Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

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.

Pathogen exposure influences immune parameters around weaning in pigs reared in commercial farms.

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 (HSLOW)- and high (HSHIGH)-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 HSLOW and HSHIGH 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 HSLOW farms as compared to their HSHIGH homologs. Also, the age-dependent evolution in γδ 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 HSLOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HSLOW piglets were more prone to secrete IL-8 than those derived from HSHIGH pigs did. Monocytes and granulocytes issued from HSLOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HSLOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HSHIGH 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 HSLOW 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.

Optimization of an O2-balanced bioartificial pancreas for type 1 diabetes using statistical design of experiment.

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 O2 supply, especially until graft neovascularization. Here, we described a methodology to design an optimal O2-balanced BAP using statistical design of experiment (DoE). A full factorial DoE was first performed to screen two O2-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 O2-carrier and islet seeding concentrations to maximize the number of viable pseudo-islets in the BAP containing an O2-generator under hypoxia. Monitoring of viability, function and maturation of neonatal pig islets for 15 days in vitro demonstrated the efficiency of the optimal O2-balanced BAP. The findings should allow the design of a more realistic BAP for humans with high islets concentration by maintaining the O2 balance in the device.

Nonhypoalbuminemic Inflammatory Bowel Disease in Dogs as Disease Model.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) is increasing worldwide, emphasizing the need of relevant models, as dogs spontaneously affected by IBD may be, for better knowledge of the disease's physiopathology. METHODS: We studied 22 client-owned dogs suffering from IBD without protein loss and 14 control dogs. Biopsies were obtained from the duodenum, ileum, and colon. Inflammatory grade was assessed by histopathology, immunohistochemistry, and chemokine analysis. The expression of Toll-like receptors (TLR) in mucosa was immunohistochemically evaluated. Antibody levels against bacterial ligands (lipopolysaccharide [LPS] and flagellin) were measured in sera using enzyme-linked immunoassay. RESULTS: Dogs with IBD showed low to severe clinical disease. Histopathologically, the gut of dogs with IBD did not exhibit significant alterations compared with controls except in the colon. The number of CD3+ T lymphocytes was decreased in the ileum and colon of dogs with IBD compared with controls, whereas the numbers of Foxp3+, CD20+, and CD204+ cells were similar in the 2 groups. Three chemokines, but no cytokines, were detected at the protein level in the mucosa, and the disease poorly affected their tissue concentrations. Dogs with IBD exhibited higher serum reactivity against LPS and flagellin than controls but similar immunoreactivity against the receptors TLR4 and TLR5. In addition, TLR2 and TLR9 showed similar expression patterns in both groups of dogs. CONCLUSIONS: Our data described dysregulated immune responses in dogs affected by IBD without protein loss. Despite fairly homogeneous dog cohorts, we were still faced with interindividual variability, and new studies with larger cohorts are needed to validate the dog as a model.

Effects of divergent selection upon adrenocortical activity on immune traits in pig.

BACKGROUND: The sustainability of farming and animal welfare requires the reconsideration of current selection schemes. In particular, implementation of new selection criteria related to animal health and welfare should help to produce more robust animals and to reduce anti-microbial use. The hypothalamo-pituitary-adrenocortical (HPA) axis plays a major role in metabolic regulation and adaptation processes and its activity is strongly influenced by genetic factors. A positive association between HPA axis activity and robustness was recently described. To explore whether selecting pigs upon HPA axis activity could increase their robustness, a divergent selection experiment was carried out in the Large White pig breed. This allowed the generation of low (HPAlo) and high (HPAhi) responders to adrenocorticotropic hormone administration. RESULTS: In this study, we compared 23 hematologic and immune parameters of 6-week-old, HPAlo and HPAhi piglets and analysed their response to a low dose of lipopolysaccharide (LPS) two weeks later. At six weeks of age, HPAhi piglets displayed greater red blood cell and leucocyte number including CD8α+ γδ cells, cytotoxic T lymphocytes, naive T helper (Th) cells and B lymphocytes as compared to HPAlo individuals. The ability of blood cells to secrete TNFα in response to LPS ex vivo was higher for HPAhi pigs. At week eight, the inflammatory response to the LPS in vivo challenge was poorly affected by the HPA axis activity. CONCLUSIONS: Divergent selection upon HPA axis activity modulated hematologic and immune parameters in 6-week-old pigs, which may confer an advantage to HPAhi pigs at weaning. However, HPAlo and HPAhi piglets did not exhibit major differences in the parameters analysed two weeks later, i. e. in 8-week-old pigs. In conclusion, chronic exposure to high cortisol levels in HPAhi pigs does not negatively impact immunity.

ß2-adrenergic stimulation of dendritic cells favors IL-10 secretion by CD4+ T cells.

Adrenergic receptor agonists and antagonists are extensively used as drugs in medicine for a broad spectrum of indications. We examined the consequences of ß2-adrenergic stimulation of murine dendritic cells (DCs) on CD4+ T cell activation. We demonstrated in vitro that treatment of LPS-matured DCs with the ß2-agonist salbutamol reduced their ability to trigger OT-II T cell proliferation specific for ovalbumin antigen. Salbutamol also induced a decrease in MHC class II molecule expression by DC through Gi protein activation. Co-culture of CD4+ T cells with salbutamol-conditioned mature DC impaired TNFα and IL-6 secretion while preserving IL-10 production by T cells. Using a vaccination protocol in mice, we showed that salbutamol favored IL-10-producing CD4+ T cells. None of these effects was observed when working with ß2-adrenoreceptor deficient mice. Finally, we suggest that ß2-adrenergic stimulation of DC could be an interesting way to shape CD4+ T cell responses for the purposes of immunotherapy.

ß2-adrenoreceptor stimulation dampens the LPS-induced M1 polarization in pig macrophages.

The cross-talk between sympatho-adreno-medullar axis and innate immunity players was mainly studied in rodents. In intensive husbandry, pigs are exposed to multiple stressors inducing repeated releases of catecholamines that bind to adrenoreceptors (AR) on target cells. Among adrenoreceptors, the ß2-AR is largely expressed by immune cells including macrophages. We report herein on the effects of catecholamines, through ß2-AR stimulation, on pig macrophage functions activated by LPS. ß2-AR stimulation of porcine macrophages prevented the LPS-induced increase in TNFα and IL-8 secretion while increasing IL-10 secretion. In contrast, treatment with a ß2-agonist had no effect on anti-microbial functions. Lastly, ß2-AR stimulation of macrophages reduced the expression of genes up-regulated by LPS. Altogether, we demonstrated that ß2-AR stimulation of porcine macrophages prevented polarization towards a pro-inflammatory phenotype. Since porcine macrophages are a suitable model for human macrophages, our results might be relevant to appreciate catecholamine effects on human macrophages.

Acute social stress-induced immunomodulation in pigs high and low responders to ACTH.

Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple stressful events such as early weaning, successive mixing, crowding and shipping. These stressors are thought to impair immune defences and might contribute, at least partly, to the prophylactic use of antibiotics. Robustness was recently defined as the ability of an individual to express a high-production potential in a wide variety of environmental conditions. Increasing robustness thus appears as a valuable option to improve resilience to stressors and could be obtained by selecting piglets upon their adrenocortical activity. In this study, we aimed at depicting the consequences of an acute social stress on the immune capacity of piglets genetically selected upon divergent hypothalamic-pituitary-adrenocortical (HPA) axis activity. For this purpose, we monitored neuroendocrine and immune parameters, in high- (HPAhi) and low- (HPAlo) responders to ACTH, just before and immediately after a one-hour mixing with unfamiliar conspecifics. As expected, stressed piglets displayed higher levels of circulating cortisol and norepinephrine. Blood cell count analysis combined to flow cytometry revealed a stress-induced leukocyte mobilization in the bloodstream with a specific recruitment of CD8α+ lymphocytes. Besides, one-hour mixing decreased LPS-induced IL-8 and TNFα secretions in whole-blood assays (WBA) and reduced mononuclear cell phagocytosis. Altogether, our data demonstrate that acute social stress alters immune competence of piglets from both groups, and bring new insights in favour of good farming practices. While for most parameters high- and low-responders to ACTH behaved similarly, HPAhi piglets displayed higher number of CD4+ CD8α- T cells, as well as increased cytokine production in WBA (LPS-induced TNFα and PIL-induced IL-8), which could confer them increased resistance to pathogens. Finally, a principal component analysis including all parameters highlighted that overall stress effects were less pronounced on piglets with a strong HPA axis. Thus, selection upon adrenocortical axis activity seems to reduce the magnitude of response to stress and appears as a good tool to increase piglet robustness.

Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes.

In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD) mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis.

Diabetes acceleration by cyclophosphamide in the non-obese diabetic mouse is associated with differentiation of immunosuppressive monocytes into immunostimulatory cells.

Cyclophosphamide (CTX) was previously shown to induce the recruitment of immunosuppressive myeloid cells in mouse. In the non-obese diabetic (NOD) mouse, which develops spontaneously type I diabetes, CTX is widely known to accelerate the autoimmune process. Our data demonstrated that CTX actually did mobilize an immunosuppressive myeloid CD11b(+) Ly-6G(-) population in the NOD mouse spleen in addition to a well-identified neutrophil CD11b(+) Ly-6G(+) population. CD11b(+) Ly-6G(-) cells, in contrast with CD11b(+) Ly-6G(+) cells, were able to inhibit in vitro mitogen-induced syngeneic T cell proliferation. CD11b(+) Ly-6G(-) cells represented a heterogeneous population mainly made of CD31(hi) cells and Ly-6C(+) monocytes. Only these last ones supported the immunosuppressive in vitro activity and resembled circulating inflammatory monocytes according to flow cytometry, cytology and RT-PCR data. Although CD11b(+) Ly-6G(-) Ly-6C(+) cells exhibited immunosuppressive function in vitro, they were not able to control the autoimmune response following CTX injection. Our data show that these CTX-induced immunosuppressive myeloid cells actually behaved as very plastic cells in vitro. Likewise, in the model of prediabetic NOD/SCID mice, CD11b(+) Ly-6G(-) Ly-6C(+) were able to differentiate into CD11c+ cells after i.v. injection. Herein, we described a new mechanism by which CTX might induce diabetes acceleration in the NOD mouse. In summary, recruited immunosuppressive cells might participate in the immunopotentiating effect of CTX on the autoimmune response by their further differentiation into immunostimulatory cells.

Easy and rapid method of zygosity determination in transgenic mice by SYBR Green real-time quantitative PCR with a simple data analysis.

Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.

Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals.

Physiological and genetic studies with the ramosus (rms) mutants in garden pea (Pisum sativum) and more axillary shoots (max) mutants in Arabidopsis (Arabidopsis thaliana) have shown that shoot branching is regulated by a network of long-distance signals. Orthologous genes RMS1 and MAX4 control the synthesis of a novel graft-transmissible branching signal that may be a carotenoid derivative and acts as a branching inhibitor. In this study, we demonstrate further conservation of the branching control system by showing that MAX2 and MAX3 are orthologous to RMS4 and RMS5, respectively. This is consistent with the long-standing hypothesis that branching in pea is regulated by a novel long-distance signal produced by RMS1 and RMS5 and that RMS4 is implicated in the response to this signal. We examine RMS5 expression and show that it is more highly expressed relative to RMS1, but under similar transcriptional regulation as RMS1. Further expression studies support the hypothesis that RMS4 functions in shoot and rootstock and participates in the feedback regulation of RMS1 and RMS5 expression. This feedback involves a second novel long-distance signal that is lacking in rms2 mutants. RMS1 and RMS5 are also independently regulated by indole-3-acetic acid. RMS1, rather than RMS5, appears to be a key regulator of the branching inhibitor. This study presents new interactions between RMS genes and provides further evidence toward the ongoing elucidation of a model of axillary bud outgrowth in pea.

MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea.

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in the MAX4 gene of Arabidopsis result in increased and auxin-resistant bud growth. Increased branching in max4 shoots is restored to wild type by grafting to wild-type rootstocks, suggesting that MAX4 is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene, RMS1. Accordingly, MAX4 and RMS1 were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.