Nonclassical CD4+CD49b+ Regulatory T Cells as a Better Alternative to Conventional CD4+CD25+ T Cells To Dampen Arthritis Severity.

Promising immunotherapeutic strategies are emerging to restore tolerance in autoimmune diseases by triggering an increase in the number and/or the function of endogenous regulatory T (Treg) cells, which actively control pathological immune responses. Evidence suggests a remarkable heterogeneity in peripheral Treg cells that warrants their better characterization in terms of phenotype and suppressive function, to determine which subset may be optimally suitable for a given clinical situation. We found that repetitive injections of immature dendritic cells expanded Foxp3-negative CD49b(+) Treg cells that displayed an effector memory phenotype. These expanded Treg cells were isolated ex vivo for transcriptome analysis and found to contain multiple transcripts of the canonical Treg signature shared mainly by CD25(+) but also by other subphenotypes. We characterized the CD49b(+) Treg cell phenotype, underscoring its similarities with the CD25(+) Treg cell phenotype and highlighting some differential expression patterns for several markers, including lymphocyte activation gene 3, KLRG1, CD103, ICOS, CTLA-4, and granzyme B. Comparison of the CD25(+) and CD49b(+) Treg cells' suppressive mechanisms, in vitro and in vivo, revealed the latter's potent suppressive activity, which was partly dependent on IL-10 secretion. Altogether, our results strongly suggest that expression of several canonical Treg cell markers and suppressive function could be Foxp3 independent, and underscore the therapeutic potential of IL-10-secreting CD49b(+) Treg cells in arthritis.

Involvement of angiopoietin-like 4 in matrix remodeling during chondrogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are considered for cartilage engineering given their ability to differentiate into chondrocytes. Chondrogenic differentiation of MSCs is currently triggered by micromass culture in the presence of a member of the TGF-ß superfamily. However, the main constituents of the cartilaginous matrix, aggrecan and type II collagen, are degraded at the end of the differentiation process through induction of matrix metallopeptidase (MMP)13. We hypothesized that MSCs undergoing chondrogenic differentiation produce an intermediate cytokine that triggers this matrix remodeling. Analysis of transcriptomic data identified angiopoietin-like 4 (ANGPTL4) as one of the most strongly up-regulated gene encoding a secreted factor during TGF-ß-induced chondrogenesis. To gain insight into the role of ANGPTL4 during chondrogenesis, we used recombinant ANGPTL4 as well as a RNA interference approach. Addition of exogenous ANGPTL4 during the course of TGF-ß-induced differentiation reduced the mRNA levels of aggrecan and type II collagen, although it increased those of MMP1 and MMP13. Accordingly, deposition of aggrecan and total collagens was diminished, whereas release of MMP1 and MMP13 was increased. Conversely, transfection of MSCs with an siRNA targeting ANGPTL4 prior to induction of chondrogenesis increased expression of type II collagen and aggrecan, whereas it repressed that of MMP1, MMP3, and MMP13. A neutralizing antibody against integrin αVß5, a known receptor for ANGPTL4, mimicked some of the effects observed after siRNA-mediated ANGPTL4 silencing. Our data provide evidence that ANGPTL4 promotes cartilage matrix remodeling by inhibiting expression of its two key components and by up-regulating the level of certain MMPs.

Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays.

BACKGROUND: Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. RESULTS: We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. CONCLUSIONS: MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse.

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.

An adaptation of the LMS method to determine expression variations in profiling data.

One of the major issues in expression profiling analysis still is to outline proper thresholds to determine differential expression, while avoiding false positives. The problem being that the variance is inversely proportional to the log of signal intensities. Aiming to solve this issue, we describe a model, expression variation (EV), based on the LMS method, which allows data normalization and to construct confidence bands of gene expression, fitting cubic spline curves to the Box-Cox transformation. The confidence bands, fitted to the actual variance of the data, include the genes devoid of significant variation, and allow, based on the confidence bandwidth, to calculate EVs. Each outlier is positioned according to the dispersion space (DS) and a P-value is statistically calculated to determine EV. This model results in variance stabilization. Using two Affymetrix-generated datasets, the sets of differentially expressed genes selected using EV and other classical methods were compared. The analysis suggests that EV is more robust on variance stabilization and on selecting differential expression from both rare and strongly expressed genes.

Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis.

Progressive loss of tissue homeostasis is a hallmark of numerous age-related pathologies, including osteoarthritis (OA). Accumulation of senescent chondrocytes in joints contributes to the age-dependent cartilage loss of functions through the production of hypertrophy-associated catabolic matrix-remodeling enzymes and pro-inflammatory cytokines. Here, we evaluated the effects of the secreted variant of the anti-aging hormone α-Klotho on cartilage homeostasis during both cartilage formation and OA development. First, we found that α-Klotho expression was detected during mouse limb development, and transiently expressed during in vitro chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Genome-wide gene array analysis of chondrocytes from OA patients revealed that incubation with recombinant secreted α-Klotho repressed expression of the NOS2 and ZIP8/MMP13 catabolic remodeling axis. Accordingly, α-Klotho expression was reduced in chronically IL1ß-treated chondrocytes and in cartilage of an OA mouse model. Finally, in vivo intra-articular secreted α-Kotho gene transfer delays cartilage degradation in the OA mouse model. Altogether, our results reveal a new tissue homeostatic function for this anti-aging hormone in protecting against OA onset and progression.

Thrombospondin-1 Partly Mediates the Cartilage Protective Effect of Adipose-Derived Mesenchymal Stem Cells in Osteoarthritis.

OBJECTIVE: Assuming that mesenchymal stem cells (MSCs) respond to the osteoarthritic joint environment to exert a chondroprotective effect, we aimed at investigating the molecular response setup by MSCs after priming by osteoarthritic chondrocytes in cocultures. METHODS: We used primary human osteoarthritic chondrocytes and adipose stem cells (ASCs) in mono- and cocultures and performed a high-throughput secretome analysis. Among secreted proteins differentially induced in cocultures, we identified thrombospondin-1 (THBS1) as a potential candidate that could be involved in the chondroprotective effect of ASCs. RESULTS: Secretome analysis revealed significant induction of THBS1 in ASCs/chondrocytes cocultures at mRNA and protein levels. We showed that THBS1 was upregulated at late stages of MSC differentiation toward chondrocytes and that recombinant THBS1 (rTHBS1) exerted a prochondrogenic effect on MSC indicating a role of THBS1 during chondrogenesis. However, compared to control ASCs, siTHBS1-transfected ASCs did not decrease the expression of hypertrophic and inflammatory markers in osteoarthritic chondrocytes, suggesting that THBS1 was not involved in the reversion of osteoarthritic phenotype. Nevertheless, downregulation of THBS1 in ASCs reduced their immunosuppressive activity, which was consistent with the anti-inflammatory role of rTHBS1 on T lymphocytes. THBS1 function was then evaluated in the collagenase-induced OA model by comparing siTHBS1-transfected and control ASCs. The protective effect of ASCs evaluated by histological and histomorphological analysis of cartilage and bone was not seen with siTHBS1-transfected ASCs. CONCLUSION: Our data suggest that THBS1 did not exert a direct protective effect on chondrocytes but might reduce inflammation, subsequently explaining the therapeutic effect of ASCs in OA.

Genomic and expression profiling of chromosome 17 in breast cancer reveals complex patterns of alterations and novel candidate genes.

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.

The transcriptomic analytical level determines the human monocyte-derived macrophage response toward either the infectious agent or the host.

Macrophages exhibit multifunctional activity and play a central role in the response to infectious agents. It is commonly accepted that the plasticity of the response of macrophages depends on the type of stimuli. Here we re-evaluate whether the macrophage response is only dependent on the stimulus. We analyzed the transcriptomic profile of monocyte-derived macrophages (MDMs) that were activated with several pathogens and multiple in vitro-stimulations. The transcriptomic data were normalized using matched-pair analysis. Further analysis showed a clustering association with (i) specific signatures of the infectious agent and its strategy as well as (ii) a preponderance of MDM overall responses related to individuals. Currently, the null hypothesis H0 is that the innate MDM response is globally associated with the pathogen. Our results reveal that the global innate MDM response is intrinsically and predominantly associated with the individual. Thus, the hypothesis is supported or negated depending on the transcriptomic analytical level. AUTHOR SUMMARY: In modern medicine, diagnosis is based on objective criteria. Scientists are focused on the common denominators indicative of an infection. Analytical studies are based on this oriented approach, which defines the null hypothesis H0: the host immune response depends on the stimulus. We observe that the macrophage response to a given pathogen represents <0.4% of the expressed transcripts. The events to which the remaining 99.6% of transcripts are associated remain unclear. We find that 10.3% of the genes modulated during the response to the stimulus are related to the individual. They represent the overall response of the host, which integrates two responses: one associated with the stimulus and the other associated with the individual.

Genetic variability in MCF-7 sublines: evidence of rapid genomic and RNA expression profile modifications.

BACKGROUND: Both phenotypic and cytogenetic variability have been reported for clones of breast carcinoma cell lines but have not been comprehensively studied. Despite this, cell lines such as MCF-7 cells are extensively used as model systems. METHODS: In this work we documented, using CGH and RNA expression profiles, the genetic variability at the genomic and RNA expression levels of MCF-7 cells of different origins. Eight MCF-7 sublines collected from different sources were studied as well as 3 subclones isolated from one of the sublines by limit dilution. RESULTS: MCF-7 sublines showed important differences in copy number alteration (CNA) profiles. Overall numbers of events ranged from 28 to 41. Involved chromosomal regions varied greatly from a subline to another. A total of 62 chromosomal regions were affected by either gains or losses in the 11 sublines studied. We performed a phylogenetic analysis of CGH profiles using maximum parsimony in order to reconstruct the putative filiation of the 11 MCF-7 sublines. The phylogenetic tree obtained showed that the MCF-7 clade was characterized by a restricted set of 8 CNAs and that the most divergent subline occupied the position closest to the common ancestor. Expression profiles of 8 MCF-7 sublines were analyzed along with those of 19 unrelated breast cancer cell lines using home made cDNA arrays comprising 720 genes. Hierarchical clustering analysis of the expression data showed that 7/8 MCF-7 sublines were grouped forming a cluster while the remaining subline clustered with unrelated breast cancer cell lines. These data thus showed that MCF-7 sublines differed at both the genomic and phenotypic levels. CONCLUSIONS: The analysis of CGH profiles of the parent subline and its three subclones supported the heteroclonal nature of MCF-7 cells. This strongly suggested that the genetic plasticity of MCF-7 cells was related to their intrinsic capacity to generate clonal heterogeneity. We propose that MCF-7, and possibly the breast tumor it was derived from, evolved in a node like pattern, rather than according to a linear progression model. Due to their capacity to undergo rapid genetic changes MCF-7 cells could represent an interesting model for genetic evolution of breast tumors.

Midgut expression of immune-related genes in Glossina palpalis gambiensis challenged with Trypanosoma brucei gambiense.

Tsetse flies from the subspecies Glossina morsitans morsitans and Glossina palpalis gambiensis, respectively, transmit Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. The former causes the acute form of sleeping sickness, and the latter provokes the chronic form. Although several articles have reported G. m. morsitans gene expression following trypanosome infection, no comparable investigation has been performed for G. p. gambiensis. This report presents results on the differential expression of immune-related genes in G. p. gambiensis challenged with T. b. gambiense. The aim was to characterize transcriptomic events occurring in the tsetse gut during the parasite establishment step, which is the crucial first step in the parasite development cycle within its vector. The selected genes were chosen from those previously shown to be highly expressed in G. m. morsitans, to allow further comparison of gene expression in both Glossina species. Using quantitative PCR, genes were amplified from the dissected midguts of trypanosome-stimulated, infected, non-infected, and self-cleared flies at three sampling timepoints (3, 10, and 20 days) after a bloodmeal. At the 3-day sampling point, transferrin transcripts were significantly up-regulated in trypanosome-challenged flies versus flies fed on non-infected mice. In self-cleared flies, serpin-2 and thioredoxin peroxidase-3 transcripts were significantly up-regulated 10 days after trypanosome challenge, whereas nitric oxide synthase and chitin-binding protein transcripts were up-regulated after 20 days. Although the expression levels of the other genes were highly variable, the expression of immune-related genes in G. p. gambiensis appears to be a time-dependent process. The possible biological significance of these findings is discussed, and the results are compared with previous reports for G. m. morsitans.

