuit analysis. PMID:28466070

Layer-specific input to distinct cell types in layer 6 of monkey primary visual cortex.

PubMed

Briggs, F; Callaway, E M

2001-05-15

Layer 6 of monkey V1 contains a physiologically and anatomically diverse population of excitatory pyramidal neurons. Distinctive arborization patterns of axons and dendrites within the functionally specialized cortical layers define eight types of layer 6 pyramidal neurons and suggest unique information processing roles for each cell type. To address how input sources contribute to cellular function, we examined the laminar sources of functional excitatory input onto individual layer 6 pyramidal neurons using scanning laser photostimulation. We find that excitatory input sources correlate with cell type. Class I neurons with axonal arbors selectively targeting magnocellular (M) recipient layer 4Calpha receive input from M-dominated layer 4B, whereas class I neurons whose axonal arbors target parvocellular (P) recipient layer 4Cbeta receive input from P-dominated layer 2/3. Surprisingly, these neuronal types do not differ significantly in the inputs they receive directly from layers 4Calpha or 4Cbeta. Class II cells, which lack dense axonal arbors within layer 4C, receive excitatory input from layers targeted by their local axons. Specifically, type IIA cells project axons to and receive input from the deep but not superficial layers. Type IIB neurons project to and receive input from the deepest and most superficial, but not middle layers. Type IIC neurons arborize throughout the cortical layers and tend to receive inputs from all cortical layers. These observations have implications for the functional roles of different layer 6 cell types in visual information processing.

Paper diagnostic for instantaneous blood typing.

PubMed

Khan, Mohidus Samad; Thouas, George; Shen, Wei; Whyte, Gordon; Garnier, Gil

2010-05-15

Agglutinated blood transports differently onto paper than stable blood with well dispersed red cells. This difference was investigated to develop instantaneous blood typing tests using specific antibody-antigen interactions to trigger blood agglutination. Two series of experiments were performed. The first related the level of agglutination and the fluidic properties of blood on its transport in paper. Blood samples were mixed at different ratios with specific and nonspecific antibodies; a droplet of each mixture was deposited onto a filter paper strip, and the kinetics of wicking and red cell separation were measured. Agglutinated blood phase separated, with the red blood cells (RBC) forming a distinct spot upon contact with paper while the plasma wicked; in contrast, stable blood suspensions wicked uniformly. The second study analyzed the wicking and the chromatographic separation of droplets of blood deposited onto paper strips pretreated with specific and nonspecific antibodies. Drastic differences in transport occurred. Blood agglutinated by interaction with one of its specific antibodies phase separated, causing a chromatographic separation. The red cells wicked very little while the plasma wicked at a faster rate than the original blood sample. Blood agglutination and wicking in paper followed the concepts of colloids chemistry. The immunoglobin M antibodies agglutinated the red blood cells by polymer bridging, upon selective adsorption on the specific antigen at their surface. The transport kinetics was viscosity controlled, with the viscosity of red cells drastically increasing upon blood agglutination. Three arm prototypes were investigated for single-step blood typing.

Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data.

PubMed

Racle, Julien; de Jonge, Kaat; Baumgaertner, Petra; Speiser, Daniel E; Gfeller, David

2017-11-13

Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org).

Epigenome overlap measure (EPOM) for comparing tissue/cell types based on chromatin states.

PubMed

Li, Wei Vivian; Razaee, Zahra S; Li, Jingyi Jessica

2016-01-11

The dynamics of epigenomic marks in their relevant chromatin states regulate distinct gene expression patterns, biological functions and phenotypic variations in biological processes. The availability of high-throughput epigenomic data generated by next-generation sequencing technologies allows a data-driven approach to evaluate the similarities and differences of diverse tissue and cell types in terms of epigenomic features. While ChromImpute has allowed for the imputation of large-scale epigenomic information to yield more robust data to capture meaningful relationships between biological samples, widely used methods such as hierarchical clustering and correlation analysis cannot adequately utilize epigenomic data to accurately reveal the distinction and grouping of different tissue and cell types. We utilize a three-step testing procedure-ANOVA, t test and overlap test to identify tissue/cell-type- associated enhancers and promoters and to calculate a newly defined Epigenomic Overlap Measure (EPOM). EPOM results in a clear correspondence map of biological samples from different tissue and cell types through comparison of epigenomic marks evaluated in their relevant chromatin states. Correspondence maps by EPOM show strong capability in distinguishing and grouping different tissue and cell types and reveal biologically meaningful similarities between Heart and Muscle, Blood & T-cell and HSC & B-cell, Brain and Neurosphere, etc. The gene ontology enrichment analysis both supports and explains the discoveries made by EPOM and suggests that the associated enhancers and promoters demonstrate distinguishable functions across tissue and cell types. Moreover, the tissue/cell-type-associated enhancers and promoters show enrichment in the disease-related SNPs that are also associated with the corresponding tissue or cell types. This agreement suggests the potential of identifying causal genetic variants relevant to cell-type-specific diseases from our identified associated enhancers and promoters. The proposed EPOM measure demonstrates superior capability in grouping and finding a clear correspondence map of biological samples from different tissue and cell types. The identified associated enhancers and promoters provide a comprehensive catalog to study distinct biological processes and disease variants in different tissue and cell types. Our results also find that the associated promoters exhibit more cell-type-specific functions than the associated enhancers do, suggesting that the non-associated promoters have more housekeeping functions than the non-associated enhancers.

Differential Activation of CD8+ Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

PubMed Central

Pauels, Hans-Gerd; Becker, Christian; Kölsch, Eckehart

1998-01-01

The involvement of counteractive CD8+ T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model. CD8+ Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cells in vivo and in vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinduced CD8+ T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γ as a suppressive factor. Whereas most longterm cultivated CD8+ ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10. CD8+ Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γ or IL-12. In contrast, ADJ-PC- 5-specific CD8+ Tc cells from immunized mice are IFN-γ producing Tc1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γ these Tc1 cells behave similar to the suppressive CD8+ T cells that are induced during early stages of ADJ-PC-5 tumorigenesis. PMID:9814607

[Molecular biology of renal cancer: bases for genetic directed therapy in advanced disease].

PubMed

Maroto Rey, José Pablo; Cillán Narvaez, Elena

2013-06-01

There has been expansion of therapeutic options in the management of metastatic renal cell carcinoma due to a better knowledge of the molecular biology of kidney cancers. There are different tumors grouped under the term renal cell carcinoma, being clear cell cancer the most frequent and accounting for 80% of kidney tumors. Mutations in the Von Hippel-Lindau gene can be identified in up to 80% of sporadic clear cell cancer, linking a genetically inheritable disease where vascular tumors are frequent, with renal cell cancer. Other histologic types present specific alterations in molecular pathways, like c-MET in papillary type I tumors, and Fumarase Hydratase in papillary type II tumors. Identification of the molecular alteration for a specific tumor may offer an opportunity for treatment selection based on biomarkers, and, in the future, for developing an engineering designed genetic treatment.

Illegitimate transcription: transcription of any gene in any cell type.

PubMed Central

Chelly, J; Concordet, J P; Kaplan, J C; Kahn, A

1989-01-01

Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Müllerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell. Images PMID:2495532

Genome-Wide Cell Type-Specific Mapping of In Vivo Chromatin Protein Binding Using an FLP-Inducible DamID System in Drosophila.

PubMed

Pindyurin, Alexey V

2017-01-01

A thorough study of the genome-wide binding patterns of chromatin proteins is essential for understanding the regulatory mechanisms of genomic processes in eukaryotic nuclei, including DNA replication, transcription, and repair. The DNA adenine methyltransferase identification (DamID) method is a powerful tool to identify genomic binding sites of chromatin proteins. This method does not require fixation of cells and the use of specific antibodies, and has been used to generate genome-wide binding maps of more than a hundred different proteins in Drosophila tissue culture cells. Recent versions of inducible DamID allow performing cell type-specific profiling of chromatin proteins even in small samples of Drosophila tissues that contain heterogeneous cell types. Importantly, with these methods sorting of cells of interest or their nuclei is not necessary as genomic DNA isolated from the whole tissue can be used as an input. Here, I describe in detail an FLP-inducible DamID method, namely generation of suitable transgenic flies, activation of the Dam transgenes by the FLP recombinase, isolation of DNA from small amounts of dissected tissues, and subsequent identification of the DNA binding sites of the chromatin proteins.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

PubMed Central

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-01

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening. PMID:26237021

Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.

PubMed

Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong

2015-06-09

Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.

Acquired Downregulation of Donor-Specific Antibody Production After ABO-Incompatible Kidney Transplantation.

PubMed

Tasaki, M; Saito, K; Nakagawa, Y; Imai, N; Ito, Y; Aoki, T; Kamimura, M; Narita, I; Tomita, Y; Takahashi, K

2017-01-01

The mechanism of long-term B cell immunity against donor blood group antigens in recipients who undergo ABO-incompatible (ABOi) living-donor kidney transplantation (LKTx) is unknown. To address this question, we evaluated serial anti-A and anti-B antibody titers in 50 adult recipients. Donor-specific antibody titers remained low (≤1:4) in 42 recipients (84%). However, antibodies against nondonor blood group antigens were continuously produced in recipients with blood type O. We stimulated recipients' peripheral blood mononuclear cells in vitro to investigate whether B cells produced antibodies against donor blood group antigens in the absence of graft adsorption in vivo. Antibodies in cell culture supernatant were measured using specific enzyme-linked immunosorbent assays (ELISAs). Thirty-five healthy volunteers and 57 recipients who underwent ABO-compatible LKTx served as controls. Antibody production in vitro against donor blood group antigens by cells from ABOi LKTx patients was lower than in the control groups. Immunoglobulin deposits were undetectable in biopsies of grafts of eight recipients with low antibody titers (≤1:4) after ABOi LKTx. One patient with blood type A1 who received a second ABOi LKTx from a type B donor did not produce B-specific antibodies. These findings suggest diminished donor-specific antibody production function in the setting of adult ABOi LKTx. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses.

PubMed

Goodwin, B J; Moore, J O; Weinberg, J B

1984-02-01

Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12-myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5-10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of various types of freshly isolated leukemia cells appear to be due to differences other than initial ligand-receptor binding.

Unique cell-type-specific patterns of DNA methylation in the root meristem.

PubMed

Kawakatsu, Taiji; Stuart, Tim; Valdes, Manuel; Breakfield, Natalie; Schmitz, Robert J; Nery, Joseph R; Urich, Mark A; Han, Xinwei; Lister, Ryan; Benfey, Philip N; Ecker, Joseph R

2016-04-29

DNA methylation is an epigenetic modification that differs between plant organs and tissues, but the extent of variation between cell types is not known. Here, we report single-base-resolution whole-genome DNA methylomes, mRNA transcriptomes and small RNA transcriptomes for six cell populations covering the major cell types of the Arabidopsis root meristem. We identify widespread cell-type-specific patterns of DNA methylation, especially in the CHH sequence context, where H is A, C or T. The genome of the columella root cap is the most highly methylated Arabidopsis cell characterized so far. It is hypermethylated within transposable elements (TEs), accompanied by increased abundance of transcripts encoding RNA-directed DNA methylation (RdDM) pathway components and 24-nt small RNAs (smRNAs). The absence of the nucleosome remodeller DECREASED DNA METHYLATION 1 (DDM1), required for maintenance of DNA methylation, and low abundance of histone transcripts involved in heterochromatin formation suggests that a loss of heterochromatin may occur in the columella, thus allowing access of RdDM factors to the whole genome, and producing an excess of 24-nt smRNAs in this tissue. Together, these maps provide new insights into the epigenomic diversity that exists between distinct plant somatic cell types.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells

NASA Astrophysics Data System (ADS)

Ha, Misook; Hong, Soondo

2016-04-01

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells.

PubMed

Ha, Misook; Hong, Soondo

2016-04-14

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.

PubMed

Lowery, Jason; Kuczmarski, Edward R; Herrmann, Harald; Goldman, Robert D

2015-07-10

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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.

WONOEP appraisal: new genetic approaches to study epilepsy

PubMed Central

Rossignol, Elsa; Kobow, Katja; Simonato, Michele; Loeb, Jeffrey A.; Grisar, Thierry; Gilby, Krista L.; Vinet, Jonathan; Kadam, Shilpa D.; Becker, Albert J.

2014-01-01

Objective New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming and optogenetic manipulations within epileptic networks are progressively unravelling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiological impacts of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. Methods This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy in Quebec, Canada. Results Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and have revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell-types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knockdown approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. Genetically-encoded cell-type labeling is also providing new means to assess the role of the non-neuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and non-coding RNAs involved in modifying gene expression following seizures. In addition, genetically-based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient iPS cells and genetically-modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Significance Genetics has considerably changed the field of epilepsy research and is paving the way for better diagnosis and therapies for patients with epilepsy. PMID:24965021

Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

PubMed Central

Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

2012-01-01

Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and breast cancer. PMID:22272226

Distinct and complementary roles for Aspergillus fumigatus-specific Tr1 and Foxp3+ regulatory T cells in humans and mice

PubMed Central

Bedke, Tanja; Iannitti, Rossana G; De Luca, Antonella; Giovannini, Gloria; Fallarino, Francesca; Berges, Carsten; Latgé, Jean-Paul; Einsele, Hermann; Romani, Luigina; Topp, Max S

2014-01-01

Unlike induced Foxp3+ regulatory T cells (Foxp3+ iTreg) that have been shown to play an essential role in the development of protective immunity to the ubiquitous mold Aspergillus fumigatus, type-(1)-regulatory T cells (Tr1) cells have, thus far, not been implicated in this process. Here, we evaluated the role of Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of A. fumigatus (Crf-1/p41) in antifungal immunity. We identified Crf-1/p41-specific latent-associated peptide+ Tr1 cells in healthy humans and mice after vaccination with Crf-1/p41+zymosan. These cells produced high amounts of interleukin (IL)-10 and suppressed the expansion of antigen-specific T cells in vitro and in vivo. In mice, in vivo differentiation of Tr1 cells was dependent on the presence of the aryl hydrocarbon receptor, c-Maf and IL-27. Moreover, in comparison to Tr1 cells, Foxp3+ iTreg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. Overall, our data show that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3+ iTreg. PMID:24820384

Cloning and Characterization of the Mouse Hepatitis Virus Receptor

DTIC Science & Technology

1991-02-11

materials. Viruses may also adhere to cell surfaces non-specifically through electrostatic interactions (Tardieu et al., 1982). Virus particles might be... viruses can utilize more than one type of receptor and that specific virus receptors may be present in low numbers on the cell surface or may be labile...known example of this type of interaction is the enhancement of virus infection by antibodies, which has been demonstrated for several viruses

Specificity, cross-talk and adaptation in Interferon signaling

NASA Astrophysics Data System (ADS)

Zilman, Anton

Innate immune system is the first line of defense of higher organisms against pathogens. It coordinates the behavior of millions of cells of multiple types, achieved through numerous signaling molecules. This talk focuses on the signaling specificity of a major class of signaling molecules - Type I Interferons - which are also used therapeutically in the treatment of a number of diseases, such as Hepatitis C, multiple sclerosis and some cancers. Puzzlingly, different Interferons act through the same cell surface receptor but have different effects on the target cells. They also exhibit a strange pattern of temporal cross-talk resulting in a serious clinical problem - loss of response to Interferon therapy. We combined mathematical modeling with quantitative experiments to develop a quantitative model of specificity and adaptation in the Interferon signaling pathway. The model resolves several outstanding experimental puzzles and directly affects the clinical use of Type I Interferons in treatment of viral hepatitis and other diseases.

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

[Embryonic stem cells. Future perspectives].

PubMed

Groebner, M; David, R; Franz, W M

2006-05-01

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

Genetic address book for retinal cell types.

PubMed

Siegert, Sandra; Scherf, Brigitte Gross; Del Punta, Karina; Didkovsky, Nick; Heintz, Nathaniel; Roska, Botond

2009-09-01

The mammalian brain is assembled from thousands of neuronal cell types that are organized in distinct circuits to perform behaviorally relevant computations. Transgenic mouse lines with selectively marked cell types would facilitate our ability to dissect functional components of complex circuits. We carried out a screen for cell type-specific green fluorescent protein expression in the retina using BAC transgenic mice from the GENSAT project. Among others, we identified mouse lines in which the inhibitory cell types of the night vision and directional selective circuit were selectively labeled. We quantified the stratification patterns to predict potential synaptic connectivity between marked cells of different lines and found that some of the lines enabled targeted recordings and imaging of cell types from developing or mature retinal circuits. Our results suggest the potential use of a stratification-based screening approach for characterizing neuronal circuitry in other layered brain structures, such as the neocortex.

Cis-regulatory landscapes of four cell types of the retina

PubMed Central

Hartl, Dominik; Jüttner, Josephine

2017-01-01

Abstract The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. PMID:29059322

Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity

PubMed Central

Bearoff, Frank; del Rio, Roxana; Case, Laure K.; Dragon, Julie A.; Nguyen-Vu, Trang; Lin, Chin-Yo; Blankenhorn, Elizabeth P.; Teuscher, Cory; Krementsov, Dimitry N.

2016-01-01

Regulation of gene expression in immune cells is known to be under genetic control, and likely contributes to susceptibility to autoimmune diseases, such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice, more accurately reflecting genetically diverse human populations, we provide an extensive characterization of the genetic regulation of gene expression in five different naïve immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control, exhibiting diverse patterns: global, cell-specific, and sex-specific. Bioinformatic analysis of the genetically-controlled transcript networks reveals reduced cell type-specificity and inflammatory activity in wild-derived PWD/PhJ mice, compared with the conventional laboratory strain C57BL/6J. Additionally, candidate MS-GWAS genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared to PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis, the principal model of MS, and skewing of the encephalitogenic T cell responses. Taken together, our results provide functional insights into the genetic regulation of the immune transcriptome, and shed light on how this in turn contributes to susceptibility to autoimmune disease. PMID:27653816

Cell type-specific translational repression of Cyclin B during meiosis in males.

PubMed

Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

2015-10-01

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

Cell-type-specific expression of NFIX in the developing and adult cerebellum.

