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Background: Diagnosis of allergies is mostly based on the patient's clinical history and allergen provocation tests. Determination of specific IgE (sIgE) profiles can be performed to support allergy diagnosis. This is commonly done in vivo by the skin prick test or in vitro with automated systems. Several platforms exist to quantify sIgE levels, but all these methods require access to specific instruments, often delaying the test's results. The IgE luciferase-linked immunosorbent assay (LuLISA) allows bioluminescent quantification of IgE against peanut in microliter samples, but this method awaits extension to other allergens. This study aimed to validate a new method, named LuLIPLEX, for multiplexed bioluminescent detection of sIgE against 20 major molecular allergens., Methods: Quantification of sIgE against 12 recombinant or purified food allergens and eight aeroallergens was performed by LuLIPLEX versus standard IgE detection methods (ImmunoCAP, ISAC, ALEX, or NOVEOS). Multiplexed detection of IgE against these 20 allergens was performed within 45 min using 50 μL of serum, plasma, or whole blood samples., Results: A head-to-head comparison between LuLIPLEX and standard IgE detection methods showed a high correlation among all allergens tested. sIgE profiles in polysensitized subjects could be determined within 45 min in serum and plasma samples, as well as using a single drop of capillary blood., Conclusions: LuLIPLEX is a rapid and sensitive method to quantify sIgE levels against multiple allergens. Given that the test is very fast and can be performed on small and inexpensive luminometers, the IgE LuLIPLEX could allow point-of-care testing of sIgE profiles in allergic subjects., (© 2024 The Author(s). Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: The seroprevalence of SARS-CoV-2 infection in the French population was estimated with a representative, repeated cross-sectional survey based on residual sera from routine blood testing. These data contained no information on infection or vaccination status, thus limiting the ability to detail changes observed in the immunity level of the population over time., Objective: Our aim is to predict the infected or vaccinated status of individuals in the French serosurveillance survey based only on the results of serological assays. Reference data on longitudinal serological profiles of seronegative, infected, and vaccinated individuals from another French cohort were used to build the predictive model., Methods: A model of individual vaccination or infection status with respect to SARS-CoV-2 obtained from a machine learning procedure was proposed based on 3 complementary serological assays. This model was applied to the French nationwide serosurveillance survey from March 2020 to March 2022 to estimate the proportions of the population that were negative, infected, vaccinated, or infected and vaccinated., Results: From February 2021 to March 2022, the estimated percentage of infected and unvaccinated individuals in France increased from 7.5% to 16.8%. During this period, the estimated percentage increased from 3.6% to 45.2% for vaccinated and uninfected individuals and from 2.1% to 29.1% for vaccinated and infected individuals. The decrease in the seronegative population can be largely attributed to vaccination., Conclusions: Combining results from the serosurveillance survey with more complete data from another longitudinal cohort completes the information retrieved from serosurveillance while keeping its protocol simple and easy to implement., (©Stéphanie Bougeard, Adeline Huneau-Salaun, Mikael Attia, Jean-Baptiste Richard, Caroline Demeret, Johnny Platon, Virginie Allain, Stéphane Le Vu, Sophie Goyard, Véronique Gillon, Sibylle Bernard-Stoecklin, Bernadette Crescenzo-Chaigne, Gabrielle Jones, Nicolas Rose, Sylvie van der Werf, Olivier Lantz, Thierry Rose, Harold Noël. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 28.11.2023.)

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cuche, Mastrogiovanni, Juzans, Laude, Ungeheuer, Krentzel, Gariboldi, Scott-Algara, Madec, Goyard, Floch, Chauveau-Le Friec, Lafaye, Renaudat, Le Bidan, Micallef, Schmutz, Mella, Novault, Hasan, Duffy, Di Bartolo and Alcover.)

