Tolerogenic dendritic cells and their role in induction of immune tolerance

Les cellules dendritiques sont des cellules du système immunitaire qui permettent d'instruire les lymphocytes T, autres cellules de ce système, pour mettre en place une réponse immunitaire adaptée afin de combattre et vaincre une infection. Ces cellules dendritiques vont reconnaître des motifs spécifiquement exprimés par des pathogènes par l'intermédiaire de récepteurs exprimés à leur surface. En détectant ces molécules, elles vont s'activer et subir diverses modifications pour pouvoir activer les lymphocytes T. Elles vont alors interagir avec les lymphocytes Τ et transférer les informations nécessaires pour que ces cellules s'activent à leur tour et produisent différentes protéines de façon à éliminer le pathogène. En fonction du type de pathogène, les informations transférées entre les cellules dendritiques et les lymphocytes seront différentes de manière à produire la réponse immunitaire la mieux adaptée pour supprimer l'élément infectieux. Dans le corps, les cellules dendritiques circulent continuellement afin de détecter les éléments étrangers. Quand elles reconnaissent une protéine étrangère, elles la phagocytent, c'est-à-dire qu'elles la mangent afin de pouvoir la présenter aux lymphocytes T. Mais quand elles phagocytent un élément étranger, elles peuvent également prendre des éléments du soi, comme par exemple quand elles phagocytent une cellule infectée par un virus. Les cellules dendritiques doivent alors être capables de différentier les molécules du soi et du non-soi de façon à ne pas induire une réponse en présentant un antigène du soi aux lymphocytes T. D'autant plus que lors de leur développement, les lymphocytes Τ qui sont capables de reconnaître le soi sont éliminés mais ce système n'est pas parfait et donc certains lymphocytes Τ auto-reactifs peuvent se trouver dans le corps. Il existe ainsi d'autres mécanismes en périphérie du site de développement pour inhiber ces lymphocytes Τ auto-reactifs. Ce sont les mécanismes de tolérance. Quand les lymphocytes Τ induisent une réponse aux antigènes du soi, cela résulte à des maladies auto-immunes. Dans mon projet de recherche, nous avons travaillé avec des lignées de cellules dendritiques, c'est-à-dire des cellules dendritiques semblables à celles que l'on peut trouver in vivo mais qui sont immortalisées, elles peuvent donc être cultiver et manipuler in vitro. Nous avons génétiquement modifiées ces lignées cellulaires pour qu'elles expriment des molécules immunosuppressives afin d'étudier comment induire une tolérance immunitaire, c'est-à-dire si l'expression de ces molécules permet d'éviter de générer une réponse immunitaire. Pour cela, nous avons utilisé des modèles murins de tumeurs et de maladies auto-immunes. Nous avons démontré que ces lignées de cellules dendritiques peuvent être un grand outil de recherche pour étudier les bénéfices de différentes molécules immuno-modulatrices afin d'induire une tolérance immunitaire à différents antigènes. - Les cellules dendritiques sont responsables de l'induction des réponses immunitaires adaptatives. Suite à une infection microbienne, les cellules dendritiques s'activent, elles induisent l'expression de molécules de costimulation à leur surface, sécrètent des cytokines et induisent la différentiation des cellules Τ effectrices et mémoires. De plus, les cellules dendritiques ont un rôle important dans l'induction et la maintenance de la tolérance immunitaire au niveau du thymus et en périphérie, en induisant l'anergie, la délétion ou la conversion des cellules Τ naïves en cellules régulatrices. Dans notre groupe, une nouvelle lignée de cellules dendritiques appelée MuTu a été crée par la culture de cellules dendritiques tumorales isolées à partir d'une rate d'une souris transgénique, dans laquelle l'expression de l'oncogène SV40 et du GFP sont sous le contrôle du promoteur CD1 le, et sont ainsi spécifiquement exprimés dans les cellules dendritiques. Ces nouvelles lignées appartiennent au sous-type des cellules dendritiques conventionnelles exprimant CD8a. Elles ont conservé leur capacité d'augmenter l'expression des marqueurs de costimulation à leur surface ainsi que le production de cytokines en réponse à des ligands des récepteurs Toll, ainsi que leur capacité à présenter des antigènes associés aux molécules du complexe majeur d'histocompatibilité (CMH) de classe I ou II pour activer la prolifération et la différentiation des lymphocytes T. En utilisant un système de transduction de lentivirus de seconde génération, ces nouvelles lignées de cellules dendritiques ont été génétiquement modifiées pour sur-exprimer des molécules immunosuppressives (IL-10, TGFP latent, TGFp actif, Activin A, Arginase 1, IDO, B7DC et CTLA4). Ces lignées permettent d'étudier de manière reproductible le rôle de ces molécules potentiellement tolérogènes sur les réponses immunitaires in vitro et in vivo. Ces lignées potentiellement tolérogènes ont été testées, tout d'abord, in vitro, pour leur capacité à inhiber l'activation des cellules dendritiques, à bloquer la prolifération des cellules Τ ou à modifier leur polarisation. Nos résultats démontrent qu'en réponse à une stimulation, la sur-expression des molécules costimulatrices et la sécrétion de molécules pro- inflammatoires est réduite quand les cellules dendritiques sur-expriment l'IL-10. La sur¬expression de TGFp sous sa forme active induit le développement de cellules régulatrices CD4+ CD25+ Foxp3+ et bloque la réponse CD8 cytotoxique tandis que la sur-expression de CTLA4 à la surface des cellules dendritiques inhibe une réponse Thl et induit des lymphocytes Τ anergiques. Ces lignées ont également été utilisées pour étudier l'induction de tolérance in vivo. Tout d'abord, nous avons étudié l'induction de tolérance dans un modèle de développement de tumeurs. En effet, quand les lignées tumorales sont transférées dans les lignées de souris C57BL/6, elles sont reconnues comme du non-soi du à l'expression de l'oncogène SV40 et du GFP et sont éliminées. Ce mécanisme d'élimination a été étudié en utilisant une lignée de cellules dendritiques modifiée pour exprimer la luciférase et qui a permis de suivre le développement des tumeurs par de l'imagerie in vivo dans des animaux vivants. Ces lignées de cellules dendritiques MuTu sont éliminées dans la souris C57BL/6 par les lymphocytes CD8 et l'action cytotoxique de la perforine. Après plusieurs injections, les cellules dendritiques sur-exprimant CTLA4 ou l'actif TGFp peuvent casser cette réponse immunitaire inhérente aux antigènes de la lignée et induire le développement de la tumeur dans la souris C57BL/6. Le développement tumoral a pu être suivi en mesurant la bioluminescence émise par des cellules dendritiques modifiées pour exprimer à la fois l'actif TGFp et la luciférase. Ces tumeurs ont pu se développer grâce à la mise en place d'un microenvironnement suppressif pour échapper à l'immunité en recrutant des cellules myéloïde suppressives, des lymphocytes CD4 régulateurs et en induisant l'expression d'une molécule inhibitrice PD-1 à la surface des lymphocytes CD8 infiltrant la tumeur. Dans un deuxième temps, ces lignées tolérogènes ont également été testées dans un modèle murin de maladies auto-immunes, appelé l'encéphalomyélite auto-immune expérimental (EAE), qui est un modèle pour la sclérose en plaques. L'EAE a été induite dans la souris par le transfert de cellules de ganglions prélevées d'une souris donneuse préalablement immunisée avec une protéine du système nerveux central, la glycoprotéine myéline oligodendrocyte (MOG) émulsifiée dans de l'adjuvant complet de Freund. La vaccination des souris donneuses et receveuses avec les cellules sur-exprimant l'actif TGFP préalablement chargées avec la protéine MOG bloque l'induction de l'EAE. Nous sommes actuellement en train de définir les mécanismes qui permettent de protéger la souris du développement de la maladie auto-immune. Dans cette étude, nous avons ainsi démontré la possibilité d'induire la tolérance in vivo et in vitro à différents antigènes en utilisant nos nouvelles lignées de cellules dendritiques et en les modifiant pour exprimer des molécules immunosuppressives. En conséquence, ces nouvelles lignées de cellules dendritiques représentent un outil pour explorer les bénéfices de différentes molécules ayant des propriétés immuno-modulatrices pour manipuler le système immunitaire vers un phénotype tolérogène. - Dendritic cells (DC) are widely recognized as potent inducers of the adaptive immune responses. Importantly, after microbial infections, DC become activated, induce co- stimulation, secrete cytokines and induce effector and memory Τ cells. DC furthermore play an important role in inducing and maintaining central and peripheral tolerance by inducing anergy, deletion or commitment of antigen-specific naïve Τ cells into regulatory Τ cells. In our group, stable MuTu DC lines were generated by culture of splenic DC tumors from transgenic mice expressing the SV40 large Τ oncogene and the GFP under DC-specific CDllc promoter. These transformed DC belong to the CD8a+ conventional DC subtype and have fully conserved their capacity to upregulate co-stimulatory markers and produce cytokines after activation with Toll Like Receptors-ligands, and to present Major Histocompatibility class-I or MHCII-restricted antigens to activate Τ cell expansion and differentiation. Using a second- generation lentiviral transduction system, these newly developed MuTu DC lines were genetically modified to overexpress immunosuppressive molecules (IL-10, latent TGFp, active TGFp, Activin A, Arginase 1, IDO, B7DC and CTLA4). This allows to reproducibly investigate the role of these potentially tolerogenic molecules on in vitro and in vivo immune responses. These potentially tolerogenic DC were tested in vitro for their ability to inhibit DC activation, to prevent Τ cell proliferation and to modify Τ cell polarization. Our results show that the upregulation of costimulatory molecules and the secretion of pro-inflammatory cytokines were reduced upon stimulation of DC overexpressing IL-10. The overexpression of active TGFP induced the development of CD4+ CD25+ Foxp3+ regulatory Τ cells and inhibited the cytotoxic CD8 Τ cell response as shown by using the OT-II Τ cell system whereas the surface expression of CTLA-4 on DC prevented the Thl response and prompted an anergic antigen-specific Τ cell response. These MuTu DC lines were also used in vivo in order to study the induction of tolerance. First we addressed the induction of tolerance in a model of tumorogenesis. The adoptively transferred tumor cell lines were cleared in C57BL/6 mice due to the foreign expression of SV40 LargeT and GFP. The mechanism of clearance of MuTu DC line into C57BL/6 mice was investigated by using luciferase-expressing DC line. These DC line allowed to follow, by in vivo imaging, the tumor development in living animals and determined that MuTu DC lines were eliminated in a perforin-mediated CD8 Τ cell dependent and CD4 Τ cell independent response. After multiple injections, DC overexpressing CTLA4 or active TGFp could break the immune response to these inherent antigens and induced DC tumorogenesis in wild type mice. The tumor outgrowth in C57BL/6 mice was nicely observed by double-transduced DC lines to express both luciferase and active TGFp. actTGFp-DC tumor was shown to recruit myeloid-derived suppressor cells, induce CD4+ CD25+ Foxp3+ regulatory Τ cells and induce the expression of the inhibitory receptor PD-1 on tumor- infiltrating CD8+ Τ cells in order to escape tumor immunity. Tolerogenic DC lines were also tested for the induction of tolerance in a murine model of autoimmune disease, the experimental autoimmune encephalitis (EAE) model for human multiple sclerosis. EAE was induced in C57BL/6 mice by the adoptive transfer of lymph node cells isolated from donor mice previously immunized by a protein specific to the central nervous system, the myelin oligodendrocyte glycoprotein (MOG) emulsified in the complete freund adjuvant. The vaccination of donor and recipient mice with MOG-pulsed actTGFP-DC line prevented EAE induction. We are still investigating how the active TGFP protect mice from EAE development. We generated tolerogenic DC lines inducing tolerance in vitro and in vivo. Thereby these MuTu DC lines represent a great tool to explore the benefits of various immuno-modulatory molecules to manipulate the immune system toward a tolerogenic phenotype.