PubMed

Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

2017-07-01

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

Microgenomic analysis reveals cell type-specific gene expression patterns between ray and fusiform initials within the cambial meristem of Populus.

PubMed

Goué, Nadia; Lesage-Descauses, Marie-Claude; Mellerowicz, Ewa J; Magel, Elisabeth; Label, Philippe; Sundberg, Björn

2008-01-01

The vascular cambium is the meristem in trees that produce wood. This meristem consists of two types of neighbouring initials: fusiform cambial cells (FCCs), which give rise to the axial cell system (i.e. fibres and vessel elements), and ray cambial cells (RCCs), which give rise to rays. There is little molecular information on the mechanisms whereby the differing characteristics of these neighbouring cells are maintained. A microgenomic approach was adopted in which the transcriptomes of FCCs and RCCs dissected out from the cambial meristem of poplar (Populus trichocarpa x Populus deltoïdes var. Boelare) were analysed, and a transcriptional database for these two cell types established. Photosynthesis genes were overrepresented in RCCs, providing molecular support for the presence of photosynthetic systems in rays. Genes that putatively encode transporters (vesicle, lipid and metal ion transporters and aquaporins) in RCCs were also identified. In addition, many cell wall-related genes showed cell type-specific expression patterns. Notably, genes involved in pectin metabolism and xyloglucan metabolism were overrepresented in RCCs and FCCs, respectively. The results demonstrate the use of microgenomics to reveal differences in biological processes in neighbouring meristematic cells, and to identify key genes involved in these processes.

Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination

PubMed Central

Dong, Xiaomin; Chen, Kenian; Cuevas-Diaz Duran, Raquel; You, Yanan; Sloan, Steven A.; Zhang, Ye; Zong, Shan; Cao, Qilin; Barres, Ben A.; Wu, Jia Qian

2015-01-01

Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases. PMID:26683846

Human neuronal changes in brain edema and increased intracranial pressure.

PubMed

Faragó, Nóra; Kocsis, Ágnes Katalin; Braskó, Csilla; Lovas, Sándor; Rózsa, Márton; Baka, Judith; Kovács, Balázs; Mikite, Katalin; Szemenyei, Viktor; Molnár, Gábor; Ozsvár, Attila; Oláh, Gáspár; Piszár, Ildikó; Zvara, Ágnes; Patócs, Attila; Barzó, Pál; Puskás, László G; Tamás, Gábor

2016-08-04

Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.

Localization of PPAR isotypes in the adult mouse and human brain

PubMed Central

Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B.; Mayfield, R. Dayne; Harris, R. Adron

2016-01-01

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain. PMID:27283430

Localization of PPAR isotypes in the adult mouse and human brain.

PubMed

Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B; Mayfield, R Dayne; Harris, R Adron

2016-06-10

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain.

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

PubMed Central

Gaillard, Dany; Xu, Mingang; Liu, Fei; Millar, Sarah E.; Barlow, Linda A.

2015-01-01

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells. PMID:26020789

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates.

PubMed

Gaillard, Dany; Xu, Mingang; Liu, Fei; Millar, Sarah E; Barlow, Linda A

2015-05-01

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.

Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

NASA Astrophysics Data System (ADS)

Bared, Kalia

The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

PubMed

Glasser, Stephan W; Eszterhas, Susan K; Detmer, Emily A; Maxfield, Melissa D; Korfhagen, Thomas R

2005-04-01

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

PubMed

Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis.

PubMed

Møller, Inge S; Gilliham, Matthew; Jha, Deepa; Mayo, Gwenda M; Roy, Stuart J; Coates, Juliet C; Haseloff, Jim; Tester, Mark

2009-07-01

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.

Type 1 diabetes vaccine candidates promote human Foxp3+Treg induction in humanized mice

PubMed Central

Serr, Isabelle; Fürst, Rainer W.; Achenbach, Peter; Scherm, Martin G.; Gökmen, Füsun; Haupt, Florian; Sedlmeier, Eva-Maria; Knopff, Annette; Shultz, Leonard; Willis, Richard A.; Ziegler, Anette-Gabriele; Daniel, Carolin

2016-01-01

Immune tolerance is executed partly by Foxp3+regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3+Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3+Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4+T cells and demonstrate efficient human insulin-specific Foxp3+Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3+Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3+Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. PMID:26975663

Antigen-driven C–C Chemokine-mediated HIV-1 Suppression by CD4+ T Cells from Exposed Uninfected Individuals Expressing the Wild-type CCR-5 Allele

PubMed Central

Furci, Lucinda; Scarlatti, Gabriella; Burastero, Samuele; Tambussi, Giuseppe; Colognesi, Claudia; Quillent, Caroline; Longhi, Renato; Loverro, Patrizia; Borgonovo, Barbara; Gaffi, Davide; Carrow, Emily; Malnati, Mauro; Lusso, Paolo; Siccardi, Antonio G.; Lazzarin, Adriano; Beretta, Alberto

1997-01-01

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1–specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32–base pair deletion in the C–C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Δ32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C–C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell–tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development. PMID:9236198

Topologically associating domains are stable units of replication-timing regulation.

PubMed

Pope, Benjamin D; Ryba, Tyrone; Dileep, Vishnu; Yue, Feng; Wu, Weisheng; Denas, Olgert; Vera, Daniel L; Wang, Yanli; Hansen, R Scott; Canfield, Theresa K; Thurman, Robert E; Cheng, Yong; Gülsoy, Günhan; Dennis, Jonathan H; Snyder, Michael P; Stamatoyannopoulos, John A; Taylor, James; Hardison, Ross C; Kahveci, Tamer; Ren, Bing; Gilbert, David M

2014-11-20

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

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

Knoper, Ryan C.; Ferrarone, John; Yan Yuhe

2009-09-01

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIHmore » 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.« less

DSCAM Localization and Function at the Mouse Cone Synapse

PubMed Central

de Andrade, Gabriel Belem; Long, Samuel S.; Fleming, Harrison; Li, Wei; Fuerst, Peter G.

2014-01-01

The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for regulation of cell number, soma spacing and cell type specific dendrite avoidance in many types of retinal ganglion and amacrine cells. In this study we assay the organization of cells making up the outer plexiform layer of the retina in the absence of Dscam. Some types of OFF bipolar cells, type 3b and type 4 bipolar cells, had defects in dendrite arborization in the Dscam mutant retina, while other cell types appeared similar to wild type. The cone synapses that these cells project their dendrites to were intact, as visualized by electron microscopy, and had a distribution and density that was not significantly different than wild type. The spacing of type 3b bipolar cell dendrites was further analyzed by Voronoi domain analysis, Density Recovery Profiling (DRP) analysis and Nearest Neighbor Analysis (NNA). Spacing was found to be significantly different when comparing wild type and mutant type 3b bipolar cell dendrites. Defects in arborization of these bipolar cells could not be attributed to the disorganization of inner plexiform layer cells that occurs in the Dscam mutant retina or an increase in cell number, as they arborized when Dscam was targeted in retinal ganglion cells only or in the bax null retina. Localization of DSCAM was assayed and the protein was localized near to cone synapses in mouse, macaque and ground squirrel retinas. DSCAM protein was detected in several types of bipolar cells, including type 3b and type 4 bipolar cells. PMID:24477985

The T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B.

PubMed

Lapinski, Philip E; Oliver, Jennifer A; Bodie, Jennifer N; Marti, Francesc; King, Philip D

2009-11-01

Adapter proteins play key roles in intracellular signal transduction through complex formation with catalytically active signaling molecules. In T lymphocytes, the role of several different types of adapter proteins in T-cell antigen receptor signal transduction is well established. An exception to this is the family of T-cell-specific adapter (TSAd) proteins comprising of TSAd, adapter protein of unknown function (ALX), SH2D4A, and SH2D4B. Only recently has the function of these adapters in T-cell signal transduction been explored. Here, we discuss advances in our understanding of the role of this family of adapter proteins in T cells. Their function as regulators of signal transduction in other cell types is also discussed.

Loss and gain of cone types in vertebrate ciliary photoreceptor evolution.

PubMed

Musser, Jacob M; Arendt, Detlev

2017-11-01

Ciliary photoreceptors are a diverse cell type family that comprises the rods and cones of the retina and other related cell types such as pineal photoreceptors. Ciliary photoreceptor evolution has been dynamic during vertebrate evolution with numerous gains and losses of opsin and phototransduction genes, and changes in their expression. For example, early mammals lost all but two cone opsins, indicating loss of cone receptor types in response to nocturnal lifestyle. Our review focuses on the comparison of specifying transcription factors and cell type-specific transcriptome data in vertebrate retinae to build and test hypotheses on ciliary photoreceptor evolution. Regarding cones, recent data reveal that a combination of factors specific for long-wavelength sensitive opsin (Lws)- cones in non-mammalian vertebrates (Thrb and Rxrg) is found across all differentiating cone photoreceptors in mice. This suggests that mammalian ancestors lost all but one ancestral cone type, the Lws-cone. We test this hypothesis by a correlation analysis of cone transcriptomes in mouse and chick, and find that, indeed, transcriptomes of all mouse cones are most highly correlated to avian Lws-cones. These findings underscore the importance of specifying transcription factors in tracking cell type evolution, and shed new light on the mechanisms of cell type loss and gain in retina evolution. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

PubMed

Du, Xiaoping; Jensen, Patricia; Goldowitz, Daniel; Hamre, Kristin M

2007-05-15

The transcription factor Math1 has been shown to be critical in the formation of hair cells (HCs) in the inner ear. However, the influence of environmental factors in HC specification suggests that cell extrinsic factors are also crucial to their development. To test whether extrinsic factors impact development of Math1-null (Math1(beta-Gal/beta-Gal)) HCs, we examined neonatal (postnatal ages P0-P4.5) Math1-null chimeric mice in which genotypically mutant and wild-type cells intermingle to form the inner ear. We provide the first direct evidence that Math1-null HCs are able to be generated and survive in the conducive chimeric environment. beta-Galactosidase expression was used to identify genetically mutant cells while cells were phenotypically defined as HCs by morphological characteristics notably the expression of HC-specific markers. Genotypically mutant HCs were found in all sensory epithelia of the inner ear at all ages examined. Comparable results were obtained irrespective of the wild-type component of the chimeric mice. Thus, genotypically mutant cells retain the competence to differentiate into HCs. The implication is that the lack of the Math1 gene in HC precursors can be overcome by environmental influences, such as cell-cell interactions with wild-type cells, to ultimately result in the formation of HCs.

Hepatocyte-specific PPARA expression exclusively promotes agonist-induced cell proliferation without influence from nonparenchymal cells

PubMed Central

Brocker, Chad N.; Yue, Jiang; Kim, Donghwan; Qu, Aijuan; Bonzo, Jessica A.

2017-01-01

Peroxisome proliferator-activated receptor-α (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of nonparenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte (Ppara△Hep)- and macrophage (Ppara△Mac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days, and response of conditional null mice was compared with conventional knockout mice (Ppara−/−). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and Ppara△Mac mice, and histological analysis revealed characteristic hepatocyte swelling; Ppara△Hep and Ppara−/− mice were protected from these effects. Ppara△Mac serum chemistries, as well as aspartate aminotransferase and alanine aminotransferase levels, matched wild-type mice. Agonist-treated Ppara△Hep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared with Ppara−/− mice, indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and Ppara△Mac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, whereas gene expression was repressed in Ppara△Hep mice. Interestingly, downregulation of inflammatory cytokines IL-15 and IL-18 was dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists. PMID:28082284

Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

PubMed Central

Von Seggern, Dan J.; Huang, Shuang; Fleck, Shonna Kaye; Stevenson, Susan C.; Nemerow, Glen R.

2000-01-01

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types. PMID:10590124

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

PubMed Central

2012-01-01

Background Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. Methods We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. Results Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. Conclusions We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific. PMID:22631225

Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data

PubMed Central

Racle, Julien; de Jonge, Kaat; Baumgaertner, Petra; Speiser, Daniel E

2017-01-01

Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org). PMID:29130882

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

NASA Astrophysics Data System (ADS)

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-04-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53-/- mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53-/- mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli.

PubMed

Munne, Pauliina M; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G

2014-04-11

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Secondary IgG responses to type III pneumococcal polysaccharide. II. Different cellular requirements for induction and elicitation.

PubMed

Braley-Mullen, H

1976-04-01

Mice primed with a thymus- (T) dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to erythrocytes (S3-RBC) produce S3-specific IgG antibody after secondary challenge with either S3 or S3-RBC. The production of IgG antibody by mice challenged with S3 was shown to be T independent since secondary responses were enhanced when mice were treated with anti-lymphocyte serum (ALS) at the time of secondary challenge with S3 and T-depleted spleen cells responded as well as unfractionated spleen cells to S3 in an adoptive transfer system. Secondary S3-specific IgG responses in mice challenged with S3-RBC were shown to be T dependent by the same criteria. The results obtained by using S3 as the antigen indicate that IgG-producing B cells (B lambda cells) can recognize and respond to antigen in the absence of helper T cells. On the other hand, T cells were required for the induction of S3-specific memory B lambda cells since mice depleted of T cells by treatment with ALS at the time of priming with S3-RBC failed to produce S3-specific IgG antibody after secondary challenge with either S3-specific IgG antibody after secondary chall-nge with either S3 or S3rbc. Since RBC-specific memory cells were induced in T-deprived mice the results suggest that T cell regulation of IgG antibody production may vary for different antigens.

Peanut-specific type 1 regulatory T cells induced in vitro from allergic subjects are functionally impaired.

PubMed

Pellerin, Laurence; Jenks, Jennifer Anne; Chinthrajah, Sharon; Dominguez, Tina; Block, Whitney; Zhou, Xiaoying; Noshirvan, Arram; Gregori, Silvia; Roncarolo, Maria Grazia; Nadeau, Kari Christine; Bacchetta, Rosa

2018-01-01

Peanut allergy (PA) is a life-threatening condition that lacks regulator-approved treatment. Regulatory T type 1 (T R 1) cells are potent suppressors of immune responses and can be induced in vivo upon repeated antigen exposure or in vitro by using tolerogenic dendritic cells. Whether oral immunotherapy (OIT) leads to antigen-specific T R 1 cell induction has not been established. We sought to determine whether peanut-specific T R 1 cells can be generated in vitro from peripheral blood of patients with PA at baseline or during OIT and whether they are functional compared with peanut-specific T R 1 cells induced from healthy control (HC) subjects. Tolerogenic dendritic cells were differentiated in the presence of IL-10 from PBMCs of patients with PA and HC subjects pulsed with the main peanut allergens of Arachis hypogaea, Ara h 1 and 2, and used as antigen-presenting cells for autologous CD4 + T cells (CD4 + T cells coincubated with tolerogenic dendritic cells pulsed with the main peanut allergens [pea-T10 cells]). Pea-T10 cells were characterized by the presence of CD49b + lymphocyte-activation gene 3 (LAG3) + T R 1 cells, antigen-specific proliferative responses, and cytokine production. CD49b + LAG3 + T R 1 cells were induced in pea-T10 cells at comparable percentages from HC subjects and patients with PA. Despite their antigen specificity, pea-T10 cells of patients with PA with or without OIT, as compared with those of HC subjects, were not anergic and had high T H 2 cytokine production upon peanut-specific restimulation. Peanut-specific T R 1 cells can be induced from HC subjects and patients with PA, but those from patients with PA are functionally defective independent of OIT. The unfavorable T R 1/T H 2 ratio is discussed as a possible cause of PA T R 1 cell impairment. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

H-2D (Rfv-1) gene influence on recovery from Friend virus leukemia is mediated by nonleukemic cells of the spleen and bone marrow

PubMed Central

1980-01-01

H-2D (Rfv-1)-associated control of recovery from FV leukemia was studied in congenic mice. In irradiation chimeras, the high recovery phenotype was transferred by cells of the spleen, bone marrow, and fetal liver. Furthermore, in cell transfers using unirradiated recipients, spleen and bone marrow cells of the high-recovery genotype were able to mediate recovery from leukemia in mice of the low-recovery genotype. Thus, the H-2D (Rfv-1) influence on recovery appeared to operate via nonleukemic cells of the spleen and bone marrow rather than via leukemic cells. The specific nonleukemic cell type(s) involved in recovery remains unknown. However, the mechanism appears to be complex and probably involves both anti-FV antibody and FV-specific cytotoxic T lymphocytes. PMID:6935387

Cell-specific vacuolar calcium storage mediated by "CAX1" regulates apoplastic calcium concentration, gas exchange, and plant productivity in "Arabidopsis"

USDA-ARS?s Scientific Manuscript database

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from "Arabidopsis thaliana" leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen...

Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

PubMed Central

Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

2014-01-01

Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

Respiratory syncytial virus induced type I IFN production by pDC is regulated by RSV-infected airway epithelial cells, RSV-exposed monocytes and virus specific antibodies.

PubMed

Schijf, Marcel A; Lukens, Michael V; Kruijsen, Debby; van Uden, Nathalie O P; Garssen, Johan; Coenjaerts, Frank E J; Van't Land, Belinda; van Bleek, Grada M

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14(+) myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

PubMed Central

Schijf, Marcel A.; Lukens, Michael V.; Kruijsen, Debby; van Uden, Nathalie O. P.; Garssen, Johan; Coenjaerts, Frank E. J.; van’t Land, Belinda; van Bleek, Grada M.

