Immuno-Endocrinologie Cellulaire et Moléculaire

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of beta-cell autoimmunity. Taking advantage of a panel of HLA-A2-restricted beta-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-gamma enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying beta-cell-reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention.

Recombinant adeno-associated virus (rAAV) vector supports long-term transgene expression from skeletal muscle in most mammals, including human. In some instances, the requirement for tight control of the transgene expression is expected. The original tetracycline-dependent system using the rtTA (Dox-on) transactivator displayed a baseline activity in the off state but improved versions are now available and need to be evaluated in a single-rAAV-vector strategy. In the present study we cloned, in three different orientations, the two expression cassettes responsible for doxycycline-mediated transgene regulation and further evaluated the basal and inducible activity of the recently described rtTA2S-S2, rtTA2S-M2, and rtTA2S-M2nls transactivators. Evaluations were conducted in vivo in mice and nonhuman primates using the respective homologous erythropoietin cDNA as a reporter gene because of its sensitive detection by ELISA. The woodchuck hepatitis virus posttranscriptional regulatory element sequence was also introduced to enhance further the stringency with respect to basal activity in the absence of inducer.

The first blood transfusions in humans were xenotransfusions, carried out by Jean-Baptiste Denis beginning in 1667. Richard Lower, Matthäus Purmann and Georges Mercklin also experimented with the use of animal blood for transfusion until this practice was forbidden in 1670, after the death of one of Denis's patients. In the middle of the 19th century, xenotransfusion was rescued from oblivion by the work of Pierre Cyprien Oré. Franz Gesellius and Oscar Hasse fervently defended xenotransfusion, but Emil Ponfick and Leonard Landois stressed the potentially harmful effects of inter-species transfusion from 1874 onward. Xenotransfusion was abandoned completely following the discovery of blood groups by Karl Landsteiner in 1900. From 2000, because of progress in xenotransplantation and the need of blood supply, xenotransfusion is again being considered. Pigs are the best potential donors. The development of alpha-1,3-galactosyltransferase gene-knockout pigs has overcome the first hurdle to xenotransfusion. The main obstacle to porcine red blood cell transfusion is now the cellular response involving macrophages or natural killer cells.

Seventy to ninety percentage of preformed xenoreactive antibodies in human serum bind to the galactose-α(1,3)-galactose Gal epitope, and the creation of Gal knockout (KO) pigs has eliminated hyperacute rejection as a barrier to xenotransplantation. Now other glycan antigens are barriers to move ahead with xenotransplantation, and the N-glycolyl neuraminic acid, Neu5Gc (or Hanganutziu-Deicher antigen), is also a major pig xenoantigen. Humans have anti-Neu5Gc antibodies. Several data indicate a strong immunogenicity of Neu5Gc in humans that may contribute to an important part in antibody-dependent injury to pig xenografts. Pig islets express Neu5Gc, which reacted with diet-derived human antibodies and mice deleted for Neu5Gc reject pancreatic islets from wild-type counterpart. However, Neu5Gc positive heart were not rejected in Neu5Gc KO mice indicating that the role of Neu5Gc-specific antibodies has to be nuanced and depend of the graft situation parameters (organ/tissue, recipient, implication of other glycan antigens). Recently generated Gal/Neu5Gc KO pigs eliminate the expression of Gal and Neu5Gc, and improve the crossmatch of humans with the pig. This review summarizes the current and recent experimental and (pre)clinical data on the Neu5Gc immunogenicity and emphasize of the potential impact of anti-Neu5Gc antibodies in limiting xenotransplantation in humans.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Since being discovered over half a century ago, mesenchymal stem cells (MSCs) have been investigated extensively to characterize their cellular and physiological influences. MSCs have been shown to possess immunosuppressive capacity through inhibiting lymphocyte activation/proliferation and proinflammatory cytokine secretion while simultaneously demonstrating limited allogenic reactivity, which subsequently led to the evaluation of therapeutic feasibility to treat inflammatory diseases. Although regulatory constraints have restricted MSC development pharmacologically, limited clinical studies have shown encouraging results using MSC infusions to treat systemic lupus erythematosus (SLE); but, more trials will have to be performed to conclusively determine the clinical efficacy of MSCs to treat SLE. Moreover, there is some data to suggest that MSCs possess tumorigenic potential and that the immunosuppressive influence can be dramatically affected by both donor variability and ex vivo expansion. Given that recent studies have found that the immunosuppressive effects of MSCs are a result, at least in part, to extracellular vesicle (EV) secretion, the use of MSC-derived EVs has been suggested as a cell-free therapeutic alternative. Despite the positive data observed using EVs isolated from human MSCs to suppress inflammatory responses in vitro and in inhibiting autoimmune disease pathogenesis in preclinical work, there are no studies to date examining EVs from MSCs to treat SLE in humans or animal models. Considering that EVs are not subject to the strict regulatory constraints of stem cell-based pharmacological development and are more readily standardized with regard to industrial scale production and storage, this review outlines the anti-inflammatory biology of MSCs and the scientific evidence supporting the potential use of EVs derived from human MSCs to treat patients with SLE.