Introduction: Alcohol use disorder (AUD) is associated with a significant disease burden in France, where alcohol use is deeply rooted in culture. However, the treatment gap is large because of several barriers, including stigmatisation and drinkers' apprehension about total abstinence. However, standardised and evidence-based interventions based on controlled-drinking for people with AUD are lacking. We aimed to assess the effectiveness of a novel community-based French therapeutic patient education (TPE) program for people with AUD named Choizitaconso., Methods: A before-after non-randomised quasi-experimental study, named ETHER, was designed and implemented with people living with AUD, over a period of 6 months. The primary outcome was percentage change in the number of alcohol-related harms experienced. Secondary outcomes were percentage changes in psycho-social patient-reported and community-validated outcomes. Participants in the intervention group (n = 34) benefited from the 10-week TPE program Choizitaconso, while the comparison group (n = 58) received standard care. The Kruskall-Wallis and chi-squared or Fisher's exact tests were used to compare before-after changes in variables in both groups. Linear regression models were used to test for the effect of study group on each outcome and to test for the effect of alcohol consumption as a confounder., Results: At 6 months, all outcomes but one either remained stable or numerically improved in both groups. Internalised stigma significantly improved in the intervention group (p = 0.026) but not in the comparison group (p = 0.207), with a significant group effect (p = 0.014)., Discussion and Conclusions: This study demonstrates the effectiveness of the Choizitaconso TPE program on community-validated outcomes, especially internalised stigma., (© 2023 The Authors. Drug and Alcohol Review published by John Wiley & Sons Australia, Ltd on behalf of Australasian Professional Society on Alcohol and other Drugs.)

Therapeutic patient education (TPE) aims to help people with chronic disease strengthen their empowerment and psychosocial skills to better manage their condition. Although TPE has great potential for addiction medicine, studies on its benefits for reducing alcohol-related harms and increasing empowerment are sparse. We conducted a qualitative study of people with alcohol use disorder (AUD) who participated in the community-based TPE programme Choizitaconso to assess their perceptions and experiences of it. Semi-structured interviews were conducted with 16 participants who had completed the TPE programme at least six months previously. The interviews were transcribed and analysed using a sequential thematic analysis. We identified four general themes: (1) the context of participation: the TPE programme could be a strategy to facilitate engagement in AUD care; (2) representations and experiences: the programme helped to "normalize" participants' relationship with alcohol use by increasing empowerment; (3) TPE strengths: improved knowledge about alcohol use, self-image, weight loss, self-stigma reduction; (4) TPE limitations: difficulty putting learning into practice after the programme ended. The Choizitaconso programme met participants' health and psychosocial expectations, strengthening their empowerment and reducing self-stigma, thereby facilitating engagement in AUD care.

Most viral vectors, including the potently immunogenic lentiviral vectors (LVs), only poorly direct antigens to the MHC-II endosomal pathway and elicit CD4 + T cells. We developed a new generation of LVs encoding antigen-bearing monomers of collectins substituted at their C-terminal domain with the CD40 ligand ectodomain to target and activate antigen-presenting cells. Host cells transduced with such optimized LVs secreted soluble collectin-antigen polymers with the potential to be endocytosed in vivo and reach the MHC-II pathway. In the murine tuberculosis model, such LVs induced efficient MHC-II antigenic presentation and triggered both CD8 + and CD4 + T cells at the systemic and mucosal levels. They also conferred a significant booster effect, consistent with the importance of CD4 + T cells for protection against Mycobacterium tuberculosis. Given the pivotal role of CD4 + T cells in orchestrating innate and adaptive immunity, this strategy could have a broad range of applications in the vaccinology field., (© 2022. The Author(s).)

Adenomatous polyposis coli (APC) is a tumor suppressor whose mutations underlie familial adenomatous polyposis (FAP) and colorectal cancer. Although its role in intestinal epithelial cells is well characterized, APC importance in T cell biology is ill defined. APC regulates cytoskeleton organization, cell polarity, and migration in various cell types. Here, we address whether APC plays a role in T lymphocyte migration. Using a series of cell biology tools, we unveiled that T cells from FAP patients carrying APC mutations display impaired adhesion and motility in constrained environments. We further dissected the cellular mechanisms underpinning these defects in APC-depleted CEM T cell line that recapitulate the phenotype observed in FAP T cells. We found that APC affects T cell motility by modulating integrin-dependent adhesion and cytoskeleton reorganization. Hence, APC mutations in FAP patients not only drive intestinal neoplasms but also impair T cell migration, potentially contributing to inefficient antitumor immunity.

Background: ETHER ("Education THEérapeutique pour la Réduction des dommages en alcoologie" or Therapeutic education for alcohol-related harm reduction) is a multicentre community-based mixed-methods study, which aims to evaluate the effectiveness of the innovative therapeutic patient education (TPE) programme 'Choizitaconso' in a sample of French people with alcohol use disorder (people with AUD). Choizitaconso teaches people with AUD psychosocial skills to help them (re)establish controlled drinking and reduce alcohol-related harms. Recruitment started in October 2019. We present here the protocol of the ETHER study., Methods: ETHER's quantitative component involves a 6-month controlled intervention study which evaluates Choizitaconso's effectiveness by comparing 30 people with AUD following the programme with a control group of 60 people with AUD not enrolled in it, using a questionnaire co-constructed by the research team and members of the people with AUD community. Thirty-four alcohol-related harms are assessed and summed to provide an individual measure of the 'harm burden' from consuming alcohol (primary outcome). Secondary outcomes are anticipated and internalized stigma, alcohol consumption measures, craving for alcohol, coping strategies, health-related quality of life, self-confidence to control or abstain from drinking, treatment self-regulation, anxiety and depressive symptoms, alcohol-related neuropsychological impairments, and capabilities (a measure of wellbeing in adults). Data will be collected in face-to-face and phone-based interviews at enrolment and 6 months later. Linear regression models will be used to assess the impact of the TPE programme on changes in the primary and secondary outcomes, while adjusting for other correlates and confounders. The study's qualitative component comprises semi-structured interviews with 16 people with AUD who have already completed the TPE programme at least 6 months before the interview. Qualitative interviews will be analysed using thematic analysis., Results and Conclusions: ETHER is the first evaluation study of an innovative TPE programme specifically designed to reduce alcohol-related harms and reach controlled drinking in France. The involvement of the people with AUD community in selecting which experienced and perceived alcohol-related harms to measure ensures that ETHER will provide healthcare staff and researchers with a relevant set of harm reduction criteria for use in future research. Finally, ETHER will provide scientific justification for implementing novel alcohol-related harm reduction approaches and champion controlled drinking as a therapeutic goal. Trial registration ClinicalTrials.gov, NCT03954054. Registered 17 May 2019-Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT03954054?cond=alcohol&cntry=FR&city=Marseille&draw=1&rank=1 ., (© 2022. The Author(s).)

Assessment of the cumulative incidence of SARS-CoV-2 infections is critical for monitoring the course and extent of the COVID-19 epidemic. Here, we report estimated seroprevalence in the French population and the proportion of infected individuals who developed neutralising antibodies at three points throughout the first epidemic wave. Testing 11,000 residual specimens for anti-SARS-CoV-2 IgG and neutralising antibodies, we find nationwide seroprevalence of 0.41% (95% CI: 0.05-0.88) mid-March, 4.14% (95% CI: 3.31-4.99) mid-April and 4.93% (95% CI: 4.02-5.89) mid-May 2020. Approximately 70% of seropositive individuals have detectable neutralising antibodies. Infection fatality rate is 0.84% (95% CI: 0.70-1.03) and increases exponentially with age. These results confirm that the nationwide lockdown substantially curbed transmission and that the vast majority of the French population remained susceptible to SARS-CoV-2 in May 2020. Our study shows the progression of the first epidemic wave and provides a framework to inform the ongoing public health response as viral transmission continues globally.

Background: During the COVID-19 lockdown period from March 17 to May 11, 2020, French authorities in Paris and its suburbs relocated people experiencing recurrent homelessness to emergency shelters, hotels, and large venues. A serological survey was done at some of these locations to assess the COVID-19 exposure prevalence in this group., Methods: We did a cross-sectional seroprevalence study at food distribution sites, emergency shelters, and workers' residences that were provided medical services by Médecins Sans Frontières in Paris and Seine-Saint-Denis in the Ile-de-France region. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody seropositivity was detected by Luciferase-Linked Immunosorbent Assay and Pseudo Neutralization Test. Sociodemographic and exposure related information was collected via a verbal questionnaire to analyse risk factors and associations with various COVID-19 symptoms., Findings: Between June 23 and July 2, 2020, 426 (52%) of 818 individuals recruited tested positive in 14 sites. Seroprevalence varied significantly by type of recruitment site (χ 2 p<0·0001), being highest among those living in workers' residences (88·7%, 95% CI 81·8-93·2), followed by emergency shelters (50·5%, 46·3-54·7), and food distribution sites (27·8%, 20·8-35·7). More than two thirds of COVID-19 seropositive individuals (68%, 95% CI 64·2-72·2; 291 of 426) did not report any symptoms during the recall period. COVID-19 seropositivity was strongly associated with overcrowding (medium density: adjusted odds ratio [aOR] 2·7, 95% CI 1·5-5·1, p=0·0020; high density: aOR 3·4, 1·7-6·9, p<0·0001)., Interpretation: These results show high exposure to SARS-CoV-2 with important variations between those at different study sites. Living in crowded conditions was the strongest factor associated with exposure level. This study underscores the importance of providing safe, uncrowded accommodation, alongside adequate testing and public health information., Funding: Médecins Sans Frontières, Epicentre, Institut Pasteur's URGENCE nouveau coronavirus fund, Total Foundation., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In this work on the design and studies of luciferins related to the blue-hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O-acetylated derivatives of imidazo[1,2-b]pyridazin-3(5 H)-one, imidazo[2,1-f][1,2,4]triazin-7(1 H)-one, imidazo[1,2-a]pyridin-3-ol, imidazo[1,2-a]quinoxalin-1(5 H)-one, benzo[f]imidazo[1,2-a]quinoxalin-3(11 H)-one, imidazo[1',2':1,6]pyrazino[2,3-c]quinolin-3(11 H)-one, and 5,11-dihydro-3 H-chromeno[4,3-e]imidazo[1,2-a]pyrazin-3-one is described thanks to extensive use of the Buchwald-Hartwig N-arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues., (© 2020 Wiley-VCH GmbH.)

Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Using high-throughput methods, we assessed herein the serological response against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 1847 participants working in three sites of an institution in Paris conurbation. In May-July 2020, 11% (95% confidence interval [CI]: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S proteins, and 9.5% (95% CI: 8.2-11.0) were neutralizer in pseudo-typed virus assays. The prevalence of seroconversion was 11.6% (95% CI: 10.2-13.2) when considering positivity in at least one assay. In 5% of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic. Anosmia (loss of smell) and ageusia (loss of taste) occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative, suggesting that the true prevalence of infection may have reached 16.6%. In sera obtained 4-8 weeks after the first sampling, anti-N and anti-S IgG titers and neutralization activity in pseudo-virus assay declined by 31%, 17%, and 53%, resulting thus in half-life of 35, 87, and 28 days, respectively. The population studied is representative of active workers in Paris. The short lifespan of the serological systemic responses suggests an underestimation of the true prevalence of infection., (© 2020 Wiley-VCH GmbH.)

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design., Competing Interests: The authors have declared that no competing interests exist.

Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4 + T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite’s virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S&#95;luci&#95;E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite’s virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals., Trial Registration: ClinicalTrials.gov 2013-0047.

Visceral leishmaniasis (VL) is a systemic disease with multifaceted clinical manifestations, including neurological signs, however, the involvement of the nervous system during VL is underestimated. Accordingly, we investigated both brain infection and inflammation in a mouse model of VL. Using bioluminescent Leishmania donovani and real-time 2D-3D imaging tools, we strikingly detected live parasites in the brain, where we observed a compartmentalized dual-phased inflammation pattern: an early phase during the first two weeks post-infection, with the prompt arrival of neutrophils and Ly6C high macrophages in an environment presenting a variety of pro-inflammatory mediators (IFN-γ, IL-1β, CXCL-10/CXCR-3, CCL-7/CCR-2), but with an intense anti-inflammatory response, led by IL-10; and a re-inflammation phase three months later, extremely pro-inflammatory, with novel upregulation of mediators, including IL-1β, TNF-α and MMP-9. These new data give support and corroborate previous studies connecting human and canine VL with neuroinflammation and blood-brain barrier disruption, and conclusively place the brain among the organs affected by this parasite. Altogether, our results provide convincing evidences that Leishmania donovani indeed infects and inflames the brain.

Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein. Two complementary methodologies were combined to follow the infectious process: in vivo imaging using luciferase-expressing Leishmania and real time RT-PCR to quantify both Leishmania and host transcripts. This approach allows us: i) to assess the clinical outcome of visceral leishmaniasis by individual monitoring of hamster weight, ii) to follow the parasite load in several organs by real time analysis of the bioluminescence in vivo and through real time quantitative PCR analysis of amastigote parasite transcript abundance ex vivo, iii) to evaluate the immunological responses triggered by the infection by quantifying hamster transcripts on the same samples and iv) to limit the number of hamsters selected for further analysis. The overall data highlight a correlation between the transcriptional cytokine signatures of hamster affected tissues and the amastigote burden fluctuations, thus providing new insights into the immunopathological process driven by L. donovani in the tissues of mammalian hosts. Finally, they suggest organ-specific immune responses., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Trypanosoma vivax is the most prevalent trypanosome species in African cattle. It is thought to be transmitted by tsetse flies after cyclical development restricted to the vector mouthparts. Here, we investigated the kinetics of T. vivax development in Glossina morsitans morsitans by serial dissections over 1 week to reveal differentiation and proliferation stages. After 3 days, stable numbers of attached epimastigotes were seen proliferating by symmetric division in the cibarium and proboscis, consistent with colonization and maintenance of a parasite population for the remaining lifespan of the tsetse fly. Strikingly, some asymmetrically dividing cells were also observed in proportions compatible with a continuous production of pre- metacyclic trypomastigotes. The involvement of this asymmetric division in T. vivax metacyclogenesis is discussed and compared to other trypanosomatids.

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

DNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived- or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA.

Leishmania/L. major was identified as the etiological agent of human localized cutaneous leishmaniasis. L. major metacyclic promastigotes/MP - the infectious form transmitted by sand flies - were enriched from axenically-derived cultures and inoculated into the dermis of mice (10(3) or 10(4) luciferase-expressing L. major MP inoculated into the C57BL/6 mouse ear pinna). Quantitative readout assays were then combined with imaging of this L. major-hosting skin site and established i) that a specific period of time - depending upon the L. major load used for the inoculation - is required for the L. major-hosting ear pinna to be continuously populated by a balanced population of functional regulatory and effector T lymphocytes, and that ii) this balance coincides with persisting low numbers of amastigotes in more or less rapidly healing skin. This approach also established that, whatever the MP inoculum load delivered to the primary site, the immune processes that reduce the L. major amastigote population also account for concomitant immunity, namely remodelling of the secondary site - where 10(4) MP were delivered - as a clinically silent niche hosting a small L. major population., (© 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

Chagas' disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.

Trypanosoma vivax, one of the leading parasites responsible for Animal African Trypanosomosis (Nagana), is generally cyclically transmitted by Glossina spp. but in areas devoid of the tsetse flies in Africa or in Latin American countries is mechanically transmitted across vertebrate hosts by other haematophagous insects, including tabanids. We followed on from our recent studies on the maintenance of this parasite in vivo and in vitro, and its genetic manipulation, by constructing a West African IL1392 T. vivax strain that stably expresses firefly luciferase and is fully virulent for immunocompetent mice. We report here on a study where murine infection with this strain was monitored in vivo using a non-invasive method. Study findings fully support the use of this strain in the assessment of parasite dynamics in vivo since a strong correlation was found between whole body light emission measured over the course of the infection and parasitemia determined microscopically. In addition, parasitemia and survival rates were very similar for mice infected by the intraperitoneal and sub-cutaneous routes, except for a longer prepatent period following sub-cutaneous inoculation with the parasite. Our results clearly show that when administered by the subcutaneous route, the parasite is retained few days in the skin close to the inoculation site where it multiplies before passing into the bloodstream. Ex vivo bioluminescence analyses of organs isolated from infected mice corroborated our previous histopathological observations with parasite infiltration into spleen, liver and lungs. Finally, our study reinforces previous observations on the presence of the parasite in the central nervous system and consequently the brain commitment in the very late phases of the experimental infection.

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.

Although Candida biofilms have been clearly identified as playing an increasingly important role in human disease, their biology and the reason for their poor susceptibility to antifungal agents remain largely unknown. Over recent years, various models have been developed in order to better characterize Candida biofilms. Here, we describe a number of rapid, inexpensive microtiter-format techniques and strategies which can be used for large-scale screening procedures aimed at identifying genes involved in Candida biofilm formation and/or potential antifungal agents with activity against pathogen cells growing under these conditions. The procedures could also be easily adapted for studying biofilm structures with a range of microscopy techniques.

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.

The fungal pathogen Candida albicans forms therapeutically challenging biofilms on biomedical implants. Using a transcript profiling approach genes whose expression is favoured upon biofilm growth compared with planktonic growth have been previously identified. Knock-out mutants for 38 of these genes were constructed, six of which showed a specific defect in biofilm formation. Among these genes, TYE7 that encodes a transcriptional activator of glycolytic genes in planktonic and biofilm growth conditions was identified as being required for the cohesiveness of biofilms. Biofilms formed by the tye7Δ knock-out mutant showed a hyperfilamentous morphology, and growth of this mutant on solid medium under hypoxia was also associated with the production of hyphae. Similar to TYE7 inactivation, inhibition of glycolysis or ATP synthesis using oxalate or an uncoupler, respectively, triggered morphogenesis when a wild-type strain was grown under hypoxia. These treatments also induced the formation of weakly cohesive, hyper-filamentous biofilms by a wild-type strain. Our data indicate that a hypoxic environment is generated within C. albicans biofilms and that continued biofilm development requires a Tye7p-dependent upregulation of glycolytic genes necessary to adapt to hypoxia and prevent uncontrolled hyphal formation. Thus, adaptation to hypoxia is an integral component of biofilm formation in C. albicans., (© 2011 Blackwell Publishing Ltd.)

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of L-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-wall-located, N- and O-glycosylated proteins. The characterization of C. albicans pga59Delta/pga59Delta, pga62Delta/pga62Delta and pga59Delta/pga59Delta pga62Delta/pga62Delta mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure.

Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Delta/yak1Delta mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Delta/yak1Delta mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1Delta/yak1Delta mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance.

Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete-specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall-degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter-balancing cell wall degradation to avoid cell lysis upon cell separation.

Background: Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized cutaneous leishmaniasis (LCL). Promising ointments are in the final stages of development. One main objective was to help optimize the treatment modalities of human LCL with WR279396, a topical formulation of aminoglycosides that was recently proven to be efficient and safe for use in humans., Methodology/principal Findings: C57BL/6 mice were inoculated in the ear with luciferase transgenic L. major and then treated with WR279396. The treatment period spanned lesion onset, and the evolution of clinical signs and bioluminescent parasite loads could be followed for several months without killing the mice. As judged by clinical healing and a 1.5-3 log parasite load decrease in less than 2 weeks, the 94% efficacy of 10 daily applications of WR279396 in mice was very similar to what had been previously observed in clinical trials. When WR279396 was applied with an occlusive dressing, parasitological and clinical efficacy was significantly increased and no rebound of parasite load was observed. In addition, 5 applications under occlusion were more efficient when done every other day for 10 days than daily for 5 days, showing that length of therapy is a more important determinant of treatment efficacy than the total dose topically applied., Conclusions/significance: Occlusion has a significant adjuvant effect on aminoglycoside ointment therapy of experimental cutaneaous leishmaniasis (CL), a concept that might apply to other antileishmanial or antimicrobial ointments. Generated in a laboratory mouse-based model that closely mimics the course of LCL in humans, our results support a schedule based on discontinuous applications for a few weeks rather than several daily applications for a few days.