2013-01-01

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease. PMID:24303065

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

PubMed Central

Sørensen, Morten Dræby; Agerholm, Inge Errebo; Christensen, Britta; Kølvraa, Steen; Kristensen, Peter

2010-01-01

Abstract Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific. PMID:20726925

The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions

PubMed Central

Macho-Fernandez, Elodie; Brigl, Manfred

2015-01-01

Natural killer T (NKT) cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR) usage and antigen specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer), and type II NKT cells that use a more diverse αβ TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αβ TCRs and CD1d-restricted T cells that use γδ or δ/αβ TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, anti-tumor immunity, and autoimmunity. PMID:26284062

Molybdenum and vanadium do not replace tungsten in the catalytically active forms of the three tungstoenzymes in the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed Central

Mukund, S; Adams, M W

1996-01-01

Three different types of tungsten-containing enzyme have been previously purified from Pyrococcus furiosus (optimum growth temperature, 100 degrees C): aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR), and glyceraldehyde-3-phosphate oxidoreductase (GAPOR). In this study, the organism was grown in media containing added molybdenum (but not tungsten or vanadium) or added vanadium (but not molybdenum or tungsten). In both cell types, there were no dramatic changes compared with cells grown with tungsten, in the specific activities of hydrogenase, ferredoxin:NADP oxidoreductase, or the 2-keto acid ferredoxin oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Compared with tungsten-grown cells, the specific activities of AOR, FOR, and GAPOR were 40, 74, and 1%, respectively, in molybdenum-grown cells, and 7, 0, and 0%, respectively, in vanadium-grown cells. AOR purified from vanadium-grown cells lacked detectable vanadium, and its tungsten content and specific activity were both ca. 10% of the values for AOR purified from tungsten-grown cells. AOR and FOR purified from molybdenum-grown cells contained no detectable molybdenum, and their tungsten contents and specific activities were > 70% of the values for the enzymes purified from tungsten-grown cells. These results indicate that P. furiosus uses exclusively tungsten to synthesize the catalytically active forms of AOR, FOR, and GAPOR, and active molybdenum- or vanadium-containing isoenzymes are not expressed when the cells are grown in the presence of these other metals. PMID:8550411

Validity and reproducibility of measurement of islet autoreactivity by T-cell assays in subjects with early type 1 diabetes.

PubMed

Herold, Kevan C; Brooks-Worrell, Barbara; Palmer, Jerry; Dosch, H Michael; Peakman, Mark; Gottlieb, Peter; Reijonen, Helena; Arif, Sefina; Spain, Lisa M; Thompson, Clinton; Lachin, John M

2009-11-01

Type 1 diabetes results from an immunemediated destruction of beta-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression. We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections. Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays. The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood.

The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements

PubMed Central

Uhlirova, Hana; Kılıç, Kıvılcım; Tian, Peifang; Sakadžić, Sava; Thunemann, Martin; Desjardins, Michèle; Saisan, Payam A.; Nizar, Krystal; Yaseen, Mohammad A.; Hagler, Donald J.; Vandenberghe, Matthieu; Djurovic, Srdjan; Andreassen, Ole A.; Silva, Gabriel A.; Masliah, Eliezer; Vinogradov, Sergei; Buxton, Richard B.; Einevoll, Gaute T.; Boas, David A.; Dale, Anders M.; Devor, Anna

2016-01-01

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed ‘bottom-up’ forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the ‘ground truth’ for the development of new tools for tackling the inverse problem—estimation of neuronal activity from multimodal non-invasive imaging data. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’. PMID:27574309

Pathogen stimulation history impacts donor-specific CD8+ T cell susceptibility to costimulation/integrin blockade-based therapy

PubMed Central

Badell, IR; Kitchens, WH; Wagener, ME; Lukacher, AE; Larsen, CP; Ford, ML

2017-01-01

Recent studies have shown that the quantity of donor-reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade-based immunosuppression. Using a murine skin graft model of CD8+ memory T cell-mediated costimulation blockade resistance, we elicited donor-reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor-reactive memory T cell response. Intriguingly, the most immunosuppression-sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi-cytokine producers. These data therefore demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor-reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation. PMID:26228897

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

Maintenance of memory-type pathogenic Th2 cells in the pathophysiology of chronic airway inflammation.

PubMed

Hirahara, Kiyoshi; Shinoda, Kenta; Endo, Yusuke; Ichikawa, Tomomi; Nakayama, Toshinori

2018-01-01

Immunological memory is critical for long-standing protection against microorganisms; however, certain antigen-specific memory CD4 + T helper (Th) cells drive immune-related pathology, including chronic allergic inflammation such as asthma. The IL-5-producing memory-type Tpath2 subset is important for the pathogenesis of chronic allergic inflammation. This memory-type pathogenic Th2 cell population (Tpath2) can be detected in various allergic inflammatory lesions. However, how these pathogenic populations are maintained at the local inflammatory site has remained unclear. We performed a series of experiments using mice model for chronic airway inflammation. We also investigated the human samples from patients with eosinophilic chronic rhinosinusitis. We recently reported that inducible bronchus-associated lymphoid tissue (iBALT) was shaped during chronic inflammation in the lung. We also found that memory-type Tpath2 cells are maintained within iBALT. The maintenance of the Tpath2 cells within iBALT is supported by specific cell subpopulations within the lung. Furthermore, ectopic lymphoid structures consisting of memory CD4 + T cells were found in nasal polyps of eosinophilic chronic rhinosinusitis patients, indicating that the persistence of inflammation is controlled by these structures. Thus, the cell components that organize iBALT formation may be therapeutic targets for chronic allergic airway inflammation.

Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY

PubMed Central

Ohashi-Ito, Kyoko; Bergmann, Dominique C.

2011-01-01

Complex organisms consist of a multitude of cell types arranged in precise spatial relation to each other. Arabidopsis roots generally exhibit radial tissue organization; however, within a tissue layer, cells are not identical. Specific vascular cell types are arranged in diametrically opposed longitudinal files that maximize the distance between them and create a bilaterally symmetric (diarch) root. Mutations in the LONESOME HIGHWAY (LHW) gene eliminate bilateral symmetry and reduce the number of cells in the center of the root, resulting in roots with only single and xylem and phloem poles. LHW does not appear to be required for the creation of any specific cell type, but coordinately controls the number of all vascular cell types by regulating the size of the pool of cells from which they arise. We cloned LHW and found that it encodes a protein with weak sequence similarity to basic helix-loop-helix (bHLH) domain proteins. LHW is a transcriptional activator in vitro. In plants, LHW is nuclear localized and is expressed in the root meristems where we hypothesize it acts independently of other known root patterning genes to promote the production of stele cells, but may also indirectly feed into established regulatory networks for the maintenance of the root meristem. PMID:17626058

The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons.

PubMed

Carroll, Elizabeth C; Jin, Lei; Mori, Andres; Muñoz-Wolf, Natalia; Oleszycka, Ewa; Moran, Hannah B T; Mansouri, Samira; McEntee, Craig P; Lambe, Eimear; Agger, Else Marie; Andersen, Peter; Cunningham, Colm; Hertzog, Paul; Fitzgerald, Katherine A; Bowie, Andrew G; Lavelle, Ed C

2016-03-15

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell-type specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing

PubMed Central

Sun, Yanjun; Nguyen, Amanda; Nguyen, Joseph; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M.; Xu, Xiangmin

2014-01-01

Summary We applied a new Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to CA1 excitatory and inhibitory neuron types in mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, entorhinal cortex and the medial septum (MS), and unexpectedly also from the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons while inhibitory CA1 neurons receive a great majority of input from GABAergic MS neurons; both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons receive much stronger input than SOM+ neurons from CA3, entorhinal cortex and MS. Differential input from CA3 to specific CA1 cell types was also demonstrated functionally using laser scanning photostimulation and whole cell recordings. PMID:24656815

Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex

PubMed Central

Allen, William E.; Kauvar, Isaac V.; Chen, Michael Z.; Richman, Ethan B.; Yang, Samuel J.; Chan, Ken; Gradinaru, Viviana; Deverman, Benjamin E.; Luo, Liqun; Deisseroth, Karl

2017-01-01

SUMMARY The successful planning and execution of adaptive behaviors in mammals may require long-range coordination of neural networks throughout cerebral cortex. The neuronal implementation of signals that could orchestrate cortex-wide activity remains unclear. Here, we develop and apply methods for cortex-wide Ca2+ imaging in mice performing decision-making behavior and identify a global cortical representation of task engagement encoded in the activity dynamics of both single cells and superficial neuropil distributed across the majority of dorsal cortex. The activity of multiple molecularly defined cell types was found to reflect this representation with type-specific dynamics. Focal optogenetic inhibition tiled across cortex revealed a crucial role for frontal cortex in triggering this cortex-wide phenomenon; local inhibition of this region blocked both the cortex-wide response to task-initiating cues and the voluntary behavior. These findings reveal cell-type-specific processes in cortex for globally representing goal-directed behavior and identify a major cortical node that gates the global broadcast of task-related information. PMID:28521139

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.

PubMed

Sharifi-Sanjani, Maryam; Meeker, Alan K; Mourkioti, Foteini

2017-09-01

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA) 3 peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.

Computational Modeling of Tissue Self-Assembly

NASA Astrophysics Data System (ADS)

Neagu, Adrian; Kosztin, Ioan; Jakab, Karoly; Barz, Bogdan; Neagu, Monica; Jamison, Richard; Forgacs, Gabor

As a theoretical framework for understanding the self-assembly of living cells into tissues, Steinberg proposed the differential adhesion hypothesis (DAH) according to which a specific cell type possesses a specific adhesion apparatus that combined with cell motility leads to cell assemblies of various cell types in the lowest adhesive energy state. Experimental and theoretical efforts of four decades turned the DAH into a fundamental principle of developmental biology that has been validated both in vitro and in vivo. Based on computational models of cell sorting, we have developed a DAH-based lattice model for tissues in interaction with their environment and simulated biological self-assembly using the Monte Carlo method. The present brief review highlights results on specific morphogenetic processes with relevance to tissue engineering applications. Our own work is presented on the background of several decades of theoretical efforts aimed to model morphogenesis in living tissues. Simulations of systems involving about 105 cells have been performed on high-end personal computers with CPU times of the order of days. Studied processes include cell sorting, cell sheet formation, and the development of endothelialized tubes from rings made of spheroids of two randomly intermixed cell types, when the medium in the interior of the tube was different from the external one. We conclude by noting that computer simulations based on mathematical models of living tissues yield useful guidelines for laboratory work and can catalyze the emergence of innovative technologies in tissue engineering.

Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

PubMed

Sripadi, Prabhakar; Shrestha, Bindesh; Easley, Rebecca L; Carpio, Lawrence; Kehn-Hall, Kylene; Chevalier, Sebastien; Mahieux, Renaud; Kashanchi, Fatah; Vertes, Akos

2010-09-07

Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3) transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC) lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1) and PC(O-34:1) plasmalogens were displaced by PC(30:0) and PC(32:0) species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3), expression-specific (Tax1 vs. Tax3) and cell-type-specific (T lymphocytes vs. kidney epithelial cells) changes in the metabolite profiles. The new insight on the affected metabolic pathways can be used to better understand the molecular mechanisms of HTLV induced transformation, which in turn can result in new treatment strategies.

Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity.

PubMed

Hrdlickova, Barbara; Kumar, Vinod; Kanduri, Kartiek; Zhernakova, Daria V; Tripathi, Subhash; Karjalainen, Juha; Lund, Riikka J; Li, Yang; Ullah, Ubaid; Modderman, Rutger; Abdulahad, Wayel; Lähdesmäki, Harri; Franke, Lude; Lahesmaa, Riitta; Wijmenga, Cisca; Withoff, Sebo

2014-01-01

Although genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). lncRNAs are known to show more cell-type specificity than protein-coding genes. We aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AIDs which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, natural killer (NK) cells, B cells, memory T cells, naive CD4(+) and naive CD8(+) T cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells). We show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory T and CD8(+) T cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved. The observed enrichment of lncRNA transcripts in AID loci implies lncRNAs play an important role in AID etiology and suggests that lncRNA genes should be studied in more detail to interpret GWAS findings correctly. The co-expression results strongly support a model in which the lncRNA and protein-coding genes function together in the same pathways.

Mechanisms of activation of mouse and human enteroendocrine cells by nutrients

PubMed Central

Symonds, Erin L; Peiris, Madusha; Page, Amanda J; Chia, Bridgette; Dogra, Harween; Masding, Abigail; Galanakis, Vasileios; Atiba, Michael; Bulmer, David; Young, Richard L; Blackshaw, L Ashley

2015-01-01

Objective Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release. Design and results mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators. Conclusions Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes. PMID:25015642

A Common histone modification code on C4 genes in maize and its conservation in Sorghum and Setaria italica.

PubMed

Heimann, Louisa; Horst, Ina; Perduns, Renke; Dreesen, Björn; Offermann, Sascha; Peterhansel, Christoph

2013-05-01

C4 photosynthesis evolved more than 60 times independently in different plant lineages. Each time, multiple genes were recruited into C4 metabolism. The corresponding promoters acquired new regulatory features such as high expression, light induction, or cell type-specific expression in mesophyll or bundle sheath cells. We have previously shown that histone modifications contribute to the regulation of the model C4 phosphoenolpyruvate carboxylase (C4-Pepc) promoter in maize (Zea mays). We here tested the light- and cell type-specific responses of three selected histone acetylations and two histone methylations on five additional C4 genes (C4-Ca, C4-Ppdk, C4-Me, C4-Pepck, and C4-RbcS2) in maize. Histone acetylation and nucleosome occupancy assays indicated extended promoter regions with regulatory upstream regions more than 1,000 bp from the transcription initiation site for most of these genes. Despite any detectable homology of the promoters on the primary sequence level, histone modification patterns were highly coregulated. Specifically, H3K9ac was regulated by illumination, whereas H3K4me3 was regulated in a cell type-specific manner. We further compared histone modifications on the C4-Pepc and C4-Me genes from maize and the homologous genes from sorghum (Sorghum bicolor) and Setaria italica. Whereas sorghum and maize share a common C4 origin, C4 metabolism evolved independently in S. italica. The distribution of histone modifications over the promoters differed between the species, but differential regulation of light-induced histone acetylation and cell type-specific histone methylation were evident in all three species. We propose that a preexisting histone code was recruited into C4 promoter control during the evolution of C4 metabolism.

CLO: The cell line ontology

PubMed Central

2014-01-01

Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

A multi-tissue type genome-scale metabolic network for analysis of whole-body systems physiology

PubMed Central

2011-01-01

Background Genome-scale metabolic reconstructions provide a biologically meaningful mechanistic basis for the genotype-phenotype relationship. The global human metabolic network, termed Recon 1, has recently been reconstructed allowing the systems analysis of human metabolic physiology and pathology. Utilizing high-throughput data, Recon 1 has recently been tailored to different cells and tissues, including the liver, kidney, brain, and alveolar macrophage. These models have shown utility in the study of systems medicine. However, no integrated analysis between human tissues has been done. Results To describe tissue-specific functions, Recon 1 was tailored to describe metabolism in three human cells: adipocytes, hepatocytes, and myocytes. These cell-specific networks were manually curated and validated based on known cellular metabolic functions. To study intercellular interactions, a novel multi-tissue type modeling approach was developed to integrate the metabolic functions for the three cell types, and subsequently used to simulate known integrated metabolic cycles. In addition, the multi-tissue model was used to study diabetes: a pathology with systemic properties. High-throughput data was integrated with the network to determine differential metabolic activity between obese and type II obese gastric bypass patients in a whole-body context. Conclusion The multi-tissue type modeling approach presented provides a platform to study integrated metabolic states. As more cell and tissue-specific models are released, it is critical to develop a framework in which to study their interdependencies. PMID:22041191

Three types of ependymal cells with intracellular calcium oscillation are characterized by distinct cilia beating properties.

PubMed

Liu, Tongyu; Jin, Xingjian; Prasad, Rahul M; Sari, Youssef; Nauli, Surya M

2014-09-01

Ependymal cells are multiciliated epithelial cells that line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia has been associated with various neurological deficits. For the first time, we report three distinct ependymal cell types, I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells have specific localizations within the third ventricle of the mouse brain. Furthermore, neither ependymal cell types nor their localizations are altered by aging. Our high-speed fluorescence imaging analysis reveals that these ependymal cells have an intracellular pacing calcium oscillation property. Our study further shows that alcohol can significantly repress the amplitude of calcium oscillation and the frequency of ciliary beating, resulting in an overall decrease in volume replacement by the cilia. Furthermore, the pharmacological agent cilostazol could differentially increase cilia beating frequency in type II, but not in type I or type III, ependymal cells. In summary, we provide the first evidence of three distinct types of ependymal cells with calcium oscillation properties. © 2014 Wiley Periodicals, Inc.

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

Autoantigen-specific regulatory T cells induced in patients with Type 1 Diabetes Mellitus by Insulin B-chain immunotherapy

PubMed Central

Orban, Tihamer; Farkas, Klara; Jalahej, Heyam; Kis, Janos; Treszl, Andras; Falk, Ben; Reijonen, Helena; Wolfsdorf, Joseph; Ricker, Alyne; Matthews, Jeffrey B.; Tchao, Nadio; Sayre, Peter; Bianchine, Pete

2009-01-01

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund’s adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499). PMID:19931408

Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry.

PubMed

Wan, Leo Q; Ronaldson, Kacey; Park, Miri; Taylor, Grace; Zhang, Yue; Gimble, Jeffrey M; Vunjak-Novakovic, Gordana

2011-07-26

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10(-185)) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.

High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

PubMed

Endo, Kei; Hayashi, Karin; Saito, Hirohide

2016-02-23

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

PubMed

Schweizer, Patrick A; Darche, Fabrice F; Ullrich, Nina D; Geschwill, Pascal; Greber, Boris; Rivinius, Rasmus; Seyler, Claudia; Müller-Decker, Karin; Draguhn, Andreas; Utikal, Jochen; Koenen, Michael; Katus, Hugo A; Thomas, Dierk

2017-10-16

Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells.

PubMed

Au, Ka-Yee; Wang, Rui-Rui; Wong, Yuen-Ting; Wong, Kam-Bo; Zheng, Yong-Tang; Shaw, Pang-Chui

2014-03-01

Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA. HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants. C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells. RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication. Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV. Copyright © 2013 Elsevier B.V. All rights reserved.

Cardiomyocyte-Specific Telomere Shortening is a Distinct Signature of Heart Failure in Humans.

PubMed

Sharifi-Sanjani, Maryam; Oyster, Nicholas M; Tichy, Elisia D; Bedi, Kenneth C; Harel, Ofer; Margulies, Kenneth B; Mourkioti, Foteini

2017-09-07

Telomere defects are thought to play a role in cardiomyopathies, but the specific cell type affected by the disease in human hearts is not yet identified. The aim of this study was to systematically evaluate the cell type specificity of telomere shortening in patients with heart failure in relation to their cardiac disease, age, and sex. We studied cardiac tissues from patients with heart failure by utilizing telomere quantitative fluorescence in situ hybridization, a highly sensitive method with single-cell resolution. In this study, total of 63 human left ventricular samples, including 37 diseased and 26 nonfailing donor hearts, were stained for telomeres in combination with cardiomyocyte- or α-smooth muscle cell-specific markers, cardiac troponin T, and smooth muscle actin, respectively, and assessed for telomere length. Patients with heart failure demonstrate shorter cardiomyocyte telomeres compared with nonfailing donors, which is specific only to cardiomyocytes within diseased human hearts and is associated with cardiomyocyte DNA damage. Our data further reveal that hypertrophic hearts with reduced ejection fraction exhibit the shortest telomeres. In contrast to other reported cell types, no difference in cardiomyocyte telomere length is evident with age. However, under the disease state, telomere attrition manifests in both young and older patients with cardiac hypertrophy. Finally, we demonstrate that cardiomyocyte-telomere length is better sustained in women than men under diseased conditions. This study provides the first evidence of cardiomyocyte-specific telomere shortening in heart failure. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

Cell-specific STORM superresolution imaging reveals nanoscale organization of cannabinoid signaling

PubMed Central

Szabó, Szilárd I.; Szabadits, Eszter; Pintér, Balázs; Woodhams, Stephen G.; Henstridge, Christopher M.; Balla, Gyula Y.; Nyilas, Rita; Varga, Csaba; Lee, Sang-Hun; Matolcsi, Máté; Cervenak, Judit; Kacskovics, Imre; Watanabe, Masahiko; Sagheddu, Claudia; Melis, Miriam; Pistis, Marco; Soltesz, Ivan; Katona, István

2014-01-01

A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell-type-, and subcellular compartment-specific manner. We therefore developed a novel approach combining cell-specific physiological and anatomical characterization with superresolution imaging, and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically-projecting GABAergic interneurons possess increased CB1 receptor number, active-zone complexity, and receptor/effector ratio compared to dendritically-projecting interneurons, in agreement with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked dramatic CB1-downregulation in a dose-dependent manner. Full receptor recovery required several weeks after cessation of Δ9-tetrahydrocannabinol treatment. These findings demonstrate that cell-type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits, and identify novel molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction. PMID:25485758

Localization of sesquiterpene lactone biosynthesis in cells of capitate glandular trichomes of Helianthus annuus (Asteraceae).

PubMed

Amrehn, Evelyn; Aschenbrenner, Anna-Katharina; Heller, Annerose; Spring, Otmar

2016-03-01

Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid.

A repetitive mutation and selection system for bacterial evolution to increase the specific affinity to pancreatic cancer cells.

PubMed

Osawa, Masaki

2018-01-01

It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia.

PubMed

Wang, Jinghua; Chen, Siyu; Xiao, Wei; Li, Wende; Wang, Liang; Yang, Shuo; Wang, Weida; Xu, Liping; Liao, Shuangye; Liu, Wenjian; Wang, Yang; Liu, Nawei; Zhang, Jianeng; Xia, Xiaojun; Kang, Tiebang; Chen, Gong; Cai, Xiuyu; Yang, Han; Zhang, Xing; Lu, Yue; Zhou, Penghui

2018-01-10

Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. The CLL-1 CAR-T cells specifically lysed CLL-1 + cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1 + myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

Hunger States Control the Directions of Synaptic Plasticity via Switching Cell Type-Specific Subunits of NMDA Receptors.

PubMed

Qi, Yong; Yang, Yunlei

2015-09-23

It remains largely unknown whether and how hunger states control activity-dependent synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD). We here report that both LTP and LTD of excitatory synaptic strength within the appetite control circuits residing in hypothalamic arcuate nucleus (ARC) behave in a manner of hunger states dependence and cell type specificity. For instance, we find that tetanic stimulation induces LTP at orexigenic agouti-related protein (AgRP) neurons in ad libitum fed mice, whereas it induces LTD in food-deprived mice. In an opposite direction, the same induction protocol induces LTD at anorexigenic pro-opiomelanocortin (POMC) neurons in fed mice but weak LTP in deprived mice. Mechanistically, we also find that food deprivation increases the expressions of NR2C/NR2D/NR3-containing NMDA receptors (NMDARs) at AgRP neurons that contribute to the inductions of LTD, whereas it decreases their expressions at POMC neurons. Collectively, our data reveal that hunger states control the directions of activity-dependent synaptic plasticity by switching NMDA receptor subpopulations in a cell type-specific manner, providing insights into NMDAR-mediated interactions between energy states and associative memory. Significance statement: Based on the experiments performed in this study, we demonstrate that activity-dependent synaptic plasticity is also under the control of energy states by regulating NMDAR subpopulations in a cell type-specific manner. We thus propose a reversible memory configuration constructed from energy states-dependent cell type-specific bidirectional conversions of LTP and LTD. Together with the distinct functional roles played by NMDAR signaling in the control of food intake and energy states, these findings reveal a new reciprocal interaction between energy states and associative memory, one that might serve as a target for therapeutic treatments of the energy-related memory disorders or vice versa. Copyright © 2015 the authors 0270-6474/15/3513171-12$15.00/0.

Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

PubMed Central

Scott, Jaclyn C.; Brackney, Doug E.; Campbell, Corey L.; Bondu-Hawkins, Virginie; Hjelle, Brian; Ebel, Greg D.; Olson, Ken E.; Blair, Carol D.

2010-01-01

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts. PMID:21049014

Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling

PubMed Central

Sims, Joshua J.; Scavone, Francesco; Cooper, Eric M.; Kane, Lesley A.; Youle, Richard J.; Boeke, Jef D.; Cohen, Robert E.

2012-01-01

Polyubiquitin (polyUb) chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyUb signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyUb signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity lysine-63-polyUb-binding proteins and demonstrate their specificity both in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyUb-linkage dependence of NF-κB activation in several cell types, inferring the essential role of lysine-63-polyUb for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic, and biochemical applications for these tools, and extension of the design strategy to other polymeric ubiquitin-like protein modifications. PMID:22306808

Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

PubMed Central

Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A.

2016-01-01

Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

Natural killer T cells are required for the development of a superantigen-driven T helper type 2 immune response in mice

PubMed Central

Nomizo, Auro; Postol, Edilberto; de Alencar, Raquel; Cardillo, Fabíola; Mengel, José

2005-01-01

We show, here, that one single injection or weekly injections of staphylococcal enterotoxin B (SEB), starting in 1-day-old newborn mice, induced a powerful immune response with a T helper type 2 (Th2) pattern, as judged by the isotype and cytokine profile, with the production of large amounts of SEB-specific immunoglobulin G1 (IgG1), detectable levels of SEB-specific IgE and increased production of interleukin-4 by spleen cells. These protocols also induced an increase in the levels of total IgE in the serum. Memory of SEB was transferred to secondary recipients by using total spleen cells from primed animals. The secondary humoral response in transferred mice was diminished if spleen cells from SEB-treated mice were previously depleted of CD3+ or Vβ8+ T cells or NK1.1+ cells. In vivo depletion of NK1.1+ cells in adult mice resulted in a marked reduction in the SEB-specific antibody response in both the primary and secondary immune responses. Additionally, purified NK1.1+ T cells were able to perform SEB-specific helper B-cell actions in vitro and in vivo. These results suggest that NK1.1+ T cells are required for the full development of humoral immunological memory, whilst making neonatal tolerance to SEB unachievable. PMID:16162272

A DNA methylation map of human cancer at single base-pair resolution

PubMed Central

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-01-01

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

Stem and progenitor cells: the premature desertion of rigorous definitions.

PubMed

Seaberg, Raewyn M; van der Kooy, Derek

2003-03-01

A current disturbing trend in stem cell biology is the abandonment of rigorous definitions of stem and progenitor cells in favor of more ambiguous, all-encompassing concepts. However, recent studies suggest that there are consistent, functional differences in the biology of these two cell types. Admittedly, it can be difficult to harmonize the in vivo and in vitro functional differences between stem and progenitor cells. Nonetheless, these distinctions between cell types should be emphasized rather than ignored, as they can be used to test specific hypotheses in neural stem cell biology.

Genetics of the connective tissue proteins: Assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids*

PubMed Central

Raj, Cholappadi V. Sundar; Church, Robert L.; Klobutcher, Lawrence A.; Ruddle, Frank H.

1977-01-01

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [3H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17. Images PMID:412188

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

PubMed Central

Ng, Wy Ching; Liong, Stella; Tate, Michelle D.; Irimura, Tatsuro; Denda-Nagai, Kaori; Brooks, Andrew G.; Londrigan, Sarah L.

2014-01-01

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca2+-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca2+-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV. PMID:24257596

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity

PubMed Central

Yonehara, Keisuke; Fiscella, Michele; Drinnenberg, Antonia; Esposti, Federico; Trenholm, Stuart; Krol, Jacek; Franke, Felix; Scherf, Brigitte Gross; Kusnyerik, Akos; Müller, Jan; Szabo, Arnold; Jüttner, Josephine; Cordoba, Francisco; Reddy, Ashrithpal Police; Németh, János; Nagy, Zoltán Zsolt; Munier, Francis; Hierlemann, Andreas; Roska, Botond

2016-01-01

Summary Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. Video Abstract PMID:26711119

Keratin 17 null mice exhibit age- and strain-dependent alopecia.

PubMed

McGowan, Kevin M; Tong, Xuemei; Colucci-Guyon, Emma; Langa, Francina; Babinet, Charles; Coulombe, Pierre A

2002-06-01

Onset of type I keratin 17 (K17) synthesis marks the adoption of an appendageal fate within embryonic ectoderm, and its expression persists in specific cell types within mature hair, glands, and nail. We report that K17 null mice develop severe alopecia during the first week postbirth, correlating with hair fragility, alterations in follicular histology, and apoptosis in matrix cells. These alterations are incompletely penetrant and normalize starting with the first postnatal cycle. Absence of a hair phenotype correlates with a genetic strain-dependent compensation by related keratins, including K16. These findings reveal a crucial role for K17 in the structural integrity of the first hair produced and the survival of hair-producing cells. Given that identical inherited mutations in this gene can cause either pachyonychia congenita or steatocystoma multiplex, the features of this mouse model suggest that this clinical heterogeneity arises from a cell type-specific, genetically determined compensation by related keratins.

ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts

PubMed Central

Tsukamoto, Yoshihiko; Omi, Naoko

2016-01-01

To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two-step synaptic connections largely narrowed down the S-cone component to SBG and some OFF MG cells. The other OFF MG cells, ON MG cells, and ON and OFF PG cells constructed M/L-cone dominant pathways. PMID:27833534

Cell Type-specific Intrinsic Perithreshold Oscillations in Hippocampal GABAergic Interneurons.

PubMed

Kang, Young-Jin; Lewis, Hannah Elisabeth Smashey; Young, Mason William; Govindaiah, Gubbi; Greenfield, Lazar John; Garcia-Rill, Edgar; Lee, Sang-Hun

2018-04-15

The hippocampus plays a critical role in learning, memory, and spatial processing through coordinated network activity including theta and gamma oscillations. Recent evidence suggests that hippocampal subregions (e.g., CA1) can generate these oscillations at the network level, at least in part, through GABAergic interneurons. However, it is unclear whether specific GABAergic interneurons generate intrinsic theta and/or gamma oscillations at the single-cell level. Since major types of CA1 interneurons (i.e., parvalbumin-positive basket cells (PVBCs), cannabinoid type 1 receptor-positive basket cells (CB 1 BCs), Schaffer collateral-associated cells (SCAs), neurogliaform cells and ivy cells) are thought to play key roles in network theta and gamma oscillations in the hippocampus, we tested the hypothesis that these cells generate intrinsic perithreshold oscillations at the single-cell level. We performed whole-cell patch-clamp recordings from GABAergic interneurons in the CA1 region of the mouse hippocampus in the presence of synaptic blockers to identify intrinsic perithreshold membrane potential oscillations. The majority of PVBCs (83%), but not the other interneuron subtypes, produced intrinsic perithreshold gamma oscillations if the membrane potential remained above -45 mV. In contrast, CB 1 BCs, SCAs, neurogliaform cells, ivy cells, and the remaining PVBCs (17%) produced intrinsic theta, but not gamma, oscillations. These oscillations were prevented by blockers of persistent sodium current. These data demonstrate that the major types of hippocampal interneurons produce distinct frequency bands of intrinsic perithreshold membrane oscillations. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

NASA Astrophysics Data System (ADS)

Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

2014-03-01

Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

Synaptology of physiologically identified ganglion cells in the cat retina: a comparison of retinal X- and Y-cells.

PubMed

Weber, A J; Stanford, L R

1994-05-15

It has long been known that a number of functionally different types of ganglion cells exist in the cat retina, and that each responds differently to visual stimulation. To determine whether the characteristic response properties of different retinal ganglion cell types might reflect differences in the number and distribution of their bipolar and amacrine cell inputs, we compared the percentages and distributions of the synaptic inputs from bipolar and amacrine cells to the entire dendritic arbors of physiologically characterized retinal X- and Y-cells. Sixty-two percent of the synaptic input to the Y-cell was from amacrine cell terminals, while the X-cells received approximately equal amounts of input from amacrine and bipolar cells. We found no significant difference in the distributions of bipolar or amacrine cell inputs to X- and Y-cells, or ON-center and OFF-center cells, either as a function of dendritic branch order or distance from the origin of the dendritic arbor. While, on the basis of these data, we cannot exclude the possibility that the difference in the proportion of bipolar and amacrine cell input contributes to the functional differences between X- and Y-cells, the magnitude of this difference, and the similarity in the distributions of the input from the two afferent cell types, suggest that mechanisms other than a simple predominance of input from amacrine or bipolar cells underlie the differences in their response properties. More likely, perhaps, is that the specific response features of X- and Y-cells originate in differences in the visual responses of the bipolar and amacrine cells that provide their input, or in the complex synaptic arrangements found among amacrine and bipolar cell terminals and the dendrites of specific types of retinal ganglion cells.

GIANT 2.0: genome-scale integrated analysis of gene networks in tissues.

PubMed

Wong, Aaron K; Krishnan, Arjun; Troyanskaya, Olga G

2018-05-25

GIANT2 (Genome-wide Integrated Analysis of gene Networks in Tissues) is an interactive web server that enables biomedical researchers to analyze their proteins and pathways of interest and generate hypotheses in the context of genome-scale functional maps of human tissues. The precise actions of genes are frequently dependent on their tissue context, yet direct assay of tissue-specific protein function and interactions remains infeasible in many normal human tissues and cell-types. With GIANT2, researchers can explore predicted tissue-specific functional roles of genes and reveal changes in those roles across tissues, all through interactive multi-network visualizations and analyses. Additionally, the NetWAS approach available through the server uses tissue-specific/cell-type networks predicted by GIANT2 to re-prioritize statistical associations from GWAS studies and identify disease-associated genes. GIANT2 predicts tissue-specific interactions by integrating diverse functional genomics data from now over 61 400 experiments for 283 diverse tissues and cell-types. GIANT2 does not require any registration or installation and is freely available for use at http://giant-v2.princeton.edu.

Langerhans Cells: the 'Yin and Yang' of HIV Restriction and Transmission.

PubMed

Mayr, Luzia; Su, Bin; Moog, Christiane

2017-03-01

Langerhans cells are specialized sentinels present in the epidermis expressing Langerin, a specific C-type lectin receptor involved in HIV capture and destruction. Recently, the specific mechanism leading to this HIV restriction was discovered. Nevertheless, Langerhans cells can be infected and the way HIV escapes this restriction needs to be unraveled. Copyright © 2017. Published by Elsevier Ltd.

Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix

PubMed Central

Su, Yuan; Shi, Yufang; Stolow, Melissa A.; Shi, Yun-Bo

1997-01-01

Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. PMID:9396758

Blockade of the interaction of leukotriene b4 with its receptor prevents development of autoimmune uveitis.

PubMed

Liao, Tianjiang; Ke, Yan; Shao, Wen-Hai; Haribabu, Bodduluri; Kaplan, Henry J; Sun, Deming; Shao, Hui

2006-04-01

To investigate the role of leukotriene B4 (LTB4) and its receptor BLT1 in the pathogenesis of mouse uveitis. Experimental autoimmune uveitis (EAU) was induced in B10RIII mice by immunization of interphotoreceptor retinoid binding protein (IRBP; peptide sequence 161-180) or in C57BL/6 (B6) mice by transfer of activated T cells specific for IRBP1-20. The animals were then treated with and without the BLT1 receptor antagonist, CP105696, at the disease onset after immunization or at day 0 or day 6 after T-cell transfer. EAU was also induced in wild-type B6 (WT) and BLT1-deficient (BLT1-/-) mice by reciprocal transfer of the T cells from B6 to BLT1-deficient mice and vise versa. Clinical signs of inflammation and ocular histology were compared. The chemotactic activity of LTB4 on naïve and IRBP-specific autoreactive T cells as well as effector leukocytes was examined. The treatment of CP105696, greatly reduced the intensity of ongoing disease. IRBP1-20-specific T cells derived from wild-type B6 mice induced only mild uveitis in syngeneic BLT1-deficient mice and that IRBP1-20-specific T cells derived from BLT1-/- mice induced milder disease in wild-type B6 mice than those derived from wild-type B6 mice, suggesting that expression of the LTB4 receptor on both activated autoreactive T cells and effector leukocytes was necessary for ocular inflammation to occur. Consistent with these data, transfer of autoreactive T cells from B6 mice to 5-lipoxygenase-deficient (5-LO-/-) mice, which have a functional defect in LTB4 expression, also failed to induce uveitis in the recipient mice. The results demonstrate a critical role for LTB4 in ocular inflammation and in the development and progression of EAU and suggest a new potential target for therapeutic intervention in this disease.

Whole transcriptome profiling of taste bud cells.

PubMed

Sukumaran, Sunil K; Lewandowski, Brian C; Qin, Yumei; Kotha, Ramana; Bachmanov, Alexander A; Margolskee, Robert F

2017-08-08

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex.