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells. CD8(+) T cells have recently been assigned a major role in beta cell injury. Consequently, the identification of autoreactive CD8(+) T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8(+) T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8(+) reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8(+) T cells from newly diagnosed T1D patients (n = 19) but not from CD8(+) T cells of healthy controls (n = 20). Among these, GAD(114-123), GAD(536-545) and IA-2(805-813) were recognized by 53%, 25%, and 42% of T1D patients, respectively.

= 16) of their maintenance energy requirements for 8 weeks. Analysis of similarity revealed a significant difference in gut microbiota β-diversity following the diet compared to week 0 in both groups while α-diversity was lower only in the HF-150 group. Firmicutes/Bacteroidetes ratio was higher in the HF-150 group compared to the HF-100 group at weeks 2 and 8. A reduction in insulin sensitivity was observed over time in the HF150 group. Neither endotoxemia nor inflammation was observed in either group, did not find supporting data for the hypothesis that the microbiota is involved in the decline of insulin sensitivity through metabolic endotoxemia and low-grade inflammation. Colonic permeability was increased at week 4 in both groups and returned to initial levels at week 8, and was associated with modifications to the expression of genes involved in colonic barrier function. The increase in intestinal permeability may have been caused by the altered intestinal microbiota and increased expression of genes encoding tight junction proteins might indicate a compensatory mechanism to restore normal permeability. Although simultaneous changes to the microbiota, barrier permeability, inflammatory, and metabolic status have not been observed, such a causal link cannot be excluded in dogs overfed on a HFD. Further studies are necessary to better understand the link between HFD, intestinal microbiota and the host.

Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell’s vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting towards EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.

Heme oxygenases (HOs) are the rate-limiting intracellular enzymes that degrade heme into carbon monoxide (CO), biliverdin and free divalent iron. Among HOs, HO-1 is the only isoform that is highly inducible in response to numerous stress factors and proinflammatory cytokines. This enzyme has shown cytoprotective, antioxidant and anti-inflammatory properties. Moreover, HO-1 and, in particular, CO also have tolerogenic actions in adaptive immune responses. HO-1 can provide immunosuppression through its expression by regulatory T cells or antigen-presenting cells. The physiological importance of HO-1 has been demonstrated in both mice and humans, and modulation of HO-1 expression has therapeutic effects in a variety of disorders involving inflammation and immune responses, including organ transplantation and cancer. Consistently, upregulation of the HO-1 pathway has a significant protective effect against spontaneous or induced autoimmune diseases, allergy and can be beneficial to graft survival. However, HO-1 may also play a role in tumorigenesis by lowering antitumor innate immune responses that control tumor growth or reduce tumor expansion. Thus, controlling HO-1 expression may be of great interest in immune intervention protocols where tolerance is desirable, such as in transplantation, or where enhanced immunogenicity is needed in the case of cancer.

