In vivo conditional Pax4 overexpression in mature islet β-cells prevents stress-induced hyperglycemia in mice.

OBJECTIVE To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129 W) in β-cells. RESEARCH DESIGN AND METHODS Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129 W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS Pax4 but not Pax4R129 W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129 W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.

Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.

The effect of host social system on parasite population genetic structure: comparative population genetics of two ectoparasitic mites and their bat hosts.

BACKGROUND: The population genetic structure of a parasite, and consequently its ability to adapt to a given host, is strongly linked to its own life history as well as the life history of its host. While the effects of parasite life history on their population genetic structure have received some attention, the effect of host social system has remained largely unstudied. In this study, we investigated the population genetic structure of two closely related parasitic mite species (Spinturnix myoti and Spinturnix bechsteini) with very similar life histories. Their respective hosts, the greater mouse-eared bat (Myotis myotis) and the Bechstein's bat (Myotis bechsteinii) have social systems that differ in several substantial features, such as group size, mating system and dispersal patterns. RESULTS: We found that the two mite species have strongly differing population genetic structures. In S. myoti we found high levels of genetic diversity and very little pairwise differentiation, whereas in S. bechsteini we observed much less diversity, strongly differentiated populations and strong temporal turnover. These differences are likely to be the result of the differences in genetic drift and dispersal opportunities afforded to the two parasites by the different social systems of their hosts. CONCLUSIONS: Our results suggest that host social system can strongly influence parasite population structure. As a result, the evolutionary potential of these two parasites with very similar life histories also differs, thereby affecting the risk and evolutionary pressure exerted by each parasite on its host.

SYSGENET: a meeting report from a new European network for systems genetics.

The first scientific meeting of the newly established European SYSGENET network took place at the Helmholtz Centre for Infection Research (HZI) in Braunschweig, April 7-9, 2010. About 50 researchers working in the field of systems genetics using mouse genetic reference populations (GRP) participated in the meeting and exchanged their results, phenotyping approaches, and data analysis tools for studying systems genetics. In addition, the future of GRP resources and phenotyping in Europe was discussed.

IL-13 induces expression of CD36 in human monocytes through PPARgamma activation.

The class B scavenger receptor CD36 is a component of the pattern recognition receptors on monocytes that recognizes a variety of molecules. CD36 expression in monocytes depends on exposure to soluble mediators. We demonstrate here that CD36 expression is induced in human monocytes following exposure to IL-13, a Th2 cytokine, via the peroxisome proliferator-activated receptor (PPAR)gamma pathway. Induction of CD36 protein was paralleled by an increase in CD36 mRNA. The PPARgamma pathway was demonstrated using transfection of a PPARgamma expression plasmid into the murine macrophage cell line RAW264.7, expressing very low levels of PPARgamma, and in peritoneal macrophages from PPARgamma-conditional null mice. We also show that CD36 induction by IL-13 via PPARgamma is dependent on phospholipase A2 activation and that IL-13 induces the production of endogenous 15-deoxy-Delta12,14-prostaglandin J2, an endogenous PPARgamma ligand, and its nuclear localization in human monocytes. Finally, we demonstrate that CD36 and PPARgamma are involved in IL-13-mediated phagocytosis of Plasmodium falciparum-parasitized erythrocytes. These results reveal a novel role for PPARgamma in the alternative activation of monocytes by IL-13, suggesting that endogenous PPARgamma ligands, produced by phospholipase A2 activation, could contribute to the biochemical and cellular functions of CD36.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Innate receptors for adaptive immunity.

Pattern recognition receptors (PRRs) are commonly known as sensor proteins crucial for the early detection of microbial or host-derived stress signals by innate immune cells. Interestingly, some PRRs are also expressed and functional in cells of the adaptive immune system. These receptors provide lymphocytes with innate sensing abilities; for example, B cells express Toll-like receptors, which are important for the humoral response. Strikingly, certain other NOD-like receptors are not only highly expressed in adaptive immune cells, but also exert functions related specifically to adaptive immune system pathways, such as regulating antigen presentation. In this review, we will focus particularly on the current understanding of PRR functions intrinsic to B and T lymphocytes; a developing aspect of PRR biology.

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in pregnant C57BL/6 mice infected with L. major at different gestational periods.

BACKGROUND AND OBJECTIVE: To assess if gestational factors affect the resistance of C57BL/6 mice to L major infection, this study determined the levels of IL-4 and IFN-gamma in popliteal lymph node cells of pregnant C57BL/6 mice infected with L. major at 16 hours, 5 days-, 10 days- and 15 days- post plug by PCR, ELISA and BIOASSAY. DESIGN/SETTING: Experimental. RESULTS: Infected pregnant C57BL/6 mice developed larger cutaneous footpad lesions compared with non-pregnant C57BL/6 mice (that showed signs of resolution 7-10 weeks after infection). But, the lesions in infected pregnant C57BL/6 mice and infected non-pregnant C57BL/6 mice were not as large as in susceptible BALB/c mice. The mean litter weight was also reduced in pregnant infected C57BL/6 mice particularly in the groups infected at later stages of pregnancy (day 10- and day 15-post plug). The levels of both IL-4 and IFN-gamma increased with gestation in pregnant infected C57BL/6 mice compared with pregnant non-infected group, while only IL-4 was raised in pregnant infected mice compared with infected non pregnant mice. CONCLUSIONS: It may be concluded that increased IL-4 in pregnant infected C57BL/6 mice caused the transient susceptibility to L major infection while reduced litter weight was associated with increased IFN-gamma. These effects were pronounced in C57BI/6 mice infected with L major in late pregnancy.

GWAS of human bitter taste perception identifies new loci and reveals additional complexity of bitter taste genetics.

Human perception of bitterness displays pronounced interindividual variation. This phenotypic variation is mirrored by equally pronounced genetic variation in the family of bitter taste receptor genes. To better understand the effects of common genetic variations on human bitter taste perception, we conducted a genome-wide association study on a discovery panel of 504 subjects and a validation panel of 104 subjects from the general population of São Paulo in Brazil. Correction for general taste-sensitivity allowed us to identify a SNP in the cluster of bitter taste receptors on chr12 (10.88- 11.24 Mb, build 36.1) significantly associated (best SNP: rs2708377, P = 5.31 × 10(-13), r(2) = 8.9%, β = -0.12, s.e. = 0.016) with the perceived bitterness of caffeine. This association overlaps with-but is statistically distinct from-the previously identified SNP rs10772420 influencing the perception of quinine bitterness that falls in the same bitter taste cluster. We replicated this association to quinine perception (P = 4.97 × 10(-37), r(2) = 23.2%, β = 0.25, s.e. = 0.020) and additionally found the effect of this genetic locus to be concentration specific with a strong impact on the perception of low, but no impact on the perception of high concentrations of quinine. Our study, thus, furthers our understanding of the complex genetic architecture of bitter taste perception.

Landscape genetics of the Alpine newt (Mesotriton alpestris) inferred from a strip-based approach

Habitat destruction and fragmentation are known to strongly affect dispersal by altering the quality of the environment between populations. As a consequence, lower landscape connectivity is expected to enhance extinction risks through a decrease in gene flow and the resulting negative effects of genetic drift, accumulation of deleterious mutations and inbreeding depression. Such phenomena are particularly harmful for amphibian species, characterized by disjunct breeding habitats. The dispersal behaviour of amphibians being poorly understood, it is crucial to develop new tools, allowing us to determine the influence of landscape connectivity on the persistence of populations. In this study, we developed a new landscape genetics approach that aims at identifying land-uses affecting genetic differentiation, without a priori assumptions about associated ecological costs. We surveyed genetic variation at seven microsatellite loci for 19 Alpine newt (Mesotriton alpestris) populations in western Switzerland. Using strips of varying widths that define a dispersal corridor between pairs of populations, we were able to identify land-uses that act as dispersal barriers (i.e. urban areas) and corridors (i.e. forests). Our results suggest that habitat destruction and landscape fragmentation might in the near future affect common species such as M. alpestris. In addition, by identifying relevant landscape variables influencing population structure without unrealistic assumptions about dispersal, our method offers a simple and flexible tool of investigation as an alternative to least-cost models and other approaches.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

A human MYBPC3 mutation appearing about 10 centuries ago results in a hypertrophic cardiomyopathy with delayed onset, moderate evolution but with a risk of sudden death.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) is a genetically heterogeneous disease. One specific mutation in the MYBPC3 gene is highly prevalent in center east of France giving an opportunity to define the clinical profile of this specific mutation. METHODS: HCM probands were screened for mutation in the MYH7, MYBPC3, TNNT2 and TNNI3 genes. Carriers of the MYBPC3 IVS20-2A>G mutation were genotyped with 8 microsatellites flanking this gene. The age of this MYBPC3 mutation was inferred with the software ESTIAGE. The age at first symptom, diagnosis, first complication, first severe complication and the rate of sudden death were compared between carriers of the IVS20-2 mutation (group A) and carriers of all other mutations (group B) using time to event curves and log rank test. RESULTS: Out of 107 HCM probands, 45 had a single heterozygous mutation in one of the 4 tested sarcomeric genes including 9 patients with the MYBPC3 IVS20-2A>G mutation. The IVS20-2 mutation in these 9 patients and their 25 mutation carrier relatives was embedded in a common haplotype defined after genotyping 4 polymorphic markers on each side of the MYBPC3 gene. This result supports the hypothesis of a common ancestor. Furthermore, we evaluated that the mutation occurred about 47 generations ago, approximately at the 10th century.We then compared the clinical profile of the IVS20-2 mutation carriers (group A) and the carriers of all other mutations (group B). Age at onset of symptoms was similar in the 34 group A cases and the 73 group B cases but group A cases were diagnosed on average 15 years later (log rank test p = 0.022). Age of first complication and first severe complication was delayed in group A vs group B cases but the prevalence of sudden death and age at death was similar in both groups. CONCLUSION: A founder mutation arising at about the 10th century in the MYBPC3 gene accounts for 8.4% of all HCM in center east France and results in a cardiomyopathy starting late and evolving slowly but with an apparent risk of sudden death similar to other sarcomeric mutations.