PubMed Central

Mato, M; Ookawara, S; Sakamoto, A; Aikawa, E; Ogawa, T; Mitsuhashi, U; Masuzawa, T; Suzuki, H; Honda, M; Yazaki, Y; Watanabe, E; Luoma, J; Yla-Herttuala, S; Fraser, I; Gordon, S; Kodama, T

1996-01-01

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8622926

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

PubMed

Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

2016-07-01

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Chromatin programming by developmentally regulated transcription factors: lessons from the study of haematopoietic stem cell specification and differentiation.

PubMed

Obier, Nadine; Bonifer, Constanze

2016-11-01

Although the body plan of individuals is encoded in their genomes, each cell type expresses a different gene expression programme and therefore has access to only a subset of this information. Alterations to gene expression programmes are the underlying basis for the differentiation of multiple cell types and are driven by tissue-specific transcription factors (TFs) that interact with the epigenetic regulatory machinery to programme the chromatin landscape into transcriptionally active and inactive states. The haematopoietic system has long served as a paradigm for studying the molecular principles that regulate gene expression in development. In this review article, we summarize the current knowledge on the mechanism of action of TFs regulating haematopoietic stem cell specification and differentiation, and place this information into the context of general principles governing development. © 2016 Federation of European Biochemical Societies.

Strength of signal: a fundamental mechanism for cell fate specification.

PubMed

Hayes, Sandra M; Love, Paul E

2006-02-01

How equipotent cells develop into complex tissues containing many diverse cell types is still a mystery. However, evidence is accumulating from different tissue systems in multiple organisms that many of the specific receptor families known to regulate cell fate decisions target conserved signaling pathways. A mechanism for preserving specificity in the cellular response that has emerged from these studies is one in which quantitative differences in receptor signaling regulate the cell fate decision. A signal strength model has recently gained support as a means to explain alphabeta/gammadelta lineage commitment. In this review, we compare the alphabeta/gammadelta fate decision with other cell fate decisions that occur outside of the lymphoid system to attain a better picture of the quantitative signaling mechanism for cell fate specification.

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT.

PubMed

Haga, Kei; Ohno, Shin-ichi; Yugawa, Takashi; Narisawa-Saito, Mako; Fujita, Masatoshi; Sakamoto, Michiie; Galloway, Denise A; Kiyono, Tohru

2007-02-01

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

PubMed

Rana, Subinoy; Elci, S Gokhan; Mout, Rubul; Singla, Arvind K; Yazdani, Mahdieh; Bender, Markus; Bajaj, Avinash; Saha, Krishnendu; Bunz, Uwe H F; Jirik, Frank R; Rotello, Vincent M

2016-04-06

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

Loss of Virus-Specific Memory T. cells in Coxsackievirus B3 and B4 Infected Mice

EPA Science Inventory

There are two major types of enteroviruses: polioviruses and non-polio enteroviruses. While vaccines have effectively eliminated poliovirus infections, no vaccine is currently available for the non-polio enteroviruses. Generation of long-term pathogen specific memory cells is cri...

Cis-regulatory landscapes of four cell types of the retina.

PubMed

Hartl, Dominik; Krebs, Arnaud R; Jüttner, Josephine; Roska, Botond; Schübeler, Dirk

2017-11-16

The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Type II NKT Cells in Inflammation, Autoimmunity, Microbial Immunity, and Cancer

PubMed Central

Marrero, Idania; Ware, Randle; Kumar, Vipin

2015-01-01

Natural killer T cells (NKT) recognize self and microbial lipid antigens presented by non-polymorphic CD1d molecules. Two major NKT cell subsets, type I and II, express different types of antigen receptors (TCR) with distinct mode of CD1d/lipid recognition. Though type II NKT cells are less frequent in mice and difficult to study, they are predominant in human. One of the major subsets of type II NKT cells reactive to the self-glycolipid sulfatide is the best characterized and has been shown to induce a dominant immune regulatory mechanism that controls inflammation in autoimmunity and in anti-cancer immunity. Recently, type II NKT cells reactive to other self-glycolipids and phospholipids have been identified suggesting both promiscuous and specific TCR recognition in microbial immunity as well. Since the CD1d pathway is highly conserved, a detailed understanding of the biology and function of type II NKT cells as well as their interplay with type I NKT cells or other innate and adaptive T cells will have major implications for potential novel interventions in inflammatory and autoimmune diseases, microbial immunity, and cancer. PMID:26136748

The relevance of TH1 and TH2 cells in immediate and nonimmediate reactions to gelatin-containing vaccine.

PubMed

Ohsaki, M; Tsutsumi, H; Kumagai, T; Yamanaka, T; Wataya, Y; Furukawa, H; Kojima, H; Saito, A; Yano, S; Chiba, S

1999-02-01

The immune mechanism of gelatin allergy, especially the participation of TH1 and TH2 cells and their cytokine secretion, has not been investigated. We investigated the characteristics of T lymphocytes from patients allergic to gelatin-containing vaccine by secondary in vitro stimulation of circulating mononuclear cells with gelatin. We studied 8 children with a history of immediate-type reactions and 8 with nonimmediate-type reactions after inoculation of gelatin-containing vaccine. The expression of IFN-gamma (TH1 ), IL-2 (TH1 ), IL-4 (TH2 ), and IL-13 (TH2 ) mRNA was examined semiquantitatively by using a reverse transcriptase PCR. IgE antibody to bovine gelatin was measured with the fluorometric ELISA system, and gelatin-specific T-cell responses were detected by an in vitro lymphocyte proliferation assay. Patients with an immediate reaction all had gelatin-specific IgE antibody, whereas others did not. However, all patients exhibited positive T-lymphocyte responses specific to gelatin. Lymphocytes from subjects with nonimmediate-type reactions generally expressed very weak or sometimes no IFN-gamma, IL-2, or IL-13 genes and especially no IL-4 gene. On the other hand, the lymphocytes of subjects with immediate-type reactions significantly expressed not only IL-4 and IL-13 but also IFN-gamma and IL-2 mRNA. Our observations suggest that both gelatin-specific TH2 and TH1 responses are involved in the pathogenesis of the immediate reaction to gelatin. The gelatin-specific IL-4 and/or IL-13 responses consistently observed in patients with an immediate reaction may be associated with the production of gelatin-specific IgE antibody.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells

PubMed Central

van den Brink, Susanne C.; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A.; Martinez Arias, Alfonso

2014-01-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call ‘gastruloids’. PMID:25371360

Dual targeted polymeric nanoparticles based on tumor endothelium and tumor cells for enhanced antitumor drug delivery.

PubMed

Gupta, Madhu; Chashoo, Gousia; Sharma, Parduman Raj; Saxena, Ajit Kumar; Gupta, Prem Narayan; Agrawal, Govind Prasad; Vyas, Suresh Prasad

2014-03-03

Some specific types of tumor cells and tumor endothelial cells represented CD13 proteins and act as receptors for Asn-Gly-Arg (NGR) motifs containing peptide. These CD13 receptors can be specifically recognized and bind through the specific sequence of cyclic NGR (cNGR) peptide and presented more affinity and specificity toward them. The cNGR peptide was conjugated to the poly(ethylene glycol) (PEG) terminal end in the poly(lactic-co-glycolic) acid PLGA-PEG block copolymer. Then, the ligand conjugated nanoparticles (cNGR-DNB-NPs) encapsulating docetaxel (DTX) were synthesized from preformed block copolymer by the emulsion/solvent evaporation method and characterized for different parameters. The various studies such as in vitro cytotoxicity, cell apoptosis, and cell cycle analysis presented the enhanced therapeutic potential of cNGR-DNB-NPs. The higher cellular uptake was also found in cNGR peptide anchored NPs into HUVEC and HT-1080 cells. However, free cNGR could inhibit receptor mediated intracellular uptake of NPs into both types of cells at 37 and 4 °C temperatures, revealing the involvement of receptor-mediated endocytosis. The in vivo biodistribution and antitumor efficacy studies indicated that targeted NPs have a higher therapeutic efficacy through targeting the tumor-specific site. Therefore, the study exhibited that cNGR-functionalized PEG-PLGA-NPs could be a promising approach for therapeutic applications to efficient antitumor drug delivery.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

PubMed

van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

2014-11-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells.

PubMed

Halim, Timotheus Y F; Rana, Batika M J; Walker, Jennifer A; Kerscher, Bernhard; Knolle, Martin D; Jolin, Helen E; Serrao, Eva M; Haim-Vilmovsky, Liora; Teichmann, Sarah A; Rodewald, Hans-Reimer; Botto, Marina; Vyse, Timothy J; Fallon, Padraic G; Li, Zhi; Withers, David R; McKenzie, Andrew N J

2018-06-19

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7ra Cre/+ Tnfsf4 fl/fl mice). Moreover, Il7ra Cre/+ Tnfsf4 fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.

T cell reactivity with allergoids: influence of the type of APC.

PubMed

Kahlert, H; Grage-Griebenow, E; Stüwe, H T; Cromwell, O; Fiebig, H

2000-08-15

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation

PubMed Central

Yu, Wenjie; Li, Xiao; Eliason, Steven; Romero-Bustillos, Miguel; Ries, Ryan J.; Cao, Huojun; Amendt, Brad A.

2017-01-01

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1−/− mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development. PMID:28746823

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation.

PubMed

Yu, Wenjie; Li, Xiao; Eliason, Steven; Romero-Bustillos, Miguel; Ries, Ryan J; Cao, Huojun; Amendt, Brad A

2017-09-01

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1 -/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

PubMed

Yoshida, Saishu; Kato, Takako; Kanno, Naoko; Nishimura, Naoto; Nishihara, Hiroto; Horiguchi, Kotaro; Kato, Yukio

2017-10-01

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

LMethyR-SVM: Predict Human Enhancers Using Low Methylated Regions based on Weighted Support Vector Machines.

PubMed

Xu, Jingting; Hu, Hong; Dai, Yang

The identification of enhancers is a challenging task. Various types of epigenetic information including histone modification have been utilized in the construction of enhancer prediction models based on a diverse panel of machine learning schemes. However, DNA methylation profiles generated from the whole genome bisulfite sequencing (WGBS) have not been fully explored for their potential in enhancer prediction despite the fact that low methylated regions (LMRs) have been implied to be distal active regulatory regions. In this work, we propose a prediction framework, LMethyR-SVM, using LMRs identified from cell-type-specific WGBS DNA methylation profiles and a weighted support vector machine learning framework. In LMethyR-SVM, the set of cell-type-specific LMRs is further divided into three sets: reliable positive, like positive and likely negative, according to their resemblance to a small set of experimentally validated enhancers in the VISTA database based on an estimated non-parametric density distribution. Then, the prediction model is obtained by solving a weighted support vector machine. We demonstrate the performance of LMethyR-SVM by using the WGBS DNA methylation profiles derived from the human embryonic stem cell type (H1) and the fetal lung fibroblast cell type (IMR90). The predicted enhancers are highly conserved with a reasonable validation rate based on a set of commonly used positive markers including transcription factors, p300 binding and DNase-I hypersensitive sites. In addition, we show evidence that the large fraction of the LMethyR-SVM predicted enhancers are not predicted by ChromHMM in H1 cell type and they are more enriched for the FANTOM5 enhancers. Our work suggests that low methylated regions detected from the WGBS data are useful as complementary resources to histone modification marks in developing models for the prediction of cell-type-specific enhancers.

Radiation and Smoking Effects on Lung Cancer Incidence by Histological Types Among Atomic Bomb Survivors

PubMed Central

Egawa, Hiromi; Furukawa, Kyoji; Preston, Dale; Funamoto, Sachiyo; Yonehara, Shuji; Matsuo, Takeshi; Tokuoka, Shoji; Suyama, Akihiko; Ozasa, Kotaro; Kodama, Kazunori; Mabuchi, Kiyohiko

2014-01-01

While the risk of lung cancer associated separately with smoking and radiation exposure has been widely reported, it is not clear how smoking and radiation together contribute to the risk of specific lung cancer histological types. With individual smoking histories and radiation dose estimates, we characterized the joint effects of radiation and smoking on type-specific lung cancer rates among the Life Span Study cohort of Japanese atomic bomb survivors. Among 105,404 cohort subjects followed between 1958 and 1999, 1,803 first primary lung cancer incident cases were diagnosed and classified by histological type. Poisson regression methods were used to estimate excess relative risks under several interaction models. Adenocarcinoma (636 cases), squamous-cell carcinoma (330) and small-cell carcinoma (194) made up 90% of the cases with known histology. Both smoking and radiation exposure significantly increased the risk of each major lung cancer histological type. Smoking-associated excess relative risks were significantly larger for small-cell and squamous-cell carcinomas than for adenocarcinoma. The gender-averaged excess relative risks per 1 Gy of radiation (for never-smokers at age 70 after radiation exposure at age 30) were estimated as 1.49 (95% confidence interval 0.1–4.6) for small-cell carcinoma, 0.75 (0.3–1.3) for adenocarcinoma, and 0.27 (0–1.5) for squamous-cell carcinoma. Under a model allowing radiation effects to vary with levels of smoking, the nature of the joint effect of smoking and radiation showed a similar pattern for different histological types in which the radiation-associated excess relative risk tended to be larger for moderate smokers than for heavy smokers. However, in contrast to analyses of all lung cancers as a group, such complicated interactions did not describe the data significantly better than either simple additive or multiplicative interaction models for any of the type-specific analyses. PMID:22862780

Identification of the cortical neurons that mediate antidepressant responses

PubMed Central

Schmidt, Eric F.; Warner-Schmidt, Jennifer; Otopalik, Benjamin G.; Pickett, Sarah B.; Greengard, Paul; Heintz, Nathaniel

2012-01-01

Summary Our understanding of current treatments for depression, and the development of more specific therapies, is limited by the complexity of the circuits controlling mood and the distributed actions of antidepressants. Although the therapeutic efficacy of SSRIs is correlated with increases in cortical activity, the cell types crucial for their action remain unknown. Here we employ bacTRAP translational profiling to show that layer 5 corticostriatal pyramidal cells expressing p11 (S100a10) are strongly and specifically responsive to chronic antidepressant treatment. This response requires p11 and includes the specific induction of Htr4 expression. Cortex-specific deletion of p11 abolishes behavioral responses to SSRI’s, but does not lead to increased depression-like behaviors. Our data identify corticostriatal projection neurons as critical for the response to antidepressants, and suggest that the regulation of serotonergic tone in this single cell type plays a pivotal role in antidepressant therapy. PMID:22632977

Regional-specific effect of fluoxetine on rapidly dividing progenitors along the dorsoventral axis of the hippocampus.

PubMed

Zhou, Qi-Gang; Lee, Daehoon; Ro, Eun Jeoung; Suh, Hoonkyo

2016-10-19

Hippocampus-dependent cognitive and emotional function appears to be regionally dissociated along the dorsoventral (DV) axis of the hippocampus. Recent observations that adult hippocampal neurogenesis plays a critical role in both cognition and emotion raised an interesting question whether adult neurogenesis within specific subregions of the hippocampus contributes to these distinct functions. We examined the regional-specific and cell type-specific effects of fluoxetine, which requires adult hippocampal neurogenesis to function as an antidepressant, on the proliferation of hippocampal neural stem cells (NSCs). Fluoxetine specifically increased proliferation of NSCs located in the ventral region of the hippocampus while the mitotic index of NSCs in the dorsal portion of the hippocampus remained unaltered. Moreover, within the ventral hippocampus, type II NSC and neuroblast populations specifically responded to fluoxetine, showing increased proliferation; however, proliferation of type I NSCs was unchanged in response to fluoxetine. Activation or inhibition of serotonin receptor 1A (5-HTR1A) recapitulated or abolished the effect of fluoxetine on proliferation of type II NSCs and neuroblast populations in the ventral hippocampus. Our study showed that the effect of fluoxetine on proliferation is dependent upon the type and the position of the NSCs along the DV axis of the hippocampus.

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates.

PubMed

Dowd, Jason E; Jubb, Anthea; Kwok, K Ezra; Piret, James M

2003-05-01

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of approximately 5 x 10(6) cells mL(-1). Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell(-1) day(-1). Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized ( approximately 40 mg L(-1)) at 0.2 nL cell(-1) day(-1). The volumetric protein productivity ( approximately 60 mg L(-1) day(-1) was maximized above 0.3 nL cell(-1) day(-1). The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.

Classification of Mouse Retinal Bipolar Cells: Type-Specific Connectivity with Special Reference to Rod-Driven AII Amacrine Pathways

PubMed Central

Tsukamoto, Yoshihiko; Omi, Naoko

2017-01-01

We confirmed the classification of 15 morphological types of mouse bipolar cells by serial section transmission electron microscopy and characterized each type by identifying chemical synapses and gap junctions at axon terminals. Although whether the previous type 5 cells consist of two or three types was uncertain, they are here clustered into three types based on the vertical distribution of axonal ribbons. Next, while two groups of rod bipolar (RB) cells, RB1, and RB2, were previously proposed, we clarify that a half of RB1 cells have the intermediate characteristics, suggesting that these two groups comprise a single RB type. After validation of bipolar cell types, we examined their relationship with amacrine cells then particularly with AII amacrine cells. We found a strong correlation between the number of amacrine cell synaptic contacts and the number of bipolar cell axonal ribbons. Formation of bipolar cell output at each ribbon synapse may be effectively regulated by a few nearby inhibitory inputs of amacrine cells which are chosen from among many amacrine cell types. We also found that almost all types of ON cone bipolar cells frequently have a minor group of midway ribbons along the axon passing through the OFF sublamina as well as a major group of terminal ribbons in the ON sublamina. AII amacrine cells are connected to five of six OFF bipolar cell types via conventional chemical synapses and seven of eight ON (cone) bipolar cell types via electrical synapses (gap junctions). However, the number of synapses is dependent on bipolar cell types. Type 2 cells have 69% of the total number of OFF bipolar chemical synaptic contacts with AII amacrine cells and type 6 cells have 46% of the total area of ON bipolar gap junctions with AII amacrine cells. Both type 2 and 6 cells gain the greatest access to AII amacrine cell signals also share those signals with other types of bipolar cells via networked gap junctions. These findings imply that the most sensitive scotopic signal may be conveyed to the center by ganglion cells that have the most numerous synapses with type 2 and 6 cells. PMID:29114208

HLA-DR4-associated T and B cell responses to specific determinants on the IA-2 autoantigen in type 1 diabetes.