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.

The composition of soft tissues in mammals can be simplified as approximately 60–65 % water, 16 % protein, 16 % fat, 1 % carbohydrate, and trillions of cells. This report brings together unpublished results from a collaborative efforts of 10 research groups over the past five years, all dedicated to producing mammalian tissues through extrusion-based bioprinting. What unified these studies was a common approach, with a shared bioink composition consisting of gelatin, alginate, and fibrinogen, and a post-printing consolidation strategy involving transglutaminase crosslinking, calcium chelation, and thrombin-mediated fibrin production. The range of Young’s moduli achievable was 0.17–105 kPa, perfectly align with of tissue properties. By consolidating the findings of these studies, it was conclusively demonstrated that bioprinting and culturing all 19 cells tested from 14 different organs was indeed achievable. These remarkable outcomes were attributed not only to the bio-inspired nature of the common bioink but also to its unique rheological properties, such as significant shear-thinning and a sufficiently high static yield stress. The majority of these cells exhibited behaviours consistent with their natural in vivo environments. Clearly identifiable microstructures and organizations showcased intricate morphogenesis mechanisms resulting in the formation of micro-tubules, micro-vessels, and micro-acini. It is now evident that microextrusion bioprinting, especially when using bio-inspired bioink formulations, represents a promising avenue for generating a wide range of mammalian soft tissues.

Hair cortisol is a stress indicator and could be used to assess the pigs’ exposure to stressors in the weeks/months prior to non-invasive hair sampling. The main aim of this study was to describe the hair cortisol concentration (HCC) variability between individuals within a batch, between farms and between batches within a farm. The secondary aim was to determine how the number of sampled pigs influences the characterization of HCC within a batch. Twenty farrow-to-finish pig farms were recruited considering the diversity of their management practices and health status (data collected). Hair was sampled in two separate batches, 8 months apart. The necks of 24 finishing pigs were clipped per batch the week prior to slaughter. To describe the variability in HCC, an analysis of the variance model was run with three explanatory variables (batch, farm and their interaction). To identify farm clusters, a principal component analysis followed by a hierarchical clustering was carried out with four active variables (means and standard deviations of the two batches per farm) and 17 supplementary variables (management practices, herd health data). We determined how the number of sampled pigs influenced the characterization of HCC within a batch by selecting subsamples of the results. HCC ranged from 0.4 to 121.6 pg/mg, with a mean of 25.9 ± 16.2 pg/mg. The variability in HCC was mainly explained by differences between pigs (57%), then between farms (24%), between batches within the same farm (16%) and between batches (3%). Three clusters of farms were identified: low homogeneous concentrations ( n = 3 farms), heterogeneous concentrations with either higher ( n = 7) or lower ( n = 10) HCC in batch 2 than in batch 1. The diversity of management practices and health statuses allowed to discuss hypotheses explaining the HCC variations observed. We highlighted the need to sample more than 24 pigs to characterize HCC in a pig batch. HCC differences between batches on six farms suggest sampling pigs in more than one batch to describe the HCC at the farm level. HCC variations described here confirm the need to study its links with exposure of pigs to stressors.

Nowadays, most pigs are raised indoors, on intensive farms providing a poor environment. In these conditions, the risk of the occurrence of damaging behaviours is high, with dramatic consequences for animal health and welfare as well as economic losses for farmers. Early-life conditions may predispose individuals to develop damaging behaviours later in life. In contrast, reinforcing affiliative behaviours between piglets before weaning might help to prevent tail-biting episodes. In this field study, we aimed at improving early-life conditions of piglets on a commercial farm by completely suppressing painful procedures and staggering their exposure to weaning stress factors. The alternative early-life management strategy combined housing in free-farrowing pens with temporary crating of the sow, socialisation during the lactation period with whole-life maintenance of the hierarchical groups, and delayed transfer to the postweaning room after sow removal. Control conditions included birth in farrowing crates, tail docking, absence of socialisation during the lactation period, abrupt weaning with immediate transfer to the postweaning room and mixing with non-littermates. We evaluated the health, welfare, and performance of alternatively raised pigs (n = 80) as compared to controls (n = 75). Visits were made throughout the lifespan of individuals to evaluate their growth and health status. Body and tail lesions were scored as proxy measures of aggressiveness and impaired welfare. Blood and bristle samples were periodically collected to evaluate stress, inflammation and immune competence. While the whole-life performance of pigs was similar among groups, the alternative early-life conditions prevented the growth slowdown usually observed after weaning. In addition, alternatively raised pigs displayed more neutrophils, eosinophils and monocytes the day after weaning, as well as higher C-Reactive Protein levels. One week later, their monocytes displayed greater phagocytic capacity. Altogether, these data suggest an enhanced innate immune competence for alternatively raised pigs around weaning. Piglets reared under alternative conditions also exhibited fewer and less severe body lesions than standard pigs, one week after weaning. In contrast, they showed more tail lesions on days 36 and 66 associated with greater levels of acute phase proteins (C-Reactive Protein and haptoglobin). To conclude, alternative early-life management better prepared piglets for weaning. However, the whole-life maintenance of early-established social groups was not sufficient to prevent the occurrence of damaging behaviours in undocked pigs.

Abstract Background Multiple antigenic stimulations are crucial to immune system training during early post-natal life. These stimulations can be either due to commensals, which accounts for the acquisition and maintenance of tolerance, or to pathogens, which triggers immunity. In pig, only few works previously explored the influence of natural exposition to pathogens upon immune competence. We propose herein the results of a multicentric, field study, conducted on 265 piglets exposed to contrasted pathogen levels in their living environment. Piglets were housed in 15 different commercial farms, sorted in two groups, low (HS LOW )- and high (HS HIGH )-health status farms, depending on their recurrent exposition to five common swine pathogens. Results Using animal-based measures, we compared the immune competence and growth performances of HS LOW and HS HIGH pigs around weaning. As expected, we observed a rise in the number of circulating leucocytes with age, which affected different cell populations. Monocyte, antigen-experienced and cytotoxic lymphocyte subpopulation counts were higher in piglets reared in HS LOW farms as compared to their HS HIGH homologs. Also, the age-dependent evolution in γ δ T cell and neutrophil counts was significantly affected by the health status. With age, circulating IFNα level decreased and IgM level increased while being greater in HS LOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HS LOW piglets were more prone to secrete IL-8 than those derived from HS HIGH pigs did. Monocytes and granulocytes issued from HS LOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HS LOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HS HIGH piglets did and exhibited a different metabolic profile. The higher cost of the immune responses associated with the low farm health status may account for lower HS LOW piglet performances. Conclusions Altogether, our data, obtained in field conditions, provide evidence that early exposure to pathogens shapes the immune competence of piglets. They also document the negative impact of an overstimulation of the immune system on piglets’ growth.

Abstract A bioartificial pancreas (BAP) encapsulating high pancreatic islets concentration is a promising alternative for type 1 diabetes therapy. However, the main limitation of this approach is O 2 supply, especially until graft neovascularization. Here, we described a methodology to design an optimal O 2 -balanced BAP using statistical design of experiment (DoE). A full factorial DoE was first performed to screen two O 2 -technologies on their ability to preserve pseudo-islet viability and function under hypoxia and normoxia. Then, response surface methodology was used to define the optimal O 2 -carrier and islet seeding concentrations to maximize the number of viable pseudo-islets in the BAP containing an O 2 -generator under hypoxia. Monitoring of viability, function and maturation of neonatal pig islets for 15 days in vitro demonstrated the efficiency of the optimal O 2 -balanced BAP. The findings should allow the design of a more realistic BAP for humans with high islets concentration by maintaining the O 2 balance in the device.

Few studies have examined sows and piglets' use of outdoor access during their first weeks of life, when reared with access to a hut and a pasture as in the organic system. We studied whether the age of the piglets and the season of the year influenced the use of an outdoor area by piglets and the sow, and the use of indoor spaces. For this, the localisation of sows and their piglets inside and outside the hut was analysed on 16 sows and their litters during the summer and 16 others during the winter, by scan sampling, on days 2, 7 and 13 after parturition in an organic herd in Denmark. When inside the hut, the number of piglets in the creep area, next to the sow or other piglets was noted. All parameters were analysed by linear mixed-effects ANOVA models. Sows were less often observed outside the hut in the wintertime and during the mornings compared to later times of the day (P<0.001). Furthermore, the sows increased their outdoor use with the age of their litter (P<0.001). The season also influenced the piglet use of outdoor areas (winter < summer; P<0.001). Piglets that were observed outside for the first time were older (8.9±0.9 d) during winter than during summer (3.5±0.5 d). The proportion of piglets observed outside increased with age, with a significant Season x Age interaction (P<0.001). The proportion of observations where the sows and piglets were outdoors together increased with the piglets’ age (P < 0.001). Sows’ outing was correlated with summer (R=0.3) and winter (R=0.2) temperatures, unlike piglets, where outings were correlated only with summer temperatures (R=0.3, P<0.001) only. The proportion of piglets observed in the creep area was significantly affected by the age with a Season x Age interaction effect (P<0.001). Piglets were rarely observed lying alone inside the hut. The proportion of piglets in contact with the sow and other piglets inside the pen was influenced by a significant Age x Season interaction effect (P<0.001). We showed that piglets given access to an outside paddock gradually increase their use with age and don’t go outside right away. Their first exit and the proportion of piglets outside depended also on the season. In general, a higher proportion of sows and piglets were observed indoor during the wintertime. Thus, sows and piglets adjust their use of outdoor paddock with season and piglet age during the first weeks of life.

Small extracellular vesicles (sEV) released by healthy beta cells are promising candidates for diabetes therapy thanks to their aptitude to modulate inflammation, to induce or maintain pancreatic function and to prevent pathogenic mechanisms. To advance the clinical development of therapeutics, there is a crucial need for scalable production methods. Stirred tank bioreactors (STR) are widely used in the industry due to their ability to provide homogeneous gas and nutrient supply, online monitoring, and efficient scale up. Anchorage-dependent cells can be cultured in STR on microcarriers or as spheroids, but may experience shear stress, which can affect sEV phenotype and function. Using pancreatic beta cells, this study identifies critical cell culturing parameters, including culture mode (monolayer vs. spheroids), medium formulation (with or without serum, glucose control), and process parameters (stirring, duration, cell density). The findings show that small spheroid culture promotes beta cell maturation without decreasing the yield of sEV per cell, despite a reduced cell surface exchange area. However, stirring increased expression of cellular stress markers and decreased cell viability. Set up of a three-step bioprocess allowed to maximize cell viability and sEV yields at high cell density over short production duration. sEV produced under these conditions maintained high purity, membrane integrity, and the aptitude to reduce T-lymphocyte proliferation and IFN-γ cytokine secretion in a mixed lymphocyte reaction. Flow cytometry analysis revealed lower CD63/CD81 ratios in STR, indicating enhanced ectosome production. Switch from high glucose expansion to low glucose production medium further allowed to direct sorting of the antigen insulin into beta-sEV. This study demonstrates the feasibility of producing functional sEV from mature beta cells cultured as small spheroids, suitable for upscale. Production of sEV in STR may be particularly beneficial for ectosome-enriched compound loading for therapeutic applications.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

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