In vitro recognition and impairment of pig islet cells by baboon immune cells: similarity to human cellular reactions.

BACKGROUND: Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. METHODS: Baboon or human peripheral blood mononuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) gamma secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. RESULTS: Baboon PBMCs strongly proliferated in response to PICs (stimulation index [SI]=24.8+/-6.9 [n=8] vs. 23.9+/-3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3<SI<63) and humans (1.8<SI<97). PBMC proliferation was inhibited in baboons and humans by anti-CD4 (% inhibition of SI: 71+/-10% and 75+/-7%, respectively) and anti-DR (75+/-35% and 80+/-6%) monoclonal antibodies (MoAbs) or by depletion of MHC class II+ cells (99+/-1% and 90+/-6%). Blocking by anti-CD8 or anti-CD16 MoAbs was weaker and variable among both animals and humans. IL-10 production by baboon and human PBMCs in response to PICs increased more than IFN-gamma production after 2 days of coculture, but the IL-10/IFN-gamma ratio was inverted after 5 days of coculture. After 7 days (and even after only 2 days) of coculture with baboon (n=8) or human (n=18) PBMCs, basal and glucose-stimulated insulin secretions from PICs were almost completely abolished (P<0.0001). The drop in insulin release could have mainly resulted from lysis of PICs, because the number of PICs decreased by 78% after 7 days of co-incubation with PBMCs. A decrease of insulin release by PBMCs was reproduced with plastic-adherent cells and was abolished by depletion of MHC class II+ cells or by addition of 100 microg/ml gadolinium (which inhibits macrophages), but not by cyclosporine. In baboons, as in humans, insulin release was also decreased after coculture of PICs with enriched T lymphocytes remixed with antigen-presenting cells (APCs). CONCLUSIONS: This study provides the first data on in vitro comparison of baboon and human cell-mediated recognition and impairment of PICs. Proliferation of PBMCs against PICs involves mainly CD4 T cells, with indirect recognition mediated by baboon or human MHC class II+ APCs. The Th2/Th1 profile of cytokines secreted in response to PICs was similar in baboon and human PBMCs. The model based on alteration of insulin secretion indicates that PIC impairment by whole mononuclear cells was strong and rapid and that a crucial role was played by MHC class II+ and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: (1) early and strong direct effect, which is potentially involved in vivo in primary nonfunction of islets aggressed by monocytes and macrophages; and (2) presentation of PIC xenoantigens, which leads to impairment by T lymphocytes possibly involved in in vivo-specific cellular rejection. The mechanisms and intensity of baboon cellular reactions to PICs in vitro were similar to those observed in humans, which suggests that the baboon is a suitable model for the study of cellular mechanisms during preclinical trials of pig islet xenografts

Progress towards the FRIB-EDM3-Frontend: A tool to provide radioactive molecules from isotope harvesting for fundamental symmetry studies

The under-construction FRIB-EDM3-instrument was designed to study polar radioactive molecules (such as RaF) in transparent cryogenic solids by laser spectroscopy. The instrument is divided into a frontend- and a backend section. The frontend accepts an aqueous sample from isotope harvesting and provides a mass-separated molecular ion beam in an ultra-high vacuum environment. In the backend, the ions are guided into alkali-metal vapor and the resulting neutrals are co-deposited in a solid argon matrix to perform laser spectroscopy. This work addresses the frontend of the instrument. The efficient ionization of harvested radioisotopes from aqueous samples is achieved with a spray-ionization method. Subsequently, the molecular ion beam is analyzed by mass-to-charge ratio by a quadrupole mass filter. To verify the feasibility of the approach, numerical simulations with the COMSOL and SIMION packages have been conducted. While the former was applied to study transport in ion funnels, the latter was used to investigate ion beam transmission through the lower pressure sections. Following promising simulation results, a first experimental setup is under construction

Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms

In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0·001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0·01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0·01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1β when mouse plastic-adherent spleen cells were cultured with PIC (P < 0·04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC–mouse cell co-incubations directly altered (P < 0·01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0·01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4+ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0·01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4+ T lymphocytes)

Design and Development of a Speech Intelligibility Test Based on Pseudowords in French: Why and How?

International audiencePurpose: The current intelligibility tests performed on speakers with atypical speech production are limited by the ability of listeners to restore distorted sequences. This results in a measure that is overvalued when compared with the real articulatory performance. In this article, we present a new intelligibility test in order to neutralize the commonly encountered bias in traditional perception-based assessments. We present the construction of the acoustic-phonetic decoding task and its first test during a perceptual judgment test of intelligibility and during a result comparison with a global perceptual evaluation. Method: We developed a very large pseudoword directory including about 90,000 forms that respect French phonotactic constraints. From this directory, we have created lists of pseudowords intended to be recorded for the constitution of the corpus. These lists are established due to an algorithm integrating predefined linguistic constraints and produced by 47 speakers (nine healthy and 38 patients). We then performed a perceptual judgment of intelligibility test with 20 listeners who transcribed these productions. Results: At the end of the data processing stage, we obtained a Perceived Phonological Deviation (PPD) score for each speaker that reflects the average number of features altered per phoneme. We then compared the PPD score with a global intelligibility score derived from a global perceptual assessment of intelligibility and of the alteration severity. Conclusions: The current findings confirm that a speech intelligibility test based on pseudowords in French achieves fine-grained PPD scores, which enables discrimination between patients and healthy speakers. Moreover, the PPD score is related to the global intelligibility score, especially in severity. Further studies are needed to better understand the discrimination power of this intelligibility test based on an acoustic-phonetic decoding task