PubMed

McLaughlin, Kerry A; Gulati, Kavita; Richardson, Carolyn C; Morgan, Diana; Bodansky, H Jonathan; Feltbower, Richard G; Christie, Michael R

2014-11-01

Autoantibodies to IA-2 in type 1 diabetes are associated with HLA-DR4, suggesting influences of HLA-DR4-restricted T cells on IA-2-specific B cell responses. The aim of this study was to investigate possible T-B cell collaboration by determining whether autoantibodies to IA-2 epitopes are associated with T cell responses to IA-2 peptides presented by DR4. T cells secreting the cytokines IFN-γ and IL-10 in response to seven peptides known to elicit T cell responses in type 1 diabetes were quantified by cytokine ELISPOT in HLA-typed patients characterized for Abs to IA-2 epitopes. T cell responses were detected to all peptides tested, but only IL-10 responses to 841-860 and 853-872 peptides were associated with DR4. Phenotyping by RT-PCR of FACS-sorted CD45RO(hi) T cells secreting IL-10 in response to these two peptides indicated that these expressed GATA-3 or T-bet, but not FOXP3, consistent with these being Th2 or Th1 memory T cells rather than of regulatory phenotype. T cell responses to the same two peptides were also associated with specific Abs: those to 841-860 peptide with Abs to juxtamembrane epitopes, which appear early in prediabetes, and those to peptide 853-872 with Abs to an epitope located in the 831-862 central region of the IA-2 tyrosine phosphatase domain. Abs to juxtamembrane and central region constructs were both DR4 associated. This study identifies a region of focus for B and T cell responses to IA-2 in HLA-DR4 diabetic patients that may explain HLA associations of IA-2 autoantibodies, and this region may provide a target for future immune intervention to prevent disease. Copyright © 2014 by The American Association of Immunologists, Inc.

Pattern multiplicity and fumarate hydratase (FH)/S-(2-succino)-cysteine (2SC) staining but not eosinophilic nucleoli with perinucleolar halos differentiate hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinomas from kidney tumors without FH gene alteration.

PubMed

Muller, Marie; Guillaud-Bataille, Marine; Salleron, Julia; Genestie, Catherine; Deveaux, Sophie; Slama, Abdelhamid; de Paillerets, Brigitte Bressac; Richard, Stéphane; Benusiglio, Patrick R; Ferlicot, Sophie

2018-02-06

Hereditary leiomyomatosis and renal cell carcinoma syndrome is characterized by an increased risk of agressive renal cell carcinoma, often of type 2 papillary histology, and is caused by FH germline mutations. A prominent eosinophilic macronucleolus with a perinucleolar clear halo is distinctive of hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cell carcinoma according to the 2012 ISUP and 2016 WHO kidney tumor classification. From an immunohistochemistry perspective, tumors are often FH-negative and S-(2-succino)-cysteine (2SC) positive. We performed a pathology review of 24 renal tumors in 23 FH mutation carriers, and compared them to 12 type 2 papillary renal cell carcinomas from FH wild-type patients. Prominent eosinophilic nucleoli with perinucleolar halos were present in almost all FH-deficient renal cell carcinomas (23/24). Unexpectedly, they were also present in 58% of type 2 papillary renal cell carcinomas from wild-type patients. Renal cell carcinoma in mutation carriers displayed a complex architecture with multiple patterns, typically papillary, tubulopapillary, and tubulocystic, but also sarcomatoid and rhabdoid. Such pattern diversity was not seen in non-carriers. FH/2SC immunohistochemistry was informative as all hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinomas were either FH- or 2SC+. For FH and 2SC immunohistochemistries taken separately, sensitivity of negative anti-FH immunohistochemistry was 87.5% and specificity was 100%. For positive anti-2SC immunohistochemistry, sensitivity, and specificity were 91.7% and 91.7%, respectively. All FH wild-type renal cell carcinoma were FH-positive, and all but one were 2SC-negative. In conclusion, multiplicity of architectural patterns, rhabdoid/sarcomatoid components and combined FH/2SC staining, but not prominent eosinophilic nucleoli with perinucleolar halos, differentiate hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinoma from type 2 papillary renal cell carcinoma with efficient FH gene. Our findings are crucial in identifying who should be referred to Cancer Genetics clinics for genetic counseling and testing.

Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen

PubMed Central

Taylor, Justin J.; Martinez, Ryan J.; Titcombe, Philip J.; Barsness, Laura O.; Thomas, Stephanie R.; Zhang, Na; Katzman, Shoshana D.; Jenkins, Marc K.

2012-01-01

B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen–specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen–specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens. PMID:23071255

Genetics Home Reference: Fuchs endothelial dystrophy

MedlinePlus

... a protein that is part of type VIII collagen. Type VIII collagen is largely found within the cornea, surrounding the endothelial cells. Specifically, type VIII collagen is a major component of a tissue at ...

Mouse Regenerating Myofibers Detected as False-Positive Donor Myofibers with Anti-Human Spectrin

PubMed Central

Rozkalne, Anete; Adkin, Carl; Meng, Jinhong; Lapan, Ariya; Morgan, Jennifer E.

2014-01-01

Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1nullmdx5cv, NOD/LtSz-scid IL2Rγnull mice, or mdx nude mice, irrespective of whether they were injected with human cells. These “reactive” clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies. PMID:24152287

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

PubMed

Utz, U; Banks, D; Jacobson, S; Biddison, W E

1996-02-01

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

The Adaptor Protein SAP Regulates Type II NKT Cell Development, Cytokine Production and Cytotoxicity Against Lymphoma1

PubMed Central

Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L.; Stein, Paul L.; Wang, Chyung-Ru

2014-01-01

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule-associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT cell TCR transgenic mouse model (24αβTg), we demonstrated that CD1d-expressing hematopoietic cells but not thymic epithelial cells meditate efficient selection of type II NKT cells. Further, we showed that SAP regulates type II NKT cell development by controlling Egr2 and PLZF expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IRF4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. PMID:25236978

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

PubMed

Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

2014-12-01

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Compartment-specific control of signaling from a DNA-sensing immune receptor.

PubMed

Engel, Alex; Barton, Gregory M

2010-11-30

Many cell signaling events are spatially organized, enabling control of specificity, amplitude, and duration. Toll-like receptor 9 (TLR9) binds to nucleic acid sequences present in bacteria or DNA viruses and initiates a signaling pathway that culminates in the transcriptional induction of genes important for host defense, such as those encoding proinflammatory cytokines and type I interferon. A specialized membrane trafficking pathway has been described that is required for a specific branch of TLR9 signaling: the production of type I interferon. Cells deficient for the clathrin adaptor complex AP-3 failed to traffic TLR9 to a specific endosomal compartment and were unable to produce type I interferon despite normal increases in the abundance of interleukin-12p40, a proinflammatory cytokine. These findings support a model in which the targets of TLR9 engagement are controlled by the compartment in which TLR9 is activated.

GABA, its receptors, and GABAergic inhibition in mouse taste buds

PubMed Central

Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D.

2012-01-01

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals — glial-like Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Using a combination of Ca2+ imaging, single cell RT-PCR, and immunostaining, we show that γ-amino butyric acid (GABA) is an inhibitory transmitter in mouse taste buds, acting on GABA-A and GABA-B receptors to suppress transmitter (ATP) secretion from Receptor cells during taste stimulation. Specifically, Receptor cells express GABA-A receptor subunits β2, δ, π, as well as GABA-B receptors. In contrast, Presynaptic cells express the GABA-Aβ3 subunit and only occasionally GABA-B receptors. In keeping with the distinct expression pattern of GABA receptors in Presynaptic cells, we detected no GABAergic suppression of transmitter release from Presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in Type I taste cells as well as by GAD67 in Presynaptic (Type III) taste cells and is stored in both those two cell types. We conclude that GABA is released during taste stimulation and possibly also during growth and differentiation of taste buds. PMID:21490220

GABA, its receptors, and GABAergic inhibition in mouse taste buds.

PubMed

Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

2011-04-13

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes.

PubMed

Rodrigo, Miguel A Merlos; Strmiska, Vladislav; Horackova, Eva; Buchtelova, Hana; Michalek, Petr; Stiborova, Marie; Eckschlager, Tomas; Adam, Vojtech; Heger, Zbynek

2018-02-01

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets. © 2017 Wiley Periodicals, Inc.

AtERF38 (At2g35700), an AP2/ERF family transcription factor gene from Arabidopsis thaliana, is expressed in specific cell types of roots, stems and seeds that undergo suberization.

PubMed

Lasserre, Eric; Jobet, Edouard; Llauro, Christel; Delseny, Michel

2008-12-01

An inverse genetic approach was used to gain insight into the role of AP2/ERF-type transcription factors genes during plant development in Arabidopsis thaliana. Here we show that the expression pattern of AtERF38, which is, among the organs tested, more intensively expressed in mature siliques and floral stems, is closely associated with tissues that undergo secondary cell wall modifications. Firstly, public microarray data sets analysis indicates that AtERF38 is coregulated with several genes involved in secondary wall thickening. Secondly, this was experimentally confirmed in different types of cells expressing a Pro(AtERF38)::GUS fusion: histochemical analysis revealed strong and specific GUS activity in outer integument cells of mature seeds, endodermal cells of the roots in the primary developmental stage and some sclerified cells of mature inflorescence stems. All of these cells are known or shown here to be characterized by a reinforced wall. The latter, which have not been well characterized to date in Arabidopsis and may be suberized, could benefit of the use of AtERF38 as a specific marker. We were not able to detect any phenotype in an insertion line in which ectopic expression of AtERF38 is caused by the insertion of a T-DNA in its promoter. Nevertheless, AtERF28 may be considered as a candidate regulator of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose, but that have at least in common accumulation of suberin-like lipid polyesters in their walls.

Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology.

PubMed

Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun; Aguet, Francois; Tacheva-Grigorova, Silvia K; Masuda, Takeshi; Yi, Hai; Lee, Sung-Uk; Xu, Jian; Teruya-Feldstein, Julie; Ericsson, Maria; Mullally, Ann; Heuser, John; Kirchhausen, Tom; Maeda, Takahiro

2015-04-01

Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera. Copyright© Ferrata Storti Foundation.

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

PubMed

Willott, E; Trenczek, T; Thrower, L W; Kanost, M R

1994-12-01

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

Bacterial Biofilms as Complex Communities

NASA Astrophysics Data System (ADS)

Vlamakis, Hera

2010-03-01

Many microbial populations form surface-associated multicellular communities known as biofilms. These multicellular communities are encased in a self-produced extracellular matrix composed of polysaccharides and proteins. Division of labor is a key feature of these communities and different cells serve distinct functions. We have found that in biofilms of the bacterium Bacillus subtilis, different cell types including matrix-producing and sporulating cells coexist and localize to distinct regions within the structured community. We were interested in understanding how these different cell types arise. Using fluorescence reporters under the control of promoters that are specific for distinct cell types we were able to follow the dynamics of differentiation throughout biofilm development. We found that a series of extracellular signals leads to differentiation of distinct cell types during biofilm formation. In addition, we found that extracellular matrix functions as a differentiation signal for timely sporulation within a biofilm and mutants unable to produce matrix were delayed in sporulation. Our results indicate that within a biofilm, cell-cell signaling is directional in that one cell type produces a signal that is sensed by another distinct cell type. Furthermore, once differentiated, cells become resistant to the action of other signaling molecules making it possible to maintain distinct cell populations over prolonged periods.

Partitioning heritability by functional annotation using genome-wide association summary statistics.

PubMed

Finucane, Hilary K; Bulik-Sullivan, Brendan; Gusev, Alexander; Trynka, Gosia; Reshef, Yakir; Loh, Po-Ru; Anttila, Verneri; Xu, Han; Zang, Chongzhi; Farh, Kyle; Ripke, Stephan; Day, Felix R; Purcell, Shaun; Stahl, Eli; Lindstrom, Sara; Perry, John R B; Okada, Yukinori; Raychaudhuri, Soumya; Daly, Mark J; Patterson, Nick; Neale, Benjamin M; Price, Alkes L

2015-11-01

Recent work has demonstrated that some functional categories of the genome contribute disproportionately to the heritability of complex diseases. Here we analyze a broad set of functional elements, including cell type-specific elements, to estimate their polygenic contributions to heritability in genome-wide association studies (GWAS) of 17 complex diseases and traits with an average sample size of 73,599. To enable this analysis, we introduce a new method, stratified LD score regression, for partitioning heritability from GWAS summary statistics while accounting for linked markers. This new method is computationally tractable at very large sample sizes and leverages genome-wide information. Our findings include a large enrichment of heritability in conserved regions across many traits, a very large immunological disease-specific enrichment of heritability in FANTOM5 enhancers and many cell type-specific enrichments, including significant enrichment of central nervous system cell types in the heritability of body mass index, age at menarche, educational attainment and smoking behavior.

Regulatory elements driving the expression of skeletal lineage reporters differ during bone development and adulthood.

PubMed

Stiers, Pieter-Jan; van Gastel, Nick; Moermans, Karen; Stockmans, Ingrid; Carmeliet, Geert

2017-12-01

To improve bone healing or regeneration more insight in the fate and role of the different skeletal cell types is required. Mouse models for fate mapping and lineage tracing of skeletal cells, using stage-specific promoters, have advanced our understanding of bone development, a process that is largely recapitulated during bone repair. However, validation of these models is often only performed during development, whereas proof of the activity and specificity of the used promoters during the bone regenerative process is limited. Here, we show that the regulatory elements of the 6kb collagen type II promoter are not adequate to drive gene expression during bone repair. Similarly, the 2.3kb promoter of collagen type I lacks activity in adult mice, but the 3.2kb promoter is suitable. Furthermore, Cre-mediated fate mapping allows the visualization of progeny, but this label retention may hinder to distinguish these cells from ones with active expression of the marker at later time points. Together, our results show that the lineage-specific regulatory elements driving gene expression during bone development differ from those required later in life and during bone repair, and justify validation of lineage-specific cell tracing and gene silencing strategies during fracture healing and bone regenerative applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

PubMed Central

Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

2006-01-01

Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

Clustering Single-Cell Expression Data Using Random Forest Graphs.

PubMed

Pouyan, Maziyar Baran; Nourani, Mehrdad

2017-07-01

Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.

Regulation of DNA replication during development

PubMed Central

Nordman, Jared; Orr-Weaver, Terry L.

2012-01-01

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication. PMID:22223677

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

PubMed Central

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-01-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53−/− mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53−/− mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers. PMID:24722541

Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.

PubMed

Tremblay, Kimberly D; Zaret, Kenneth S

2005-04-01

The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.

Presynaptic (Type III) cells in mouse taste buds sense sour (acid) taste.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Stimac, Robert; Roper, Stephen D

2008-06-15

Taste buds contain two types of cells that directly participate in taste transduction - receptor (Type II) cells and presynaptic (Type III) cells. Receptor cells respond to sweet, bitter and umami taste stimulation but until recently the identity of cells that respond directly to sour (acid) tastants has only been inferred from recordings in situ, from behavioural studies, and from immunostaining for putative sour transduction molecules. Using calcium imaging on single isolated taste cells and with biosensor cells to identify neurotransmitter release, we show that presynaptic (Type III) cells specifically respond to acid taste stimulation and release serotonin. By recording responses in cells isolated from taste buds and in taste cells in lingual slices to acetic acid titrated to different acid levels (pH), we also show that the active stimulus for acid taste is the membrane-permeant, uncharged acetic acid moiety (CH(3)COOH), not free protons (H(+)). That observation is consistent with the proximate stimulus for acid taste being intracellular acidification, not extracellular protons per se. These findings may also have implications for other sensory receptors that respond to acids, such as nociceptors.

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Targeting Therapy Resistant Tumor Vessels

DTIC Science & Technology

2007-05-01

Porkka K, Laakko- nen P, Ruoslahti E. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell...anti-angiogenic therapy. Markers of such vessels will be useful in developing strategies for complete destruction of breast cancer vasculature, and in...express specific markers , and that these lymphatic markers are tumor type specific and distinct from blood vessel markers in the same tumors. The

Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

PubMed

Dubayle, Jean; Vialle, Sandrine; Schneider, Diane; Pontvianne, Jérémy; Mantel, Nathalie; Adam, Olivier; Guy, Bruno; Talaga, Philippe

2015-03-10

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

PubMed

Bell, Charlotte R; MacHugh, Niall D; Connelley, Timothy K; Degnan, Kathryn; Morrison, W Ivan

2015-07-09

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells used in vaccine production, coupled with a powerful adjuvant, are responsible for the generation of pathogenic alloantibodies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell- derived-suppressor factors

PubMed Central

1979-01-01

Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti- B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper. PMID:312894

Developing Laryngeal Muscle of Xenopus laevis as a Model System: Androgen-Driven Myogenesis Controls Fiber Type Transformation

PubMed Central

Nasipak, Brian; Kelley, Darcy B.

