Characterization of cytokine profiles and double-positive lymphocyte subpopulations in normal bovine lungs

Objective - To characterize cytokine profiles and lymphocyte subpopulations in lung parenchyma and bronchoalveolar lavage (BAL) fluid from normal bovine lungs. Animals - Eight 12- to 18-month-old cattle. Procedure - Cell populations in BAL fluid and collagenase-digested lung parenchyma were analyzed by flow cytometry and monoclonal antibodies. Proportions of total cell populations were determined, using Giemsa-stained cytospots. Distribution of lymphocytes within the lung parenchyma was analyzed by immunohistochemistry, and cytokine mRNA species in the parenchyma were characterized by use of reverse transcriptase-polymerase chain reaction analysis. Results - Cytokine profiles indicated high amounts of mRNA for interleukins 6 and 10 and transforming growth factor β. In the BAL fluid and lung parenchyma, macrophages were the predominant cell type, although the proportion was lower in the parenchyma. Lymphocytes made up approximately 3% of both cell populations. Common to both lung compartments was the predominance of CD2 and γδ T cells over B lymphocytes. There were more CD8 T cells than CD4 T cells in both compartments. The γδ cells made up approximately 9% of the lymphocyte populations. Two-color flow cytometry revealed CD8 γδ T cell and CD8CD5 populations that were unique to BAL fluid. In the BAL fluid and parenchyma, most CD4 and CD8 T cells expressed high amounts of CD44, a characteristic of memory T cells. The γδ T cells were CD44, as were B cells in the lung parenchyma. The B cells from BAL fluid expressed high amounts of CD44. Immunohistologic analysis of lung tissue revealed bronchus-associated lymphoid tissue structures with distinctive germinal center organization of B cells encompassed by CD4 T cells. Conclusions - Results provided normal values for comparison with those of other species and with the bovine respiratory tract response to disease

The paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ~45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ~10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated. Author Summary Top Ciliates are unicellular eukaryotes that rearrange their genomes at every sexual generation when a new somatic macronucleus, responsible for gene expression, develops from a copy of the germline micronucleus. In Paramecium, assembly of a functional somatic genome requires precise excision of interstitial DNA segments, the Internal Eliminated Sequences (IES), involving a domesticated piggyBac transposase, PiggyMac. To study IES origin and evolution, we sequenced germline DNA and identified 45,000 IESs. We found that at least some of these unique-copy elements are decayed Tc1/mariner transposons and that IES insertion is likely an ongoing process. After insertion, elements decay rapidly by accumulation of deletions and substitutions. The 93% of IESs shorter than 150 bp display a remarkable size distribution with a periodicity of 10 bp, the helical repeat of double-stranded DNA, consistent with the idea that evolution has only retained IESs that can form a double-stranded DNA loop during assembly of an excision complex. We propose that the ancient domestication of a piggyBac transposase, which provided a precise excision mechanism, enabled transposons to subsequently invade Paramecium coding sequences, a fraction of the genome that does not usually tolerate parasitic DNA. Citation: Arnaiz O, Mathy N, Baudry C, Malinsky S, Aury J-M, et al. (2012) The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences. PLoS Genet 8(10): e1002984. doi:10.1371/journal.pgen.1002984 Editor: Harmit S. Malik, Fred Hutchinson Cancer Research Center, United States of America Received: March 16, 2012; Accepted: August 9, 2012; Published: October 4, 2012 Copyright: © Arnaiz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the ANR BLAN08-3_310945 “ParaDice,” the ANR 2010 BLAN 1603 “GENOMAC,” a CNRS ATIP-Plus grant to MB (2010–2011), and an “Equipe FRM” grant to EM. The sequencing was carried out at the Genoscope - Centre National de Séquençage (Convention GENOSCOPE-CEA number 128/AP 2007_2008/CNRS number 028666). CDW and AM were supported by Ph.D. fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript