Dynamic imaging reveals surface exposure of virulent Leishmania amastigotes during pyroptosis of infected macrophages.

Leishmania spp. are obligate intracellular parasites that infect phagocytes, notably macrophages. No information is available on how Leishmania parasites respond to pyroptosis of their host cell, which is known to limit microbial infection. Here, we analyzed the pyroptotic process and the fate of intracellular amastigotes at the single-cell level using high-content real-time imaging. Bone marrow-derived macrophages were infected with virulent Leishmania amazonensis amastigotes and sequentially treated with lipopolysaccharide and ATP to induce pyroptosis. Real-time monitoring identified distinct pyroptotic phases, including rapid decay of the parasitophorous vacuole (PV), progressive cell death and translocation of the luminal PV membrane to the cell surface in 40% of macrophages, resulting in the extracellular exposure of amastigotes that remained anchored to PV membranes. Electron microscopy analyses revealed an exclusive polarized orientation of parasites, with the anterior pole exposed toward the extracellular milieu, and the parasite posterior pole attached to the PV membrane. Exposed parasites retained their full infectivity towards naïve macrophages suggesting that host cell pyroptosis may contribute to parasite dissemination.

From Drug Screening to Target Deconvolution: a Target-Based Drug Discovery Pipeline Using Leishmania Casein Kinase 1 Isoform 2 To Identify Compounds with Antileishmanial Activity.

Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses.

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.

Simultaneous multi-parametric analysis of Leishmania and of its hosting mammal cells: A high content imaging-based method enabling sound drug discovery process.

Leishmaniasis is a vector-borne disease for which only limited therapeutic options are available. The disease is ranked among the six most important tropical infectious diseases and represents the second-largest parasitic killer in the world. The development of new therapies has been hampered by the lack of technologies and methodologies that can be integrated into the complex physiological environment of a cell or organism and adapted to suitable in vitro and in vivo Leishmania models. Recent advances in microscopy imaging offer the possibility to assess the efficacy of potential drug candidates against Leishmania within host cells. This technology allows the simultaneous visualization of relevant phenotypes in parasite and host cells and the quantification of a variety of cellular events. In this review, we present the powerful cellular imaging methodologies that have been developed for drug screening in a biologically relevant context, addressing both high-content and high-throughput needs. Furthermore, we discuss the potential of intra-vital microscopy imaging in the context of the anti-leishmanial drug discovery process.

Pharmacological assessment defines Leishmania donovani casein kinase 1 as a drug target and reveals important functions in parasite viability and intracellular infection.

Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.

[Leishmania epidemiology, diagnosis, chemotherapy and vaccination approaches in the international network of Pasteur Institutes]. / Les leishmanioses vues au travers du réseau international des Instituts Pasteur.

Protozoan parasites of the genus Leishmania generate severe human diseases termed leishmaniases. Due to their frequency and the severity of certain clinical forms, these diseases represent a major public health problem and limit the economic growth in various developing countries. The presence of Pasteur Institutes in countries with endemic leishmaniasis has provided important incentives to develop a strong public health agenda in the Pasteur scientific community with respect to this important disease. A concerted effort is now coordinated through the recently created LeishRIIP platform (www.leishriip.org), which aims to identify synergies and complementary expertise between the eleven members of the international network of Pasteur Institutes working on various aspects of the disease including epidemiology, diagnosis, chemotherapy and vaccination.

A combined luciferase imaging and reverse transcription polymerase chain reaction assay for the study of Leishmania amastigote burden and correlated mouse tissue transcript fluctuations.

Laboratory mice display features of bona fide hosts for parasites such as Leishmania major and Leishmania donovani. Characterizing the amastigote population size fluctuations and the mouse transcript abundance accounting for these fluctuations demands the capacity to record in real time and integrate quantitative multiparametric datasets from the host tissues where these processes occur. To this end, two technologies, luciferase-expressing Leishmania imaging and a very sensitive quantitative analysis of both Leishmania and mouse transcripts, were combined. After the inoculation of either L. major or L. donovani, the amastigote population size fluctuations - increase, plateau and reduction - were monitored by bioluminescence. It allowed a limited number of mice to be selected for further analysis of both mouse and amastigote transcripts using the real-time quantitative polymerase chain reaction assay we set up. The illustrative examples displayed in the present analysis highlight a correlation between the transcriptional signatures displayed by mouse tissues with the amastigote burden fluctuations. We argue that these two combined technologies will have the potential to provide further insights on complex phenotypes driven by Leishmania developmental programs in the tissues of the mammal hosts.

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.

BACKGROUND: Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. RESULTS: We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). CONCLUSIONS: The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.

Going ballistic: Leishmania nuclear subversion of host cell plasticity.

Intracellular parasites have evolved intricate strategies to subvert host cell functions for their own survival. These strategies are particularly damaging to the host if the infection involves immune cells, as illustrated by protozoan parasites of the genus Leishmania that thrive inside mononuclear phagocytic cells, causing devastating immunopathologies. While the impact of Leishmania infection on host cell phenotype and functions has been well documented, the regulatory mechanisms underlying host cell subversion were only recently investigated. Here we summarize the current knowledge on how Leishmania infection affects host nuclear activities and propose thought-provoking new concepts on the reciprocal relationship between epigenetic and transcriptional regulation in host cell phenotypic plasticity, its potential subversion by the intracellular parasite, and its relevance for host-directed therapy.

Trifloxystrobin blocks the growth of Theileria parasites and is a promising drug to treat Buparvaquone resistance.

Theileria parasites are responsible for devastating cattle diseases, causing major economic losses across Africa and Asia. Theileria spp. stand apart from other apicomplexa parasites by their ability to transform host leukocytes into immortalized, hyperproliferating, invasive cells that rapidly kill infected animals. The emergence of resistance to the theilericidal drug Buparvaquone raises the need for new anti-Theileria drugs. We developed a microscopy-based screen to reposition drugs from the open-access Medicines for Malaria Venture (MMV) Pathogen Box. We show that Trifloxystrobin (MMV688754) selectively kills lymphocytes or macrophages infected with Theileria annulata or Theileria parva parasites. Trifloxystrobin treatment reduced parasite load in vitro as effectively as Buparvaquone, with similar effects on host gene expression, cell proliferation and cell cycle. Trifloxystrobin also inhibited parasite differentiation to merozoites (merogony). Trifloxystrobin inhibition of parasite survival is independent of the parasite TaPin1 prolyl isomerase pathway. Furthermore, modeling studies predicted that Trifloxystrobin and Buparvaquone could interact distinctly with parasite Cytochrome B and we show that Trifloxystrobin was still effective against Buparvaquone-resistant cells harboring TaCytB mutations. Our study suggests that Trifloxystrobin could provide an effective alternative to Buparvaquone treatment and represents a promising candidate for future drug development against Theileria spp.

Effects of Structurally Different HDAC Inhibitors against Trypanosoma cruzi, Leishmania, and Schistosoma mansoni.

Neglected tropical diseases (NTDs), including trypanosomiasis, leishmaniasis, and schistosomiasis, result in a significant burden in terms of morbidity and mortality worldwide every year. Current antiparasitic drugs suffer from several limitations such as toxicity, no efficacy toward all of the forms of the parasites' life cycle, and/or induction of resistance. Histone-modifying enzymes play a crucial role in parasite growth and survival; thus, the use of epigenetic drugs has been suggested as a strategy for the treatment of NTDs. We tested structurally different HDACi 1-9, chosen from our in-house library or newly synthesized, against Trypanosoma cruzi, Leishmania spp, and Schistosoma mansoni. Among them, 4 emerged as the most potent against all of the tested parasites, but it was too toxic against host cells, hampering further studies. The retinoic 2'-aminoanilide 8 was less potent than 4 in all parasitic assays, but as its toxicity is considerably lower, it could be the starting structure for further development. In T. cruzi, compound 3 exhibited a single-digit micromolar inhibition of parasite growth combined with moderate toxicity. In S. mansoni, 4's close analogs 17-20 were tested in new transformed schistosomula (NTS) and adult worms displaying high death induction against both parasite forms. Among them, 17 and 19 exhibited very low toxicity in human retinal pigment epithelial (RPE) cells, thus being promising compounds for further optimization.

Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe.

Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

Infectivity and Drug Susceptibility Profiling of Different Leishmania-Host Cell Combinations.

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and >10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani.

Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface. SIGNIFICANCE: Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC-based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the "proteomic reprogramming" of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis.

Targeting Macrophage Histone H3 Modification as a Leishmania Strategy to Dampen the NF-κB/NLRP3-Mediated Inflammatory Response.

Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery.

Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation.

Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells-a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between trans-acting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.

Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes.

BACKGROUND: Mammal macrophages (MPhi) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MPhi. RESULTS: Using BALB/c mouse bone marrow-derived MPhi loaded or not with amastigotes, we analyzed the transcriptional signatures of MPhi 24 h later, when the amastigote population was growing. Total RNA from MPhi cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR). A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02) involving several genes (1.95 to 4.30 fold change values), and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. CONCLUSION: Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MPhi lipid and polyamine pathways. Moreover, these MPhi hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

The Immunomodulatory Effect of IrSPI, a Tick Salivary Gland Serine Protease Inhibitor Involved in Ixodes ricinus Tick Feeding.

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process-taking up to several days-and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs-especially salivary glands-during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

AffyGCQC: a web-based interface to detect outlying genechips with extreme studentized deviate tests.

Affymetrix GeneChip oligonucleotide arrays are dedicated to analyzing gene expression differences across distinct experimental conditions. Data production for such arrays is an elaborate process with many potential sources of variability unrelated to biologically relevant gene expression variations. Therefore, rigorous data quality assessment is fundamental throughout the process for downstream biologically meaningful analyses. We have developed a program named AffyGCQC, which is the acronym for a bioinformatics tool designed to perform Affymetrix GeneChip Quality Control. This program implements a graphical representation of QC metrics recommended by Affymetrix for GeneChip oligonucleotide array technology. Most importantly, it performs extreme studentized deviate statistical tests for the set of arrays being compared in a given experiment, thus providing an objective measure for outlier detection. AffyGCQC has been designed as an easy-to-use Web-based interface (online supplementary information: http://www.transcriptome.ens.fr/AffyGCQC/; contact: affygcqc@biologie.ens.fr).