2014-01-01

The developmental programs that contribute to myogenic stem cell proliferation and muscle fiber differentiation control fiber numbers and twitch type. In this study, we describe the use of an experimental model system—androgen-regulated laryngeal muscle of juvenile clawed frogs, Xenopus laevis—to examine the contribution of proliferation by specific populations of myogenic stem cells to expression of the larynx-specific myosin heavy chain isoform, LM. Androgen treatment of juveniles (Stage PM0) resulted in up-regulation of an early (Myf-5) and a late (myogenin) myogenic regulatory factor; the time course of LM up-regulation tracked that of myogenin. Myogenic stem cells stimulated to proliferate by androgen include a population that expresses Pax-7, a marker for the satellite cell myogenic stem cell population. Since androgen can switch muscle fiber types from fast to slow even in denervated larynges, we developed an ex vivo culture system to explore the relation between proliferation and LM expression. Cultured whole larynges maintain sensitivity to androgen, increasing in size and LM expression. Blockade of cell proliferation with cis-platin prevents the switch from slow to fast twitch muscle fibers as assayed by ATPase activity. Blockade of cell proliferation in vivo also resulted in inhibition of LM expression. Thus, both in vivo and ex vivo, inhibition of myogenic stem cell proliferation blocks androgen-induced LM expression and fiber type switching in juveniles. PMID:21954146

A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research

PubMed Central

Kockel, Lutz; Huq, Lutfi M.; Ayyar, Anika; Herold, Emma; MacAlpine, Elle; Logan, Madeline; Savvides, Christina; Kim, Grace E. S.; Chen, Jiapei; Clark, Theresa; Duong, Trang; Fazel-Rezai, Vahid; Havey, Deanna; Han, Samuel; Jagadeesan, Ravi; Kim, Eun Soo Jackie; Lee, Diane; Lombardo, Kaelina; Piyale, Ida; Shi, Hansen; Stahr, Lydia; Tung, Dana; Tayvah, Uriel; Wang, Flora; Wang, Ja-Hon; Xiao, Sarah; Topper, Sydni M.; Park, Sangbin; Rotondo, Cheryl; Rankin, Anne E.; Chisholm, Townley W.; Kim, Seung K.

2016-01-01

Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila. However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals. PMID:27527793

A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research.

PubMed

Kockel, Lutz; Huq, Lutfi M; Ayyar, Anika; Herold, Emma; MacAlpine, Elle; Logan, Madeline; Savvides, Christina; Kim, Grace E S; Chen, Jiapei; Clark, Theresa; Duong, Trang; Fazel-Rezai, Vahid; Havey, Deanna; Han, Samuel; Jagadeesan, Ravi; Kim, Eun Soo Jackie; Lee, Diane; Lombardo, Kaelina; Piyale, Ida; Shi, Hansen; Stahr, Lydia; Tung, Dana; Tayvah, Uriel; Wang, Flora; Wang, Ja-Hon; Xiao, Sarah; Topper, Sydni M; Park, Sangbin; Rotondo, Cheryl; Rankin, Anne E; Chisholm, Townley W; Kim, Seung K

2016-10-13

Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals. Copyright © 2016 Kockel et al.

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea.

PubMed

Huang, Lin-Chien; Thorne, Peter R; Housley, Gary D; Montgomery, Johanna M

2007-08-01

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

PubMed

Myer, A; Wagner, D S; Vivian, J L; Olson, E N; Klein, W H

1997-05-15

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

PubMed Central

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-01-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.

PubMed

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-08-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

PubMed

Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells

PubMed Central

Varga, Balázs V; Hádinger, Nóra; Gócza, Elen; Dulberg, Vered; Demeter, Kornél; Madarász, Emília; Herberth, Balázs

2008-01-01

Background The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. Results In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. Conclusion The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification. PMID:18808670

Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

NASA Astrophysics Data System (ADS)

Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

2016-06-01

A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions

PubMed Central

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

2015-01-01

Dendritic cells (DCs) were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T-cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks, and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization, and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation, and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes, and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types, organs, and species. PMID:26082777

Radiation-Induced Cytogenetic Damage as a Predictor of Cancer Risk for Protons and Fe Ions

NASA Technical Reports Server (NTRS)

Williams, Jerry R.

1999-01-01

We have successfully completed the series of experiments planned for year 1 and the first part of year 2 measuring the induction of chromosome aberrations induced in multiple cell types by three model space radiations: Fe-ions, protons and photons. Most of these data have now been compiled and a significant part subjected to detailed data analyses, although continuing data analysis is an important part of our current and future efforts. These analyses are directed toward defining the patterns of chromosomal damage induction by the three radiations and the extent to which such patterns are dependent on the type of cell irradiated. Our studies show significant differences, both quantitatively and qualitatively, between response of different cell types to these radiations however there is an overall pattern that characterizes each type of radiation in most cell lines. Thus our data identifies general dose-response patterns for each radiation for induction of multiple types of chromosomal aberrations but also identifies significant differences in response between some cell types. Specifically, we observe significant resistance for induction of aberrations in rat mammary epithelial cells when they are irradiated in vivo and assayed in vitro. Further, we have observed some remarkable differences in susceptibility to certain radiation-induced aberrations in cells whose genome has been modulated for two cancer- relevant genes, TP53 and CDKNIA. This data, if confirmed, may represent the first evidence of gene-specific differences in cellular metabolism of damage induced by densely-ionizing radiation that confers substantial sensitivity to protons compared to photons.

Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

PubMed Central

Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

2016-01-01

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

PubMed Central

Maguire, Colin T.; Demarest, Bradley L.; Hill, Jonathon T.; Palmer, James D.; Brothman, Arthur R.; Yost, H. Joseph; Condic, Maureen L.

2013-01-01

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage. PMID:23326421

Distribution of L-type calcium channels in rat thalamic neurones.

PubMed

Budde, T; Munsch, T; Pape, H C

1998-02-01

One major pathway for calcium entry into neurones is through voltage-activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high-voltage activated calcium currents. The use of specific L-type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L-type channels to the total high-voltage-activated calcium current. In order to localize L-type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L-type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L-type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L-type channels in more central somatic regions. The differential localization of L-type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium-dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.

Serial measurement of type-specific human papillomavirus load enables classification of cervical intraepithelial neoplasia lesions according to occurring human papillomavirus-induced pathway.

PubMed

Verhelst, Stefanie; Poppe, Willy A J; Bogers, Johannes J; Depuydt, Christophe E

2017-03-01

This retrospective study examined whether human papillomavirus (HPV) type-specific viral load changes measured in two or three serial cervical smears are predictive for the natural evolution of HPV infections and correlate with histological grades of cervical intraepithelial neoplasia (CIN), allowing triage of HPV-positive women. A cervical histology database was used to select consecutive women with biopsy-proven CIN in 2012 who had at least two liquid-based cytology samples before the diagnosis of CIN. Before performing cytology, 18 different quantitative PCRs allowed HPV type-specific viral load measurement. Changes in HPV-specific load between measurements were assessed by linear regression, with calculation of coefficient of determination (R) and slope. All infections could be classified into one of five categories: (i) clonal progressing process (R≥0.85; positive slope), (ii) simultaneously occurring clonal progressive and transient infection, (iii) clonal regressing process (R≥0.85; negative slope), (iv) serial transient infection with latency [R<0.85; slopes (two points) between 0.0010 and -0.0010 HPV copies/cell/day], and (v) transient productive infection (R<0.85; slope: ±0.0099 HPV copies/cell/day). Three hundred and seven women with CIN were included; 124 had single-type infections and 183 had multiple HPV types. Only with three consecutive measurements could a clonal process be identified in all CIN3 cases. We could clearly demonstrate clonal regressing lesions with a persistent linear decrease in viral load (R≥0.85; -0.003 HPV copies/cell/day) in all CIN categories. Type-specific viral load increase/decrease in three consecutive measurements enabled classification of CIN lesions in clonal HPV-driven transformation (progression/regression) and nonclonal virion-productive (serial transient/transient) processes.

Origin and specification of type II neuroblasts in the Drosophila embryo.

PubMed

Álvarez, José-Andrés; Díaz-Benjumea, Fernando J

2018-04-05

In Drosophila , neural stem cells or neuroblasts (NBs) acquire different identities according to their site of origin in the embryonic neuroectoderm. Their identity determines the number of times they will divide and the types of daughter cells they will generate. All NBs divide asymmetrically, with type I NBs undergoing self-renewal and generating another cell that will divide only once more. By contrast, a small set of NBs in the larval brain, type II NBs, divides differently, undergoing self-renewal and generating an intermediate neural progenitor (INP) that continues to divide asymmetrically several more times, generating larger lineages. In this study, we have analysed the origin of type II NBs and how they are specified. Our results indicate that these cells originate in three distinct clusters in the dorsal protocerebrum during stage 12 of embryonic development. Moreover, it appears that their specification requires the combined action of EGFR signalling and the activity of the related genes buttonhead and Drosophila Sp1 In addition, we also show that the INPs generated in the embryo enter quiescence at the end of embryogenesis, resuming proliferation during the larval stage. © 2018. Published by The Company of Biologists Ltd.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

PubMed Central

Pascal, Laura E; True, Lawrence D; Campbell, David S; Deutsch, Eric W; Risk, Michael; Coleman, Ilsa M; Eichner, Lillian J; Nelson, Peter S; Liu, Alvin Y

2008-01-01

Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers. PMID:18501003

[Electrophysiological study on rat conduit pulmonary artery smooth muscle cells under normoxia and acute hypoxia].

PubMed

Hu, Ying; Zou, Fei; Cai, Chun-Qing; Wu, Hang-Yu; Yun, Hai-Xia; Chen, Yun-Tian; Jin, Guo-En; Ge, Ri-Li

2006-10-25

The present study was designed to investigate the electrophysiological characteristics of rat conduit pulmonary artery smooth muscle cells (PASMCs) and the response to acute hypoxia. PASMCs of the 1st to 2nd order branches in the conduit pulmonary arteries were obtained by enzymatic isolation. The PASMCs were divided into acute hypoxia preconditioned group and normoxia group. Hypoxia solutions were achieved by bubbling with 5% CO2 plus 95% N2 for at least 30 min before cell perfusion. Potassium currents were compared between these two groups using whole-cell patch clamp technique. The total outward current of PASMCs was measured under normoxia condition when iBTX [specific blocking agent of large conductance Ca-activated K(+) (BK(Ca)) channel] and 4-AP [specific blocking agent of delayed rectifier K(+) (K(DR)) channel] were added consequently into bath solution. PASMCs were classified into three types according to their size, shape and electrophysiological characteristics. Type I cells are the smallest with spindle shape, smooth surface and discrete perinuclear bulge. Type II cells show the biggest size with banana-like appearance. Type III cells have the similar size with type I, and present intermediary shape between type I and type II. iBTX had little effect on the total outward current in type I cells, while 4-AP almost completely blocked it. Most of the total outward current in type II cells was inhibited by iBTX, and the remaining was sensitive to 4-AP. In type III cells, the total outward current was sensitive to both iBTX and 4-AP. Acute hypoxia reduced the current in all three types of cells: (1614.8+/-62.5) pA to (892.4+/-33.6) pA for type I cells (P<0.01); (438.3+/-42.8) pA to (277.5+/-44.7) pA for type II cells (P<0.01); (1 042.0+/-37.2) pA to (613.6+/-23.8) pA for type III (P<0.01), and raised the resting membrane potentials (E(m)) in all these three types of cells: (-41.6+/-1.6) mV to (-18.6+/-1.5) mV (P<0.01), (-42.3+/-3.8) mV to (-30.6+/-3.0) mV (P<0.01), (-43.3+/-1.6) mV to (-28.4+/-1.4) mV (P<0.01), for type I, II, III cells, respectively. These results suggest that acute hypoxia suppresses the potassium current and improves the E(m) in PASMCs. These effects may be involved in the modulation of constriction/relaxation of conduit artery under acute hypoxia. Different distribution of K(DR) and BK(Ca) channels in these three types of PASMCs might account for their different constriction/relaxation response to acute hypoxia.

METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

PubMed Central

Escamez, Sacha; André, Domenique; Zhang, Bo; Bollhöner, Benjamin; Pesquet, Edouard; Tuominen, Hannele

2016-01-01

ABSTRACT We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells. PMID:26740571

Myxoma Virus dsRNA Binding Protein M029  Inhibits the Type I IFN-Induced Antiviral State in a  Highly Species-Specific Fashion.

PubMed

Rahman, Masmudur M; McFadden, Grant

2017-02-02

Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN)-induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s) of this species-specific inhibition of  type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA)  binding  protein  that  inhibits  protein  kinase  R (PKR),  can  also  antagonize the type I IFN-induced  antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre-infected with MYXV,  IFN-induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s)  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species-specific fashion.

Autoimmune Neuromuscular Disorders

PubMed Central

Kraker, Jessica; Živković, Saša A

2011-01-01

Autoimmune neuromuscular disorders affecting peripheral nerves, neuromuscular junction or muscle have a wide clinical spectrum with diverse pathogenetic mechanisms. Peripheral nervous system may be targeted in the context of complex immune reactions involving different cytokines, antigen-presenting cells, B cells and different types of T cells. Various immunomodulating and cytotoxic treatments block proliferation or activation of immune cells by different mechanisms attempting to control the response of the immune system and limit target organ injury. Most treatment protocols for autoimmune neuromuscular disorders are based on the use of corticosteroids, intravenous immunoglobulins and plasmapheresis, with cytotoxic agents mostly used as steroid-sparing medications. More recently, development of specific monoclonal antibodies targeting individual cell types allowed a different approach targeting specific immune pathways, but these new treatments are also associated with various adverse effects and their long-term efficacy is still unknown. PMID:22379454

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

PubMed Central

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes.

PubMed

Hanon, E; Hall, S; Taylor, G P; Saito, M; Davis, R; Tanaka, Y; Usuku, K; Osame, M; Weber, J N; Bangham, C R

2000-02-15

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax(11-19)-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo. (Blood. 2000;95:1386-1392)

Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome.

PubMed

Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

2017-02-07

The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach

PubMed Central

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions. PMID:29033978

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach.

PubMed

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions.

Fibrinogen and fibrin based micro and nano scaffolds incorporated with drugs, proteins, cells and genes for therapeutic biomedical applications

PubMed Central

Rajangam, Thanavel; An, Seong Soo A

2013-01-01

Over the past two decades, many types of natural and synthetic polymer-based micro- and nanocarriers, with exciting properties and applications, have been developed for application in various types of tissue regeneration, including bone, cartilage, nerve, blood vessels, and skin. The development of suitable polymers scaffold designs to aid the repair of specific cell types have created diverse and important potentials in tissue restoration. Fibrinogen (Fbg)- and fibrin (Fbn)-based micro- and nanostructures can provide suitable natural matrix environments. Since these primary materials are abundantly available in blood as the main coagulation proteins, they can easily interact with damaged tissues and cells through native biochemical interactions. Fbg- and Fbn-based micro and nanostructures can also be consecutively furnished/or encapsulated and specifically delivered, with multiple growth factors, proteins, and stem cells, in structures designed to aid in specific phases of the tissue regeneration process. The present review has been carried out to demonstrate the progress made with micro and nanoscaffold applications and features a number of applications of Fbg- and Fbn-based carriers in the field of biomaterials, including the delivery of drugs, active biomolecules, cells, and genes, that have been effectively used in tissue engineering and regenerative medicine. PMID:24106425

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

PubMed

Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

2014-10-31

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons

PubMed Central

Pinto, Ashwin; Gillard, Samantha; Moss, Fraser; Whyte, Kathryn; Brust, Paul; Williams, Mark; Stauderman, Ken; Harpold, Michael; Lang, Bethan; Newsom-Davis, John; Bleakman, David; Lodge, David; Boot, John

1998-01-01

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the α1A cell line but not in the α1B, α1C, α1D, and α1E cell lines, establishing the α1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs. PMID:9653186

Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice.

PubMed

Klymiuk, Nikolai; van Buerck, Lelia; Bähr, Andrea; Offers, Monika; Kessler, Barbara; Wuensch, Annegret; Kurome, Mayuko; Thormann, Michael; Lochner, Katharina; Nagashima, Hiroshi; Herbach, Nadja; Wanke, Rüdiger; Seissler, Jochen; Wolf, Eckhard

2012-06-01

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

PubMed

Danner, S; Lohse, M J

1997-07-16

Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

PubMed

Rogers, Danny A; Schor, Nina F

2013-03-10

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

[Occupational lung cancer. A comparison between humans and experimental animals].

PubMed

Adachi, S; Takemoto, K

1987-09-01

Many epidemiological and experimental studies have suggested that the respiratory tract is one of the most sensitive organs to environmental carcinogens. Nevertheless there is little evidence to determine the relationship between a specific environmental carcinogen and a cell type of lung cancer, because the cell types of lung cancer and their relative frequencies are highly complex compared with those of other organs and tissues. In the present paper, occupational lung-cancer characteristics, which are the clearest in the relation between cause and effect in human lung cancers, were reviewed in comparison with the results of animal experiments concerned with occupational lung carcinogens. Through accumulation of histopathological examinations of the lung cancer cases, the following relationships between cause and cell type were conjectured: chromium and squamous cell carcinoma; asbestos and adenocarcinoma; nickel and squamous cell carcinoma; beryllium and small cell carcinoma; bis (chloromethyl) ether and small cell carcinoma; mustard gas and squamous cell or small cell carcinoma; vinyl chloride and large cell or adenocarcinoma; radionuclides and small cell carcinoma. The relation pertaining to arsenic, benzotrichloride and tar could not be conjectured because of insufficient cases and information in the histological diagnosis. On the other hand, the carcinogenicity of these substances in occupational exposure has been confirmed by animal experiments administered intratracheally or by inhalation studies under relatively higher concentration. As a result of recent refinements of inhalation study, all-day and life-span exposure to extremely low concentrations, such as microgram/m3 orders, of certain substances has been possible. The characteristics of lung tumors occurring in these animals are rather different from those of human. For example, in mouse, almost all of the malignant lung tumors developed by carcinogens are adenocarcinomas and it is rare to find the squamous cell carcinoma. Moreover, small cell carcinoma and large cell carcinoma have not known to occur in the lungs of rats and mice. Therefore, future research should focus elucidating the specific relationship between cause and cell type of human lung cancer by means of animal experiments on lung cancer that give attention to the specificities of each experimental animal and the origin of the resultant lung tumor.

Induction of pneumococcal polysaccharide-specific mucosal immune responses by oral immunization.

