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Witso E.,Norwegian Institute of Public Health | Tapia G.,Norwegian Institute of Public Health | Cinek O.,Charles University | Pociot F.M.,Glostrup Research Institute | And 2 more authors.
PLoS ONE | Year: 2011

Interferon induced with helicase C domain 1 (IFIH1) senses and initiates antiviral activity against enteroviruses. Genetic variants of IFIH1, one common and four rare SNPs have been associated with lower risk for type 1 diabetes. Our aim was to test whether these type 1 diabetes-associated IFIH1 polymorphisms are associated with the occurrence of enterovirus infection in the gut of healthy children, or influence the lack of association between gut enterovirus infection and islet autoimmunity. After testing of 46,939 Norwegian newborns, 421 children carrying the high risk genotype for type 1 diabetes (HLA-DR4-DQ8/DR3-DQ2) as well as 375 children without this genotype were included for monthly fecal collections from 3 to 35 months of age, and genotyped for the IFIH1 polymorphisms. A total of 7,793 fecal samples were tested for presence of enterovirus RNA using real time reverse transcriptase PCR. We found no association with frequency of enterovirus in the gut for the common IFIH1 polymorphism rs1990760, or either of the rare variants of rs35744605, rs35667974, rs35337543, while the enterovirus prevalence marginally differed in samples from the 8 carriers of a rare allele of rs35732034 (26.1%, 18/69 samples) as compared to wild-type homozygotes (12.4%, 955/7724 samples); odds ratio 2.5, p = 0.06. The association was stronger when infections were restricted to those with high viral loads (odds ratio 3.3, 95% CI 1.3-8.4, p = 0.01). The lack of association between enterovirus frequency and islet autoimmunity reported in our previous study was not materially influenced by the IFIH1 SNPs. We conclude that the type 1 diabetes-associated IFIH1 polymorphisms have no, or only minor influence on the occurrence, quantity or duration of enterovirus infection in the gut. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection. © 2011 Witsø et al.


Sorensen J.S.,Aarhus University Hospital | Vaziri-Sani F.,Skåne University Hospital | Maziarz M.,University of Washington | Kristensen K.,Aarhus University Hospital | And 6 more authors.
Diabetes Research and Clinical Practice | Year: 2012

Aims: To test if islet autoantibodies at diagnosis of type 1 diabetes (T1DM) and after 3-6 years with T1D predict residual beta-cell function (RBF) after 3-6 years with T1D. Methods: T1D children (n= 260, median age at diagnosis 9.4, range 0.9-14.7 years) were tested for GAD65, IA-2, ZnT8R, ZnT8W and ZnT8Q autoantibodies (A) at diagnosis, and 3-6 years after diagnosis when also fasting and stimulated RBF were determined. Results: For every 1-year increase in age at diagnosis of TID, the odds of detectable C-peptide increased 1.21 (1.09, 1.34) times for fasting C-peptide and 1.28 (1.15, 1.42) times for stimulated C-peptide. Based on a linear model for subjects with no change in IA-2A levels, the odds of detectable C-peptide were 35% higher than for subjects whose IA-2A levels decreased by half (OR = 1.35 (1.09, 1.67), p= 0.006); similarly for ZnT8WA (OR = 1.39 (1.09, 1.77), p= 0.008) and ZnT8QA (OR = 1.55 (1.06, 2.26) p= 0.024). Such relationship was not detected for GADA or ZnT8RA. All OR adjusted for confounders. Conclusions: Age at diagnosis with T1D was the major predictor of detectable C-peptide 3-6 years post-diagnosis. Decreases in IA-2A, and possibly ZnT8A, levels between diagnosis and post-diagnosis were associated with a reduction in RBF post-diagnosis. © 2012 Elsevier Ireland Ltd.


Palmisano G.,University of Southern Denmark | Jensen S.,University of Southern Denmark | Le Bihan M.-C.,University of Southern Denmark | Le Bihan M.-C.,French Institute of Health and Medical Research | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2012

Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular bodies with the plasma membrane, respectively. They are thought to be involved in many significant biological processes such as cell to cell communication, rescue from apoptosis, and immunological responses. Here we report for the first time a quantitative study of proteins from β-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of β-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from β-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, α-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting ser-ine- threonine kinase 1, and intercellular adhesion molecule 1. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Povlsen G.K.,Glostrup Research Institute | Waldsee R.,Lund University | Ahnstedt H.,Lund University | Kristiansen K.A.,Glostrup Research Institute | And 2 more authors.
Experimental Brain Research | Year: 2012

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ETB) and 5-hydroxytryptamine IB (5-HTIB) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ETBand 5-HT 1Breceptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2 h MCA occlusion followed by reperfusion for 1 or 24 h. Alternatively, MCAs from naïve rats were cultured for 1 or 24 h. ETBand 5-HT1Breceptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ETBand 5-HT 1Breceptors were strongly upregulated and slightly downregulated, respectively, 24 h after experimental stroke or organ culture. ET Breceptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT1Breceptor- mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels. © Springer-Verlag 2012.


Ramachandran R.,Copenhagen University | Bhatt D.K.,Copenhagen University | Ploug K.B.,Copenhagen University | Hay-Schmidt A.,Glostrup Research Institute | And 3 more authors.
Cephalalgia | Year: 2014

Background and aim: Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. Materials and methods: The femoral vein was catheterised in rats and GTN was infused (4 mg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP andWestern blot for measuring nNOS protein expression. The effect of olcegepant, L-nitro-arginine methyl ester (L-NAME) and neurokinin (NK)-1 receptor antagonist L-733060 were analysed on Fos activation. Results: GTN-treated rats showed a significant increase of nNOS and CGRP in dura mater and CGRP in the trigeminal nucleus caudalis (TNC). Upregulation of Fos was observed in TNC four hours after the infusion. This activation was inhibited by pre-treatment with olcegepant. Pre-treatment with L-NAME and L-733060 also significantly inhibited GTN induced Fos expression. Conclusion: The present study indicates that blockers of CGRP, NOS and NK-1 receptors all inhibit GTN induced Fos activation. These findings also predict that pre-treatment with olcegepant may be a better option than post-treatment to study its inhibitory effect in GTN migraine models. © 2013 International Headache Society.


Pedersen M.H.F.,Technical University of Denmark | Baun M.,Glostrup Research Institute
Journal of Labelled Compounds and Radiopharmaceuticals | Year: 2012

In the interest of developing efficient methods for tritium labelling peptides, we here demonstrate the successful labelling of PACAP-38 (pituitary adenylate cyclase-activating polypeptide), a 38-mer peptide, using a synthetic diiodinated PACAP-38 precursor. In this example, we employ standard hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system. © 2011 John Wiley & Sons, Ltd.


Sandhu H.,Glostrup Research Institute | Xu C.B.,Lund University | Edvinsson L.,Glostrup Research Institute | Edvinsson L.,Lund University
Toxicology and Applied Pharmacology | Year: 2010

Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ETB) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-ΚB) mediate the upregulation of contractile endothelin receptors in the cerebral arteries.Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSP with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-ΚB specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ETB receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ETB receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively.Results show that organ culture per se induced transcriptional upregulation of contractile ETB receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ETB receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ETB receptors. Thus, the MAPK-mediated upregulation of contractile ETB receptors in cerebral arteries might be a pharmacological target for the treatment of smoke-associated cerebral vascular disease like stroke. © 2010 Elsevier Inc.


Gupta S.,Glostrup Research Institute | Bhatt D.K.,Glostrup Research Institute | Boni L.J.,Glostrup Research Institute | Olesen J.,Glostrup Research Institute
Cephalalgia | Year: 2010

Intravital microscopy on a closed cranial window allows one to measure change in the diameter of cranial blood vessels after intravenous (i.v.) administration of pharmacodynamic substances. Putative targets being pursued in migraine are large vasodilating peptide molecules such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase polypeptide (PACAP)-38. High i.v. doses are required to study their craniovascular pharmacology. Unfortunately, this leads to a drop in blood pressure (BP) that subsequently causes blood vessels to dilate by autoregulation. Hence it is difficult to decipher what effect is caused by direct receptor agonist interaction or contributed by autoregulation. In the present study we infused substances with an ingenious indwelling catheter in the common carotid artery in rats. Intracarotidly seven-, 12-and 17-fold lower doses of CGRP, PACAP-38 and capsaicin were required, respectively, compared with i.v. infusion to induce the same dilation in dural artery. Dilating intracarotid (i.c.) doses caused no or a minimal fall in BP, whereas equi-responsive i.v. doses caused a marked BP reduction. The CGRP blocking potential of olcegepant was amplified by > 20 times on i.c. infusion. Pial artery responses to CGRP did not change with i.c. infusion, demonstrating that dilations after i.v. CGRP are mediated by autoregulation rather than through specific receptors. We applied CGRP topically, which induced concentration-dependent dural vasodilation, but no effect on pial artery or on BP. In conclusion, this new approach offers an improvement of the existing model by allowing more accurate assessment of effects of pharmaca on the cranial vasculature without inducing significant systemic effects. © International Headache Society 2009.


PubMed | Glostrup Research Institute
Type: Comparative Study | Journal: Toxicology and applied pharmacology | Year: 2011

The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET(B), serotonin 5-HT(1B), and thromboxane A(2) TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET(B), 5-HT(1B), and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25ng nicotine/ml) lowered the 5-HT(1B) receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250ng nicotine/ml) increased the ET(B) receptor-mediated contraction. The combined 5-HT(1B) and 5-HT(2A) receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or nicotine (250ng nicotine/ml). Only the DSS group showed that the increase of 5-HT(1B) receptor-mediated contraction occurred at the transcriptional level, demonstrated by an increased mRNA expression for the receptor. Thus, snus and nicotine alter the GPCR expression in the cerebral arteries, which may be involved in cerebral vascular disease.


PubMed | Glostrup Research Institute
Type: Journal Article | Journal: Experimental brain research | Year: 2012

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries after stroke. Here, we evaluate changes of ET(B) and 5-HT(1B) receptors, intracellular calcium levels, and calcium channel expression in rat middle cerebral artery (MCA) after focal cerebral ischemia and in vitro organ culture, a proposed model of vasoconstrictor receptor changes after stroke. Rats were subjected to 2h MCA occlusion followed by reperfusion for 1 or 24h. Alternatively, MCAs from nave rats were cultured for 1 or 24h. ET(B) and 5-HT(1B) receptor-mediated contractions were evaluated by wire myography. Receptor and channel expressions were measured by real-time PCR and immunohistochemistry. Intracellular calcium was measured by FURA-2. Expression and contractile functions of ET(B) and 5-HT(1B) receptors were strongly upregulated and slightly downregulated, respectively, 24h after experimental stroke or organ culture. ET(B) receptor-mediated contraction was mediated by calcium from intracellular and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential canonical calcium channels, but not voltage-operated calcium channels.

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