Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis

© The Author(s) 2017. This article is an open access publication. Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Purpose. Progress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.Methods. Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. Results. Cell health, morphology and lipid content were comparable (p<0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.Conclusions. A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.Peer reviewedFinal Published versio

Translation regulation of the Human Immunodeficiency Virus type 1

Viruses are among the ultimate conquerors. Even the exploits of Genghis Khan and Alexander the Great seem pale when compared to viral feats. To give but one example, over the last 50 years, the human immunodeficiency virus (HIV) has swept across six continents, now claiming several million lives yearly. Despite sustained intense research efforts, there is still no treatment to eradicate HIV infection.For billions of years, viruses evolved strategies to enter and take control of organisms to generate progeny viruses. Eukaryotic cell viruses developed various means of hijacking the cellular protein synthesis machinery. Understanding these mechanisms opens a unique window of opportunity: that of eventually being able to specifically inhibit virus protein production. In this context, we investigated how HIV-1 translation is regulated. This work initially characterizes an RNA structural element in the HIV-1 leader able to directly recruit the protein synthesis machinery, i.e. an internal ribosome entry site (IRES). This element is capable of driving protein synthesis during the G2/M cell-cycle phase when cap-dependent translation is inhibited.Several virus families use IRESs. IRES-dependent translation usually involves a subset of the factors implicated in cellular protein synthesis. However, toeprinting studies suggest that HIV-1 requires factors different from the canonical translation initiation factors to initiate protein synthesis. HeLa cell protein fractionation studies identified p97, an eIF4G homolog, its apoptotic cleavage product, p86, and a novel protein, ropp120, as putative HIV-1 transactivators. Further testing revealed that these proteins do not directly stimulate HIV-1 leader dependent translation. Experiments also showed that La autoantigen, another likely HIV-1 IRES transactivator candidate, does not directly stimulate HIV-1 dependent translation.The last portion of this work investigates the interplay of the HIV-1 IRES with cap structures, polyA tails and the HIV-1 3'UTR region since these elements are present on the viral genomic RNA. We found that the HIV-1 leader does not synergize nor does it interfere with the translation stimulation mediated by the cap, the polyA and the HIV-1 3'UTR. Data presented herein suggest that the HIV-1 leader is an IRES able to shunt initiation complexes from the cap structure to drive protein synthesis

The Leader of Human Immunodeficiency Virus Type 1 Genomic RNA Harbors an Internal Ribosome Entry Segment That Is Active during the G(2)/M Phase of the Cell Cycle

The 5′ leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA contains highly structured domains involved in key steps of the viral life cycle. These RNA domains inhibit cap-dependent protein synthesis. Here we report that the HIV-1 5′ leader harbors an internal ribosome entry site (IRES) capable of driving protein synthesis during the G(2)/M cell cycle phase in which cap-dependent initiation is inhibited. The HIV-1 IRES was delineated with bicistronic mRNAs in in vitro and ex vivo assays. The HIV-1 leader IRES spans nucleotides 104 to 336 and partially overlaps the major determinants of genomic RNA packaging. These data strongly suggest that, as for HIV-1 transcription, IRES-mediated translation initiation could play an important role in virus replication during virus-induced G(2)/M cell cycle arrest

Two types of human TCR differentially regulate reactivity to self and non-self antigens.

peer reviewedBased on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are reported as disease-associated. Thus, 10%-30% of most frequent cord blood TCRs are associated with common pathogens and autoantigens. TDT-dependent TCRs present distinct structural features and are less shared among subjects. TDT-dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP