An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major.

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.

Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

Perinuclear Mlp proteins down-regulate gene expression in response to a defect in mRNA export

Résumé Une caractéristique des cellules eucaryotes est le confinement du matériel génétique (ADN/DNA) dans le noyau. Pour décoder cette information, un ARN messager (mRNA) est d'abord transcrit sous forme d'un ARN prémessager (pré-mRNA). Ce-dernier doit subir plusieurs étapes de maturation pour aboutir à une particule ribonucléoprotéique (mRNP) qui sera exportée vers le cytoplasme et traduite en protéine. La protéine de levure Mex67p et son homologue humain TAP sont des récepteurs d'export médiant la translocation du mRNP au travers des complexes du pore nucléaire (NPC). Mex67p/TAP ne se lient pas directement au mRNA, mais nécessitent la présence de protéines adaptatrices, telles que Yra1p et son homologue humain REF1. Afin d'identifier de nouveaux facteurs impliqués dans l'export des mRNPs ou de nouvelles fonctions pour Yra1p, nous avons effectué un crible génétique avec un mutant thermosensible de Yra1p, GFP-yra 1 -8. Ce mutant présente un défaut d'export des mRNAs et une diminution des niveaux de transcrits du gène rapporteur LacZ ainsi que de certains transcrits endogènes. Nous avons trouvé que la perte de Mlp2p, ou d'une protéine hautement similaire, Mlp1p, restaure la croissance du mutant GFP-yra1-8 à température restrictive. Mlp1p et Mlp2p sont des protéines nucléaires, dont l'homologue humain est TPR. Les Mlp (myosin¬like proteins) ainsi que TPR forment des structures filamenteuses ancrées aux NPC. Bien que la fonction des Mlp ne soit pas clairement définie, un rôle dans la biogenèse et la surveillance des mRNPs a été récemment proposé. Notre étude montre que la perte des Mlp, non seulement restaure la croissance de GFP-yra1-8, mais augmente aussi les niveaux des transcrits LacZ et facilite leur apparition dans le cytoplasme. Des expériences d'immunoprécipitations de la chromatine révèlent que Mlp2p diminue le taux de synthèse du transcrit LacZ dans GFP-yra1-8. Des analyses du transcriptome montrent que Mlp2p réduit aussi les niveaux d'une population de transcrits endogènes dans le mutant. Finalement, des localisations in situ suggèrent que la transcription du rapporteur LacZ a lieu à la périphérie du noyau, à proximité des Mlp. Ainsi, les protéines Mlp pourraient préférentiellement diminuer la transcription de gènes exprimés à la périphérie nucléaire. Nous montrons aussi que Yra1p interagit génétiquement avec Nab2p une protéine liée au mRNA et impliquée dans son export, mais non avec d'autres protéines également impliquées dans l'export des mRNAs. Les résultats obtenus soutiennent un modèle où les protéines Yra1p et Nab2p sont nécessaires à l'arrimage des mRNPs sur la plate-forme des Mlp. Si ces signaux manquent ou sont défectueux, les mRNPs ne peuvent pas poursuivre leur trajet vers le canal central du NPC. Ce bloc induirait par la suite une diminution de la transcription d'une population de gènes potentiellement localisée à la périphérie nucléaire. Dans son ensemble, cette étude suggère que les protéines Mlp établissent un lien entre la transcription de certains mRNAs et leur export au travers du pore nucléaire. Summary A hallmark of the eukaryotic cell is the packaging of DNA in the nucleus. To decode the genetic information, a messenger RNA (mRNA) is first synthesized as a pre-mRNA molecule, which undergoes different maturation steps resulting in an mRNP (messenger RNA ribonucleoprotein), which can be actively transported to the cytoplasm and translated into a protein. Yeast Mex67p and its human homologue TAP are export receptors mediating mRNP translocation through the nuclear pore complex (NPC). The recruitment of Mex67p/TAP to mRNA is mediated by mRNA export adaptors of the evolutionarily conserved REF (RNA and Export Factor binding) family: yeast Yra1p and human REF1. To uncover new functions of Yra1p or new factors implicated in mRNA export, we performed a genetic screen with a themiosensitive (ts) yra1 mutant, GFP-yra1-8. This mutant exhibits mRNA export defects and a decrease in the levels of LacZ reporter and certain endogenous transcripts. We found that the loss of Mlp2p, or the related Mlp1p protein, substantially rescues the growth defect of the GFP-yra1 -8 mutant. Mlp1p and M1p2p are large non-essential proteins, homologous to human TPR, proposed to form intra-nuclear filamentous structures anchored at the NPC. Their role is not clearly defined, but they have been implicated in mRNP biogenesis and surveillance. Our study shows that loss of Mlp proteins not only restores growth of GFP-yra1-8, but also rescues LacZ mRNA levels and increases their appearance in the cytoplasm. Chromatin immunoprecipitation and pulse chase experiments indicate that Mlp2p down-regulates LacZ mRNA synthesis in GFP-yra1-8. DNA micro- array analyses reveal that Mlp2p also reduces the levels of a subset of cellular transcripts in the yra1 mutant strain. In situ localizations suggest that LacZ transcription occurs at the nuclear periphery, in close proximity to Mlp proteins. Thus, Mlp proteins may preferentially down-regulate genes expressed at the nuclear periphery. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlp proteins rescues the growth defect of yra1 and nab2, but not other mRNA export mutants. The data support a model in which Nab2p and Yra1p are required for rnRNP docking to the Mlp platform. Lack of these signals prevents mRNPs from crossing the Mlp gate. This block may then negatively feed-back on the transcription of a subset of genes, potentially located at the nuclear envelope. Overall, this study suggests that perinuclear Mlp proteins establish a link between mRNA transcription and export.

