[CD3-specific antibody-induced T-cell apoptosis and apoptotic cell uptake lead to CD3-specific antibody-induced tolerance]

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Apoptotic cell-mediated suppression of streptococcal cell wall-induced arthritis is associated with alteration of macrophage function and local regulatory T-cell increase: a potential cell-based therapy?

International audienceINTRODUCTION: Experimental streptococcal cell wall (SCW)-induced arthritis is characterized by two successive phases of the disease. The acute phase occurs early and is associated with an inflammatory process and neutrophil infiltration into the synovium. The second chronic phase is related to effector T-cell activation and the dysregulation of macrophage function. Creation of an immunomodulatory environment has been attributed to apoptotic cells themselves, apoptotic cell uptake by phagocytes as well as a less sensibility of phagocytes capturing apoptotic bodies to activation. Therefore we evaluated the potential of apoptotic cell injection to influence the course of inflammation in SCW-induced arthritis in rats. METHODS: Rat apoptotic thymocytes were injected intraperitoneally (2 x 108) in addition to an arthritogenic dose of systemic SCW in LEW female rats. Control rats received SCW immunization and PBS. Rats were then followed for arthritis occurrence and circulating cytokine detection. At sacrifice, regulatory T cells (Tregs) and macrophages were analyzed. RESULTS: Apoptotic cell injection profoundly suppressed joint swelling and destruction typically observed during the acute and chronic phases of SCW-induced arthritis. Synovial inflammatory cell infiltration and bone destruction were also markedly suppressed. Ex vivo experiments revealed reduced levels of TNF in cultures of macrophages from rats challenged with SCW in the presence of apoptotic thymocytes as well as reduced macrophage response to lipopolysaccharide. Moreover, apoptotic cell injection induced higher Foxp3+ Tregs in the lymphoid organs, especially in the draining lymph nodes. CONCLUSIONS: Our data indicate that apoptotic cells modulate macrophage function and result in Treg generation/increase. This may be involved in inhibition of inflammation and amelioration of arthritis. This highlights and confirms previous studies showing that in vivo generation of Tregs using apoptotic cell injection may be a useful tool to prevent and treat inflammatory autoimmune responses

Les fibules gallo-romaines des « Champs des Fougères » à Mandeure (Doubs) : un mobilier cultuel dans l’est de la France

Depuis le début des années 2000, les prospections géophysiques et les fouilles récentes entamées sur la ville antique de Mandeure (Doubs) ont permis la découverte et l’exploration d’un nouveau sanctuaire, localisé au lieu-dit « Champs des Fougères ». Sa fouille, bien que partielle, a permis de recueillir de nombreuses séries de mobiliers. Au total, ce sont 202 fibules ou fragments de fibules qui ont été découverts dans l’ensemble clos du sanctuaire. L’importance numérique du corpus et la qualité de conservation des structures ont favorisé un traitement conjoint de la typo-chronologie ainsi que des modalités de dépôts et de sélection. En vue d’exploiter ces données contextuelles remarquables, l’étude écarte de façon délibérée le mobilier issu des fouilles anciennes. Ainsi, ce site offre l’occasion d’étudier plus largement la place et l’usage des fibules dans un lieu de culte gallo-romain de l’est de la Gaule.Since the early 2000s, geophysical surveys and recent excavations in the ancient city of Mandeure (Doubs) have led to the discovery and exploration of a new sanctuary located in the hamlet of “Champs des Fougères”. Its partial excavation has yielded numerous artifact assemblages. A total of 202 fibulae or fragments has been discovered within the perimeter of the sanctuary. The abundance of artifacts and the good state of preservation of the structures enables a combined analysis of the typo-chronology and the nature and selection of grave goods. To exploit these remarkable data to their fullest, the artifacts collected through early excavations are deliberately left out of this study. This site therefore provides an opportunity to more broadly study the role and use of fibulae in a Gallo-Roman ritual context in eastern Gaul.Seit Beginn der 2000er Jahre werden in Epomanduodurum, dem antiken Mandeure (Departement Doubs), geophysikalische Prospektionen und vor kurzem auch Ausgrabungen vorgenommen. Dabei wurde auf der Flur Champs des Fougères ein neues Heiligtum entdeckt und erforscht. Obwohl wurde es nicht vollständig ergraben wurde, kamen zahlreiche Fundreihen zutage. Insgesamt wurden in diesem Heiligtum 202 Fibeln oder Fibelfragmente entdeckt. Dieser zahlenmäßig bedeutende Korpus und der hervorragende Erhaltungszustand der Strukturen haben eine Untersuchung der Typochronologie sowie der Depot- und Auswahlkriterien begünstigt. Bei der Auswertung dieser bemerkenswerten Befunddaten wurde das Mobiliar aus Altgrabungen von vorne herein ausgeschlossen. Dieses Heiligtum bietet im weiteren Sinn Gelegenheit die Rolle und die Verwendung der Fibeln in einem gallo-römischen Kultplatz Ostgalliens zu untersuchen

Development of a contactless capacitive immunosensor

In the present work, a label-free, contactless and capacitive immunosensor is developed using impedance spectroscopy, in the aim to perform low-cost immunoassays. Chapter 1 puts this work in perspective with some existing techniques, while a presentation of impedance theory used in this work is carried out in chapter 2. In Chapter 3, numerical simulations using a commercial finite element method software is carried out. The response of coplanar and face-to-face designs using an insulating layer is studied with respect to a frequency ranging from 100 Hz to 10 MHz . Two levels of capacitance were observed across the frequency range : the low frequency capacitance created by the insulating layer and the high frequency capacitance created by the solution. These capacitances depend on parameters like the solution conductivity, the distance between the electrodes, the electrodes width or the insulating layer thickness. A dimensionless parameter is defined to evaluate the quality of the geometry at high frequencies. Microchips using a coplanar design are developed in Chapter 4. They are composed of two silver electrodes drilled in a PET sheet by laser photoablation. The design of both holder and of the microchips is optimized to increase as much as possible the signal-to-noise ratio. Bovine Serum Albumin is detected by a variation of the channel conductivity. Chapter 5 introduces the design of a sensor using electrodes made of a mass market aluminium foil. The study of the frequency response of the electrodes led to the creation of a discrete analytical model. The electrodes are then mounted into a holder using a face-to-face or coplanar design. The system is characterized through the variation of several geometrical parameters (height of fluid in the reservoir, electrode surface area, solution conductivity, ...). The coplanar design is also optimized to be able to work in a holder equipped with a fluidic channel. Finally, the ability of the aluminium electrodes based sensor to monitor an adsorption is studied in Chapter 6. The resonance is used to detect the adsorption of proteins like BSA on the electrodes using coplanar and face-to-face designs. The adsorption is found to follow a Langmuir isotherm and an adsorption equilibrium constant is extracted. The second adsorbate layer is detected using a coplanar design, enabling the achievement of a immunoassay

Apoptotic cell infusion treats ongoing collagen-induced arthritis, even in the presence of methotrexate, and is synergic with anti-TNF therapy

International audienceAbstractBackgroundApoptotic cell-based therapies have been proposed to treat chronic inflammatory diseases. The aim of this study was to investigate the effect of intravenous (i.v.) apoptotic cell infusion in ongoing collagen-induced arthritis (CIA) and the interaction of this therapy with other treatments used in rheumatoid arthritis (RA), including methotrexate (MTX) or anti-TNF therapy.MethodsThe effects of i.v. apoptotic cell infusion were evaluated in a CIA mouse model in DBA/1 mice immunized with bovine type II collagen. The number and functions of antigen-presenting cells (APC), regulatory CD4+ T cells (Treg), and circulating anti-collagen auto-antibodies were analyzed in CIA mice.ResultsTreatment of arthritic mice with i.v. apoptotic cell infusion significantly reduced the arthritis clinical score. This therapeutic approach modified T cell responses against the collagen auto-antigen with selective induction of collagen-specific Treg. In addition, we observed that APC from apoptotic-cell-treated animals were resistant to toll-like receptor ligand activation and favored ex vivo Treg induction, indicating APC reprogramming. Apoptotic cell injection-induced arthritis modulation was dependent on transforming growth factor (TGF)-β, as neutralizing anti-TGF-β antibody prevented the effects of apoptotic cells. Methotrexate did not interfere, while anti-TNF therapy was synergic with apoptotic-cell-based therapy.ConclusionOverall, our data demonstrate that apoptotic-cell-based therapy is efficient in treating ongoing CIA, compatible with current RA treatments, and needs to be evaluated in humans in the treatment of RA

L14. Immunomodulatory properties of apoptotic cells.

International audienc

Increased production of soluble CTLA-4 in patients with spondylarthropathies correlates with disease activity

International audienceINTRODUCTION: Spondylarthropathies (SpA) are characterized by abnormal immune responses including T cell activation. Cytotoxic T lymphocyte associated molecule-4 (CTLA-4) is involved in down-regulating immune responses. A soluble form of CTLA-4 (sCTLA-4), resulting from an alternative splicing, has been identified and was found increased in several autoimmune diseases. Here, we evaluated circulating levels of sCTLA-4 as a marker of immune dysregulation in SpA. Intracellular CTLA-4 and levels of CTLA-4 transcript expression in peripheral blood lymphocytes (PBL) were also studied. METHODS: Sera from 165 patients with SpA were evaluated for sCTLA-4 measurements. Results were compared with those from 71 patients with rheumatoid arthritis (RA) and 88 healthy subjects. In 32 patients with SpA, 22 patients with RA and 15 healthy controls, we analyzed the intracellular CTLA-4 expression in CD4+ T cells, CD8+ T cells, activated (HLA-DR+Foxp3-) CD4+ T cells, CD4+ regulatory (CD25+Foxp3+) T cells and in CD3 negative cells by flow cytometry. Expression of the full length (coding for membrane CTLA-4) and spliced form (coding for sCTLA-4) of CTLA-4 transcripts in PBL were analyzed by quantitative real-time polymerase chain reaction (QRT-PCR). RESULTS: High levels of sCTLA-4 were found in the SpA group compared to the RA group and healthy controls (P < 0.0001). Soluble CTLA-4 serum levels strongly correlated with clinical index of disease activity BASDAI (r = 0.42, P < 0.0001) and C-reactive protein (CRP) levels (r = 0.17, P = 0.037). In contrast to RA patients, SpA patients did not exhibit changes in intracellular CTLA-4 expression in the different PBL subsets tested. Finally, the SpA group showed a preferential expression of the spliced CTLA-4 mRNA (P = 0.0014) in PBL. CONCLUSIONS: SpA patients exhibit high levels of circulating sCTLA-4 that may result from an alternative splicing of CTLA-4 transcripts. This may influence immune activation and regulation in SpA

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence.

International audienceBACKGROUND: Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology. METHODS: Chimerism state, circulating anticardiolipin, and antidouble stranded DNA autoantibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. RESULTS: We observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies, and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of autoantibodies. CONCLUSION: Recipient B cell persistence may contribute to the frequency and/or severity of cGVHD after RICT

Electrospray Micromixer Chip for On-Line Derivatization and Kinetic Studies

An electrospray microchip for mass spectrometry comprising an integrated passive mixer to carry out on-chip chemical derivatizations is described. The microchip fabricated using UV-photoablation is composed of two microchannels linked together by a liquid junction. Downstream of this liquid junction, a mixing unit made of parallel oblique grooves is integrated to the microchannel in order to create flow perturbations. Several mixer designs are evaluated. The mixer efficiency is investigated both by fluorescence study and mass spectrometric monitoring of the tagging reaction of cysteinyl peptides with 1,4-benzoquinone. The comparisons with a microchip without a mixing unit and a kinetic model are used to assess the efficiency of the mixer showing tagging kinetics close to that of bulk reactions in an ideally mixed reactor. As an ultimate application, the electrospray micromixer is implemented in a LC-MS workflow. Online derivatization of albumin tryptic peptides after a reversed-phase separation and counting of their cysteines drastically enhance the protein identification

Factors Produced by Macrophages Eliminating Apoptotic Cells Demonstrate Pro-Resolutive Properties and Terminate Ongoing Inflammation

Unresolved inflammation is a common feature in the pathogenesis of chronic inflammatory/autoimmune diseases. The factors produced by macrophages eliminating apoptotic cells during resolution are crucial to terminate inflammation, and for subsequent tissue healing. We demonstrated here that the factors produced by macrophages eliminating apoptotic cells were sufficient to reboot the resolution of inflammation in vivo, and thus definitively terminated ongoing chronic inflammation. These factors were called SuperMApo and revealed pro-resolutive properties and accelerated acute inflammation resolution, as attested by both increased phagocytic capacities of macrophages and enhanced thioglycollate-induced peritonitis resolution. Activated antigen-presenting cells exposed to SuperMApo accelerated their return to homeostasis and demonstrated pro-regulatory T cell properties. In mice with ongoing collagen-induced arthritis, SuperMApo injection resolved and definitively terminated chronic inflammation. The same pro-resolving properties were observed in human settings in addition to xenogeneic colitis and graft-vs.-host disease modulation, highlighting SuperMApo as a new therapeutic opportunity to circumvent inflammatory diseases