PubMed

VanCott, J L; Kobayashi, T; Yamamoto, M; Pillai, S; McGhee, J R; Kiyono, H

1996-04-01

Liposome and cholera toxin (CT) are considered to be effective antigen delivery vehicles and adjuvants for mucosal vaccines. The effect of these antigen delivery systems on adjuvant responses to mucosally administered pneumococcal polysaccharide (Pnup) was investigated in this study. Both mucosal (e.g. oral) and systemic (i.p.) immunization of mice with purified preparations of Pnup type 23F induced antigen-specific IgM responses in sera. Interestingly, oral immunization of as little as 10 micrograms of Pnup type 23F was sufficient to induce systemic IgM responses. Pnup-specific IgM antibodies peaked by day 7 and no booster responses were evident after a second dose on day 14. In order to examine whether IgG and IgA Pnup-specific immune responses are induced by mucosal immunization, the mucosal adjuvant CT was mixed with Pnup type 23 as an oral vaccine. Co-oral administration of CT and Pnup type 23F resulted in the induction of Pnup-specific faecal IgA antibodies. These results were confirmed by detecting antigen-specific IgA-spot-forming cells in mononuclear cell suspensions prepared from the intestine of immunized mice. These findings suggest that oral immunization with Pnup in the presence of mucosal adjuvants, such as CT, could induce Pnup-specific IgA responses whereas Pnup alone did not. In an attempt to further enhance antigen-specific antibody responses, Pnup type 23F was encapsulated in liposomes and used as mucosal vaccine. However, immunogenicity of Pnup was not improved.

Mucinous breast carcinoma with tall columnar cells.

PubMed

Tsoukalas, N; Kiakou, M; Tolia, M; Kostakis, I D; Galanopoulos, M; Nakos, G; Tryfonopoulos, D; Kyrgias, G; Koumakis, G

2018-05-01

Mucinous carcinoma of the breast represents 1%-4% of all breast cancers. The World Health Organization classification divides this type of tumour into three different subtypes: mucinous carcinoma, mucinous carcinoma with tall columnar cells (mucinous cystadenocarcinoma and columnar cell mucinous carcinoma) and signet ring cell carcinoma. A 74-year-old woman presented a tumour with inflammatory features in the upper outer quadrant of her left breast, 7 cm in diameter. The core biopsy showed infiltrating ductal carcinoma of no specific type. The tumour-node-metastasis clinical staging was T4cN3M0 (Stage IIIC). She received neoadjuvant chemotherapy, underwent left mastectomy with radical axillary resection and subsequently received radiotherapy and chemotherapy. The histological examination of the surgical specimen revealed two solid tumors in the tail of Spence, which corresponded to adenocarcinoma with high columnar cells. The patient died 16 months after the diagnosis, suffering from pulmonary metastases and anterior chest wall infiltration. A review of the literature revealed only 21 reports of mucinous carcinoma of the breast with tall columnar cells, including our case. This is only the third time that the specific histological type of columnar cell mucinous carcinoma has been reported in the literature.

Applications and mechanisms of immunotherapy in allergic rhinitis and asthma.

PubMed

Kappen, Jasper H; Durham, Stephen R; Veen, Hans In 't; Shamji, Mohamed H

2017-01-01

Clinical and immunologic tolerance are hallmarks of successful allergen immunotherapy (AIT). Clinical benefits such as reduced symptoms, pharmacotherapy intake and improvement of quality of life persist following cessation of treatment. Successful AIT is associated with suppression of allergic inflammatory cells such as mast cells, eosinophils and basophils in target organs. Furthermore, AIT down-regulates type 2 innate lymphoid cells and allergen-specific type 2 T-helper (Th2) cells. The immunologic tolerant state following AIT is associated with the induction of distinct phenotypes of regulatory T-cells (T-regs) including interleukin (IL)-10-, IL-35- and transforming growth factor (TGF)-β- producing T-regs and FoxP3 + T-regs. B-cell responses, including the induction of IL-10 + regulatory B-cells (B-regs) and the production of IgG4-associated blocking antibodies are also induced following successful AIT. These events are associated with the suppression of antigen-specific Th2 responses and delayed immune deviation in favour of Th1 type responses. Insight into the mechanisms of AIT has allowed identification of novel biomarkers with potential to predict the clinical response to AIT and also novel therapeutic strategies for more effective and safer AIT.

Expansion of donor-derived hematopoietic stem cells with PIGA mutation associated with late graft failure after allogeneic stem cell transplantation.

PubMed

Mochizuki, Kanako; Sugimori, Chiharu; Qi, Zhirong; Lu, Xuzhang; Takami, Akiyoshi; Ishiyama, Ken; Kondo, Yukio; Yamazaki, Hirohito; Okumura, Hirokazu; Nakao, Shinji

2008-09-01

A small population of CD55(-)CD59(-) blood cells was detected in a patient who developed donor-type late graft failure after allogeneic stem cell transplantation (SCT) for treatment of aplastic anemia (AA). Chimerism and PIGA gene analyses showed the paroxysmal nocturnal hemoglobinuria (PNH)-type granulocytes to be of a donor-derived stem cell with a thymine insertion in PIGA exon 2. A sensitive mutation-specific polymerase chain reaction (PCR)-based analysis detected the mutation exclusively in DNA derived from the donor bone marrow (BM) cells. The patient responded to immunosuppressive therapy and achieved transfusion independence. The small population of PNH-type cells was undetectable in any of the 50 SCT recipients showing stable engraftment. The de novo development of donor cell-derived AA with a small population of PNH-type cells in this patient supports the concept that glycosyl phosphatidylinositol-anchored protein-deficient stem cells have a survival advantage in the setting of immune-mediated BM injury.

Characterization of TLX expression in neural stem cells and progenitor cells in adult brains.

PubMed

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression. Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

PubMed Central

Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

2012-01-01

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells. PMID:22952666

Context-dependent control of alternative splicing by RNA-binding proteins

PubMed Central

Fu, Xiang-Dong; Ares, Manuel

2015-01-01

Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

β-cell-specific IL-2 therapy increases islet Foxp3+Treg and suppresses type 1 diabetes in NOD mice.

PubMed

Johnson, Mark C; Garland, Alaina L; Nicolson, Sarah C; Li, Chengwen; Samulski, R Jude; Wang, Bo; Tisch, Roland

2013-11-01

Interleukin-2 (IL-2) is a critical cytokine for the homeostasis and function of forkhead box p3-expressing regulatory T cells (Foxp3(+)Tregs). Dysregulation of the IL-2-IL-2 receptor axis is associated with aberrant Foxp3(+)Tregs and T cell-mediated autoimmune diseases such as type 1 diabetes. Treatment with recombinant IL-2 has been reported to enhance Foxp3(+)Tregs and suppress different models of autoimmunity. However, efficacy of IL-2 therapy is dependent on achieving sufficient levels of IL-2 to boost tissue-resident Foxp3(+)Tregs while avoiding the potential toxic effects of systemic IL-2. With this in mind, adeno-associated virus (AAV) vector gene delivery was used to localize IL-2 expression to the islets of NOD mice. Injection of a double-stranded AAV vector encoding IL-2 driven by a mouse insulin promoter (dsAAVmIP-IL2) increased Foxp3(+)Tregs in the islets but not the draining pancreatic lymph nodes. Islet Foxp3(+)Tregs in dsAAVmIP-IL2-treated NOD mice exhibited enhanced fitness marked by increased expression of Bcl-2, proliferation, and suppressor function. In contrast, ectopic IL-2 had no significant effect on conventional islet-infiltrating effector T cells. Notably, β-cell-specific IL-2 expression suppressed late preclinical type 1 diabetes in NOD mice. Collectively, these findings demonstrate that β-cell-specific IL-2 expands an islet-resident Foxp3(+)Tregs pool that effectively suppresses ongoing type 1 diabetes long term.

BIM determines the number of merocytic dendritic cells, a cell type that breaks immune tolerance.

PubMed

Audiger, Cindy; Lesage, Sylvie

2018-05-13

In contrast to conventional dendritic cells (cDC), when merocytic dendritic cells (mcDC) present antigens derived from apoptotic bodies, T-cell anergy is reversed rather than induced, a process that promotes autoimmunity. Interestingly, mcDC are present in higher proportion in type 1 diabetes-prone NOD mice than in autoimmune-resistant B6 and BALB/c mice, and the Insulin-dependent diabetes (Idd)13 locus is linked to mcDC proportion. Therefore, mcDC are notably associated with susceptibility to autoimmune diabetes. To identify which gene determines the proportion and absolute number of mcDC, we undertook a candidate gene approach by selecting relevant candidates within the Idd13 locus. We find that neither β2m nor Sirpa appear to influence the proportion of mcDC. Instead, we show that Bim effectively modulates mcDC number in a hematopoietic-intrinsic manner. We also demonstrate that Bim-deficiency does not impact other cDC subsets and appears to play a specific role in determining the proportion and absolute number of mcDC by promoting their survival. Together, these data demonstrate that Bim specifically modulates the number of mcDC. Identifying factors that facilitate apoptosis of mcDC by increasing BIM activity in a cell type-specific manner may help prevent autoimmunity. © 2018 Australasian Society for Immunology Inc.

The Neonatal Fc Receptor (FcRn) Enhances Human Immunodeficiency Virus Type 1 (HIV-1) Transcytosis across Epithelial Cells

PubMed Central

Gupta, Sandeep; Gach, Johannes S.; Becerra, Juan C.; Phan, Tran B.; Pudney, Jeffrey; Moldoveanu, Zina; Joseph, Sarah B.; Landucci, Gary; Supnet, Medalyn Jude; Ping, Li-Hua; Corti, Davide; Moldt, Brian; Hel, Zdenek; Lanzavecchia, Antonio; Ruprecht, Ruth M.; Burton, Dennis R.; Mestecky, Jiri; Anderson, Deborah J.; Forthal, Donald N.

2013-01-01

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure. PMID:24278022

2011 Rita Schaffer lecture: nanoparticles for intracellular nucleic acid delivery.

PubMed

Green, Jordan J

2012-07-01

Nanoparticles are a promising technology for delivery of new types of therapeutics. A polymer library approach has allowed engineering of polymeric particles that are particularly effective for the delivery of DNA and siRNA to human cells. Certain chemical structural motifs, degradable linkages, hydrophobicity, and biophysical properties are key for successful intracellular delivery. Small differences to biomaterial structure, and especially the type of degradable linkage in the polymers, can be critical for successful delivery of siRNA vs. DNA. Furthermore, subtle changes to biomaterial structure can facilitate cell-type gene delivery specificity between human brain cancer cells and healthy cells as well as between human retinal endothelial cells and epithelial cells. These polymeric nanoparticles are effective for nucleic acid delivery in a broad range of human cell types and have applications to regenerative medicine, ophthalmology, and cancer among many other biomedical research areas.

Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

PubMed

Méndez, E; Kawanishi, T; Clemens, K; Siomi, H; Soldan, S S; Calabresi, P; Brady, J; Jacobson, S

1997-12-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.

Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

PubMed

Kehrmann, Jan; Tatura, Roman; Zeschnigk, Michael; Probst-Kepper, Michael; Geffers, Robert; Steinmann, Joerg; Buer, Jan

2014-07-01

The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells. © 2014 John Wiley & Sons Ltd.

Unique volatolomic signatures of TP53 and KRAS in lung cells

PubMed Central

Davies, M P A; Barash, O; Jeries, R; Peled, N; Ilouze, M; Hyde, R; Marcus, M W; Field, J K; Haick, H

2014-01-01

Background: Volatile organic compounds (VOCs) are potential biomarkers for cancer detection in breath, but it is unclear if they reflect specific mutations. To test this, we have compared human bronchial epithelial cell (HBEC) cell lines carrying the KRASV12 mutation, knockdown of TP53 or both with parental HBEC cells. Methods: VOC from headspace above cultured cells were collected by passive sampling and analysed by thermal desorption gas chromatography mass spectrometry (TD-GC–MS) or sensor array with discriminant factor analysis (DFA). Results: In TD-GC–MS analysis, individual compounds had limited ability to discriminate between cell lines, but by applying DFA analysis combinations of 20 VOCs successfully discriminated between all cell types (accuracies 80–100%, with leave-one-out cross validation). Sensor array detection DFA demonstrated the ability to discriminate samples based on their cell type for all comparisons with accuracies varying between 77% and 93%. Conclusions: Our results demonstrate that minimal genetic changes in bronchial airway cells lead to detectable differences in levels of specific VOCs identified by TD-GC–MS or of patterns of VOCs identified by sensor array output. From the clinical aspect, these results suggest the possibility of breath analysis for detection of minimal genetic changes for earlier diagnosis or for genetic typing of lung cancers. PMID:25051409

Adjuvant-specific regulation of long-term antibody responses by ZBTB20

PubMed Central

Wang, Yinan

2014-01-01

The duration of antibody production by long-lived plasma cells varies with the type of immunization, but the basis for these differences is unknown. We demonstrate that plasma cells formed in response to the same immunogen engage distinct survival programs depending on the adjuvant. After alum-adjuvanted immunization, antigen-specific bone marrow plasma cells deficient in the transcription factor ZBTB20 failed to accumulate over time, leading to a progressive loss of antibody production relative to wild-type controls. Fetal liver reconstitution experiments demonstrated that the requirement for ZBTB20 was B cell intrinsic. No defects were observed in germinal center numbers, affinity maturation, or plasma cell formation or proliferation in ZBTB20-deficient chimeras. However, ZBTB20-deficient plasma cells expressed reduced levels of MCL1 relative to wild-type controls, and transgenic expression of BCL2 increased serum antibody titers. These data indicate a role for ZBTB20 in promoting survival in plasma cells. Strikingly, adjuvants that activate TLR2 and TLR4 restored long-term antibody production in ZBTB20-deficient chimeras through the induction of compensatory survival programs in plasma cells. Thus, distinct lifespans are imprinted in plasma cells as they are formed, depending on the primary activation conditions. The durability of vaccines may accordingly be improved through the selection of appropriate adjuvants. PMID:24711582

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

PubMed

Alikhan, Mir; Song, Joo Y; Sohani, Aliyah R; Moroch, Julien; Plonquet, Anne; Duffield, Amy S; Borowitz, Michael J; Jiang, Liuyan; Bueso-Ramos, Carlos; Inamdar, Kedar; Menon, Madhu P; Gurbuxani, Sandeep; Chan, Ernest; Smith, Sonali M; Nicolae, Alina; Jaffe, Elaine S; Gaulard, Philippe; Venkataraman, Girish

2016-10-01

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

On-chip activation and subsequent detection of individual antigen-specific T cells

PubMed Central

Song, Qing; Han, Qing; Bradshaw, Elizabeth M.; Kent, Sally C.; Raddassi, Khadir; Nilsson, Björn; Nepom, Gerald T.; Hafler, David A.; Love, J. Christopher

2010-01-01

The frequencies of antigen-specific CD4+ T cells in samples of human tissue has been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or Type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC Class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples. PMID:20000848

Two MCAT elements of the SM alpha-actin promoter function differentially in SM vs. non-SM cells.

PubMed

Swartz, E A; Johnson, A D; Owens, G K

1998-08-01

Transcriptional activity of the smooth muscle (SM) alpha-actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM alpha-actin promoter are two MCAT motifs, binding sites for transcription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM alpha-actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significance. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resolution gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts from the different cell types, although no single band was unique to SM. Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple reactive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-related protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCAT elements may contribute to cell type-specific regulation of the SM alpha-actin gene. However, it remains to be determined whether the differential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins or to unique (cell type specific) combinatorial interactions of the MCAT elements with other cis-elements and trans-factors.

A peptide-major histocompatibility complex II chimera favors survival of pancreatic beta-islets grafted in type 1 diabetic mice.

PubMed

Casares, Sofia; Lin, Marvin; Zhang, Nan; Teijaro, John R; Stoica, Cristina; McEvoy, Robert; Farber, Donna L; Bona, Constantin; Brumeanu, Teodor D

2008-06-27

Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.

Breaking self-tolerance during autoimmunity and cancer immunity: Myeloid cells and type I IFN response regulation.

PubMed

Tarbell, Kristin V; Egen, Jackson G

2018-02-02

The generation and regulation of innate immune signals are key determinants of autoimmune pathogenesis. Emerging evidence suggests that parallel processes operating in the setting of solid tumors can similarly determine the balance between tolerance and immunity and ultimately the effectiveness of the antitumor immune response. In both contexts, self-specific responses start with innate immune cell activation that leads to the initial break in self-tolerance, which can be followed by immune response amplification and maturation through innate-adaptive crosstalk, and finally immune-mediated tissue/tumor destruction that can further potentiate inflammation. Of particular importance for these processes is type I IFN, which is induced in response to endogenous ligands, such as self-nucleic acids, and acts on myeloid cells to promote the expansion of autoreactive or tumor-specific T cells and their influx into the target tissue. Evidence from the study of human disease pathophysiology and genetics and mouse models of disease has revealed an extensive and complex network of negative regulatory pathways that has evolved to restrain type I IFN production and activity. Here, we review the overlapping features of self- and tumor-specific immune responses, including the central role that regulators of the type I IFN response and innate immune cell activation play in maintaining tolerance, and discuss how a better understanding of the pathophysiology of autoimmunity can help to identify new approaches to promote immune-mediated tumor destruction. ©2018 Society for Leukocyte Biology.

A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types

PubMed Central

Campbell, John N.; Macosko, Evan Z.; Fenselau, Henning; Pers, Tune H.; Lyubetskaya, Anna; Tenen, Danielle; Goldman, Melissa; Verstegen, Anne M.J.; Resch, Jon M.; McCarroll, Steven A.; Rosen, Evan D.; Lowell, Bradford B.; Tsai, Linus

2017-01-01

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility, and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a novel leptin-sensing neuronal population, multiple AgRP and POMC subtypes, and an orexigenic somatostatin neuronal population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinctly responsive subtypes of AgRP and POMC neurons. Finally, integrating our data with human GWAS data implicates two previously unknown neuronal subtypes in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred. PMID:28166221

Invadopodia formation in blood clots: Not so SLUGgish after all.

PubMed

Knowles, Lynn M; Maranchie, Jodi K; Pilch, Jan

2014-01-01

Blood clotting specifically supports the metastatic dissemination of malignant cells to the lung. We have recently demonstrated that 2 tumor types that are prone to form lung metastases, renal cell carcinoma and soft tissue sarcoma, share specific adhesive mechanisms that support the invasion and colonization of blood clots in the pulmonary vasculature.