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

Expression of genes encoding the pre-TCR and CD3 complex during thymus development.

The mature TCR is composed of a clonotypic heterodimer (alpha beta or gamma delta) associated with the invariant CD3 components (gamma, delta, epsilon and zeta). There is now considerable evidence that more immature forms of the TCR-CD3 complex (consisting of either CD3 alone or CD3 associated with a heterodimer of TCR beta and pre-T alpha) can be expressed at the cell surface on early thymocytes. These pre-TCR complexes are believed to be necessary for the ordered progression of early T cell development. We have analyzed in detail the expression of both the pre-TCR and CD3 complex at various stages of adult thymus development. Our data indicate that all CD3 components are already expressed at the mRNA level by the earliest identifiable (CD4lo) thymic precursor. In contrast, genes encoding the pre-TCR complex (pre-T alpha and fully rearranged TCR beta) are first expressed at the CD44loCD25+CD4-CD8- stage. Detectable surface expression of both CD3 and TCR beta are delayed relative to expression of the corresponding genes, suggesting the existence of other (as yet unidentified) components of the pre-TCR complex.

Circulating miRNAs: a new generation of anti-doping biomarkers

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. Cell-free miRNAs detected in blood plasma are used as specific and sensitive markers of physiological processes and some diseases. Circulating miRNAs are highly stable in body fluids, for example plasma. Therefore, profiles of circulating miRNAs have been investigated for potential use as novel, non-invasive anti-doping biomarkers. This review describes the biological mechanisms underlying the variation of circulating miRNAs, revealing that they have great potential as a new class of biomarker for detection of doping substances. The latest developments in extraction and profiling technology, and the technical design of experiments useful for anti-doping, are also discussed. Longitudinal measurements of circulating miRNAs in the context of the athlete biological passport are proposed as an efficient strategy for the use of these new markers. The review also emphasizes potential challenges for the translation of circulating miRNAs from research into practical anti-doping applications.

Nonenzymatic lipid peroxidation reprograms gene expression and activates defense markers in Arabidopsis tocopherol-deficient mutants.

Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.

Differential neuroglycan C expression during retinal degeneration in Rpe65-/- mice.

PURPOSE: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65-/- mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression. METHODS: Retinas from C57/Bl6 wild-type and Rpe65-/- mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry. RESULTS: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65-/- retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65-/- mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected. CONCLUSIONS: During retinal degeneration in Rpe65-/- mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.

Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA.

BACKGROUND: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. METHODS: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. RESULTS: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. CONCLUSIONS: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Dairy calcium supplementation in overweight or obese persons: its effect on markers of fat metabolism

BACKGROUND: Dairy calcium supplementation has been proposed to increase fat oxidation and to inhibit lipogenesis. OBJECTIVE: We aimed to investigate the effects of calcium supplementation on markers of fat metabolism. DESIGN: In a placebo-controlled, crossover experiment, 10 overweight or obese subjects who were low calcium consumers received 800 mg dairy Ca/d for 5 wk. After 4 wk, adipose tissue was taken for biopsy for analysis of gene expression. Respiratory exchange, glycerol turnover, and subcutaneous adipose tissue microdialysis were performed for 7 h after consumption of 400 mg Ca or placebo, and the ingestion of either randomized slow-release caffeine (SRC; 300 mg) or lactose (500 mg). One week later, the test was repeated with the SRC or lactose crossover. RESULTS: Calcium supplementation increased urinary calcium excretion by 16% (P = 0.017) but did not alter plasma parathyroid hormone or osteocalcin concentrations. Resting energy expenditure (59.9 +/- 3.0 or 59.6 +/- 3.3 kcal/h), fat oxidation (58.4 +/- 2.5 or 53.8 +/- 2.2 mg/min), plasma free fatty acid concentrations (0.63 +/- 0.02 or 0.62 +/- 0.03 mmol/L), and glycerol turnover (3.63 +/- 0.41 or 3.70 +/- 0.38 micromol . kg(-1) . min(-1)) were similar with or without calcium, respectively. SRC significantly increased free fatty acid concentrations, resting fat oxidation, and resting energy expenditure. During microdialysis, epinephrine increased dialysate glycerol concentrations by 250% without and 254% with calcium. Expression of 7 key metabolic genes in subcutaneous adipose tissue was not affected by calcium supplementation. CONCLUSION: Dairy calcium supplementation in overweight subjects with habitually low calcium intakes failed to alter fat metabolism and energy expenditure under resting conditions and during acute stimulation by caffeine or epinephrine

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.