Les spirochètes dans tous leurs etats [A short journey through spirochetal infections]

Spirochetal infections present with a variety of clinical syndromes and epidemiologic features. Diagnosis remains challenging for the clinician because of the often protean clinical presentation and poor performance of stan-dard microbiological tests. We present 3 clinical cases, illustrating interesting or unusual features of these infections. First, we present a case of leptospirosis acquired in Switzerland after a rat bite. We then present a case of early disseminated Lyme disease with multiple erythema migrans, lymphopenia, thrombocytopenia and liver enzyme elevation. Finally, we present a case of secondary syphilis in an HIV-positive man, complicated by sensorineural deafness. For each case we highlight and discuss the specific epidemiological, clinical and therapeutic features.

Buccal dental microwear and stable isotopes of El Collado: A mesolithic site from Spain,

This study examines the correlation between buccal dental microwear and stable isotopes. The buccal surface of post-canine teeth casts from El Collado, the largest Mesolithic site in Spain, were examined under Scanning Electron Microscope; photomicrographs were taken from the middle third of the buccal surface with magnification 100X. Only six individuals passed the criteria for buccal dental microwear analysis. The photomicrographs were treated by adobe Photoshop 8.01 to cover an area 0.56 mm² of middle third of buccal surface, the output photomicrographs were digitized using Sigmascan Pro 5 by SPSS. Then the correlation between buccal microwear pattern and stable isotopes of the same individuals, of the previous study of Guixe et al., 2006, was examined using a Pearson test. Statistical analysis revealed that there is no significant correlation between stable isotopes and buccal dental microwear of the people of the Mesolithic site of El Collado. The historical and archaeological documentation suggest that the Mesolithic people tended to consume marine food. Fish-drying techniques were used during the Mesolithic period which allowed the introduction of dust and sand to the fish. These abrasive particles affected the buccal dental microwear pattern, so that no correlation between the isotopes and microwear may be expected. This also suggests that the buccal dental microwear pattern exceeds dietary reconstruction to reconstruct food processing techniques.