Promyelocytic leukemia zinc-finger induction signs mesenchymal stem cell commitment: identification of a key marker for stemness maintenance?

INTRODUCTION: Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and bone tissue engineering given their ability to differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question about the identification of regulatory pathways determining the differentiation fate of MSCs into chondrocyte or osteoblast. METHODS: Chondrogenesis, osteoblastogenesis, and adipogenesis of human and mouse MSC were induced by using specific inductive culture conditions. Expression of promyelocytic leukemia zinc-finger (PLZF) or differentiation markers in MSCs was determined by RT-qPCR. PLZF-expressing MSC were implanted in a mouse osteochondral defect model and the neotissue was analyzed by routine histology and microcomputed tomography. RESULTS: We found out that PLZF is not expressed in MSCs and its expression at early stages of MSC differentiation is the mark of their commitment toward the three main lineages. PLZF acts as an upstream regulator of both Sox9 and Runx2, and its overexpression in MSC enhances chondrogenesis and osteogenesis while it inhibits adipogenesis. In vivo, implantation of PLZF-expressing MSC in mice with full-thickness osteochondral defects resulted in the formation of a reparative tissue resembling cartilage and bone. CONCLUSIONS: Our findings demonstrate that absence of PLZF is required for stemness maintenance and its expression is an early event at the onset of MSC commitment during the differentiation processes of the three main lineages.

FOXO3A regulation by miRNA-29a Controls chondrogenic differentiation of mesenchymal stem cells and cartilage formation.

Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.

p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis.

INTRODUCTION: Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). METHODS: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. RESULTS: p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. CONCLUSIONS: We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Differential activity of type I interferon subtypes for dendritic cell differentiation.

The type I interferon (IFN) family comprises 15 cytokines (in human 13α, 1ß, 1ω), which exert several cellular functions through binding to a common receptor. Despite initial activation of the same Jak/Stat signalling pathway, the cellular response may differ depending on type I IFN subtype. We investigated the activity of six type I IFN subtypes - IFNα1, α2, α8, α21, ω and ß- to promote the differentiation of dendritic cells (DC). Transcriptome analyses identified two distinct groups, the IFNα/ω-DC and the IFNß-DC. In addition, the expression level of seven chemokines and several cell surface markers characteristic of DC distinguished IFNα-DC and IFNß-DC. These differences are unlikely to impact the efficacy of T cell functional response since IFNα2-DC and IFNß-DC were equipotent in inducing the proliferation and the polarization of allogenic naïve CD4 T cells into Th1 cells and in stimulating autologous antigen specific CD4 or CD8 T cells. Of the functional parameters analysed, the only one that showed a modest differential was the phagocytic uptake of dead cells which was higher for IFNα2-DC.

Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells.

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.

Intertwining threshold settings, biological data and database knowledge to optimize the selection of differentially expressed genes from microarray.

BACKGROUND: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed. METHODOLOGY/PRINCIPAL FINDINGS: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes. CONCLUSIONS/SIGNIFICANCE: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

"Pathogeno-proteomics".

Many scientists working on pathogens (viruses, bacteria, fungi, parasites) are betting heavily on data generated by longitudinal genomic-transcriptomic-proteomic studies to explain biochemical host-vector-pathogen interactions and thus to contribute to disease control. Availability of genome sequences of various organisms, from viruses to complex metazoans, led to the discovery of the functions of the genes themselves. The postgenomic era stimulated the development of proteomic and bioinformatics tools to identify the locations, functions, and interactions of the gene products in tissues and/or cells of living organisms. Because of the diversity of available methods and the level of integration they promote, proteomics tools are potentially able to resolve interesting issues specific not only to host-vector-pathogen interactions in cell immunobiology, but also to ecology and evolution, population biology, and adaptive processes. These new analytical tools, as all new tools, contain pitfalls directly related to experimental design, statistical treatment, and protein identification. Nevertheless, they offer the potency of building large protein-protein interaction networks for in silico analysis of novel biological entities named "interactomes," a way of modeling host-vector-pathogen interactions to define new interference strategies.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.