Simultaneous gene expression profiling in human macrophages infected with Leishmania major parasites using SAGE

<p>Abstract</p> <p>Background</p> <p><it>Leishmania </it>(<it>L</it>) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MΦ) striving to eliminate the pathogen and the parasite struggling for its own survival.</p> <p>To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MΦs (MDM) infection by <it>L. major </it>metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used.</p> <p>Results</p> <p>After extracting mRNA from resting human MΦs, <it>Leishmania</it>-infected human MΦs and <it>L. major </it>parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MΦs' and 3,666 tags to <it>L. major </it>parasites transcripts. We focused on these, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MΦs genes, belonging to key immune response proteins (e.g., IFNγ pathway, S100 and chemokine families) and (ii) a group of <it>Leishmania </it>genes showing a preferential expression at the parasite's intra-cellular developing stage.</p> <p>Conclusion</p> <p>Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in <it>Leishmania</it>-infected human MΦs. The findings presented in this work suggest that the <it>Leishmania </it>parasite modulates key transcripts in human MΦs that may be beneficial for its establishment and survival. Furthermore, these results provide an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes and indicate a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes will be useful in future rounds of data mining and gene annotation.</p

Transcriptome annotation using tandem SAGE tags

Analysis of several million expressed gene signatures (tags) revealed an increasing number of different sequences, largely exceeding that of annotated genes in mammalian genomes. Serial analysis of gene expression (SAGE) can reveal new Poly(A) RNAs transcribed from previously unrecognized chromosomal regions. However, conventional SAGE tags are too short to identify unambiguously unique sites in large genomes. Here, we design a novel strategy with tags anchored on two different restrictions sites of cDNAs. New transcripts are then tentatively defined by the two SAGE tags in tandem and by the spanning sequence read on the genome between these tagged sites. Having developed a new algorithm to locate these tag-delimited genomic sequences (TDGS), we first validated its capacity to recognize known genes and its ability to reveal new transcripts with two SAGE libraries built in parallel from a single RNA sample. Our algorithm proves fast enough to experiment this strategy at a large scale. We then collected and processed the complete sets of human SAGE tags to predict yet unknown transcripts. A cross-validation with tiling arrays data shows that 47% of these TDGS overlap transcriptional active regions. Our method provides a new and complementary approach for complex transcriptome annotation

Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis (HAT). Infection can last for months or even years without major signs or symptoms of infection, but if left untreated, sleeping sickness is always fatal. In the present study, different T. b. gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro. These isolates belong to the homogeneous Group 1 of T. b. gambiense, which is known to induce a chronic infection in humans. In spite of this, these isolates induced infections ranging from chronic to silent in mice, with variations in parasitaemia, mouse lifespan, their ability to invade the CNS and to elicit specific immune responses. In addition, during infection, an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis. The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection, which will be very useful for the treatment and the diagnosis of the disease

Rôle des lymphocytes T Vgamma9Vdelta2 dans l'infection par la bactérie "Brucella"

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis

In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis

BACKGROUND: Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid-laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment. METHODS AND RESULTS: Human scFv (single-chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co-immunoprecipitated with the selected scFv-Fc followed by mass spectrometry for target identification. Near-infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647-labeled scFv-Fc-2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nanoobjects for theranostic applications. One scFv-Fc clone (P3) displayed the highest cross-reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin-3, a β-galactoside-binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti-galectin-3 antibody confirmed this specificity. P3 scFv-Fc-2c specifically targeted atherosclerotic plaques in the Apoe −/− mouse model. CONCLUSIONS: These results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin-3 was proposed as a high-value biomarker for the assessment of coronary and carotid atherosclerosis

Reactivity patterns of immunoreactive invariant trypanosome proteins during BALB/c mice infections.

<p>Four mice were infected with either a low (10³) or a high (10⁶) load of <i>Tbg</i>945b, <i>Tbg</i>1122b, <i>Tbg</i>1166b, <i>Tbg</i>1135b or <i>Tbg</i>1135c isolates and their sera collected at different time points were tested by Western blotting (1/100 dilution) against a strip loaded with recombinant protein: 0.5 µg PFR and ISG75, 1 µg ISG65, ISG64 and TgsGP and 2 µg calflagin. The data are representative of one immunoblot out of 4 mice tested. NI represents the control sera before infection.</p