Prospective assessment of trocar-specific morbidity in laparoscopy.

BACKGROUND: The purpose of the present study was to challenge the hypothetical advantage of single port laparoscopy (SPL) over conventional laparoscopy by measuring prospectively the morbidity specifically related to conventional trocar sites (TS). METHODS: From November 2010 to December 2011, 300 patients undergoing various laparoscopic procedures were enrolled. Patient, surgery, and trocar characteristics were recorded. We evaluated at three time points (in-hospital and at 1 and 6 months postoperatively) specifically for each TS, pain (Visual Analog Scale), morbidity (infection, hematoma, hernia), and cosmesis (Patient Scar Assessment Score; PSAS). Patients designated their "worst TS," and a composite endpoint "bad TS" was defined to include any adverse outcome at a TS. RESULTS: We analyzed 1,074 TS. Follow-up was >90 %. Pain scores of >3/10 at 1 and 6 months postoperatively, were reported by 3 and 1 % of patients at the 5 mm TS and by 9 and 1 % at the larger TS, respectively (5 mm TS vs larger TS; p = 0.001). Pain was significantly lower for TS located in the lower abdomen than for the upper abdomen or the umbilicus (p = 0.001). The overall complication rate was <1 % and significantly lower for the 5 mm TS (hematoma p = 0.046; infection p = 0.0001). No hernia was found. The overall PSAS score was low and significantly lower for the 5 mm TS (p = 0.0001). Significant predictors of "bad TS" were larger TS (p = 0.001), umbilical position (p = 0.0001), emergency surgery (p = 0.0001), accidental trocar exit (p = 0.022), fascia closure (p = 0.006), and specimen extraction site (p = 0.0001). CONCLUSIONS: Specific trocar morbidity is low and almost negligible for 5 mm trocars. The umbilicus appears to be an unfavorable TS.

Site quality curves for birch stands in north-western Spain

Abstract

Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

Bayesian Markov-chain-Monte-Carlo inversion of time-lapse cross hole ground-penetrating radar data to characterize the vadose zone at the Arrenaes field site, Denmark

The ground-penetrating radar (GPR) geophysical method has the potential to provide valuable information on the hydraulic properties of the vadose zone because of its strong sensitivity to soil water content. In particular, recent evidence has suggested that the stochastic inversion of crosshole GPR traveltime data can allow for a significant reduction in uncertainty regarding subsurface van Genuchten-Mualem (VGM) parameters. Much of the previous work on the stochastic estimation of VGM parameters from crosshole GPR data has considered the case of steady-state infiltration conditions, which represent only a small fraction of practically relevant scenarios. We explored in detail the dynamic infiltration case, specifically examining to what extent time-lapse crosshole GPR traveltimes, measured during a forced infiltration experiment at the Arreneas field site in Denmark, could help to quantify VGM parameters and their uncertainties in a layered medium, as well as the corresponding soil hydraulic properties. We used a Bayesian Markov-chain-Monte-Carlo inversion approach. We first explored the advantages and limitations of this approach with regard to a realistic synthetic example before applying it to field measurements. In our analysis, we also considered different degrees of prior information. Our findings indicate that the stochastic inversion of the time-lapse GPR data does indeed allow for a substantial refinement in the inferred posterior VGM parameter distributions compared with the corresponding priors, which in turn significantly improves knowledge of soil hydraulic properties. Overall, the results obtained clearly demonstrate the value of the information contained in time-lapse GPR data for characterizing vadose zone dynamics.

Prévention des infections chez les voyageurs immunosupprimés [Prevention of infection in immunocompromised travelers].

Every year there are more immunocompromised patients with a better quality of life and, therefore, that travel more frequently. While traveling, patients may be exposed to several infections, such as traveler's diarrhea or malaria, which can be associated with a high rate of complications in this population. An appropriate strategy for the prevention of travel-related infections is essential, including education about hygiene measures, vaccinations and prescription of a tailored antimicrobial prophylaxis/stand-by treatment, according to the type of immunosuppression. Potential drug interactions, particularly between antimalaric and immunosuppressive drugs, must also be considered for decision taking. Collaboration between the general practitioner and the travel medicine and infectious diseases specialists is highly recommended to improve the management of these patients.

Les infections endo-artérielles, complications redoutables des infections à salmonelles non typhi [Endarterial infections, redoubtable complications of non-typhi salmonella infections].

Three cases are reported of salmonella aortitis observed in three men aged 55, 60 and 48 years, the last of whom had a prosthetic aortic valve and ascending aorta. The microorganisms were S. typhi murium, S. paratyphi B, and S. wien. Despite antibiotic treatment two patients died of perforating aortitis. The third patient developed S. wien gastroenteritis a few days after surgical replacement of the aortic valve and the ascending aorta. Five years later he presented with several bacteremic episodes due to S. wien, which recurred despite several courses of cotrimoxazole treatment. He has now been asymptomatic for over one year under prolonged cotrimoxazole treatment. Since vascular infection may occur following non typhi salmonellosis in 5% of patients over 50, or who have underlying endothelial lesions, the question arises as to whether non typhi S. gastroenteritis should be treated with antibiotics in these high risk patients, in contrast to present recommendations.

Letter health consultation : Doty Landfill site, Camanche, Iowa EPA Facility ID: IAD980497556 (2008)

The Doty Landfill encompasses 13 acres of land and is located in the southeastern quarter of Section 29, Township 81 North, Range 6 East, Clinton County, Iowa. The site was used as a landfill for municipal solid waste from 1970 to 1975. In addition, local residents have expressed concern that other chemical-or pesticide waste had been disposed at the site. Previous site investigations had been completed in 1992 and in 2005. In October 2007 water samples from private wells located in the vicinity of the Doty Landfill site were collected and analyzed for dissolved metals. Two of the water samples obtained from drinking water wells contained dissolved arsenic above the US EPA Maximum Contaminant Level (MCL) for arsenic of 10 μg/L (micrograms per liter) or 10 ppb (parts per billion). The water samples in question contained dissolved arsenic at concentrations of 19.3 and 14.9 μg/L or 19.3 and 14.9 ppb.

Health consultation : Review of on-site air monitoring data during the removal action at Le Mars coal gas site, Le Mars coal gas plant Le Mars, Plymouth County, Iowa EPA facility ID: IA0001032556 (2005)

The Iowa Department of Public Health (IDPH), Hazardous Waste Site Health Assessment Program was asked by the US Environmental Protection Agency (EPA) to review a round of air sampling data. The air data was collected and analyzed during a removal action at the Le Mars Coal Gas Site in Le Mars, Iowa. EPA asked IDPH to determine from the air data if additional monitoring is necessary throughout the removal action to protect nearby residents from exposure.

Health consultation : jet gas spill site, Lowell, Henry County, Iowa (2004)

An attorney representing seven property owners in Lowell, Iowa, requested the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program to perform a health consultation for the Jet Gas Spill Site. The attorney asked the IDPH to provide comments concerning the potential and likely health effects to her clients due to exposure from the spill. The specific issues to be addressed are listed in this health consultation. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.

Health consultation : Climbing Hill groundwater contaminiation site, Climbing Hill, Woodbury County, Iowa (2004)

The Iowa Department of Natural Resources (IDNR) asked the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program to perform a health consultation for the Climbing Hill, Iowa, groundwater contamination site. IDNR wants to know if the site poses a public health hazard. The information in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations. Climbing Hill is an unincorporated town in Woodbury County, Iowa, approximately 15 miles southeast of Sioux City. The town has approximately 120 residents. All of the residents and most businesses within the town use private wells to supply their drinking water. The local restaurant has an individual well that is classified as a public water supply system because it has the potential to serve more than 25 people in a day. Several wells in the town have become contaminated with gasoline and diesel fuel leaking from two underground storage tanks. All of the wells are roughly 75–80 feet deep (R. Cardinale, IDNR, Underground Storage Tank Section, personal communication, January 30, 2004).

Health consultation : Hills, Iowa perchlorate groundwater contamination site, Hills, Johnson County, EPA facility ID: IAN 000704368 (2004)

The U. S. Environmental Protection Agency (EPA) Region 7, as well as concerned citizens, asked the Iowa Department of Public Health (IDPH) Hazardous Waste Site Health Assessment Program to perform a health consultation for the Hills, Iowa Perchlorate Groundwater Contamination Site. Specifically, IDPH was asked to determine if EPA’s action of providing bottled water to residents whose private wells had concentrations of greater than 18 μg/L (micrograms per liter) or 18 parts per billion (ppb) perchlorate is protective of public health, and to address some community health concerns. The information included in this health consultation was current at the time of writing. Data that emerges later could alter this document’s conclusions and recommendations.