Value of the radiolabelled GLP-1 receptor antagonist exendin(9-39) for targeting of GLP-1 receptor-expressing pancreatic tissues in mice and humans

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans.

Association between variants of the leptin receptor gene (LEPR) and overweight: a systematic review and an analysis of the CoLaus study

Background Three non-synonymous single nucleotide polymorphisms (Q223R, K109R and K656N) of the leptin receptor gene (LEPR) have been tested for association with obesity-related outcomes in multiple studies, showing inconclusive results. We performed a systematic review and meta-analysis on the association of the three LEPR variants with BMI. In addition, we analysed 15 SNPs within the LEPR gene in the CoLaus study, assessing the interaction of the variants with sex. Methodology/Principal Findings We searched electronic databases, including population-based studies that investigated the association between LEPR variants Q223R, K109R and K656N and obesity- related phenotypes in healthy, unrelated subjects. We furthermore performed meta-analyses of the genotype and allele frequencies in case-control studies. Results were stratified by SNP and by potential effect modifiers. CoLaus data were analysed by logistic and linear regressions and tested for interaction with sex. The meta-analysis of published data did not show an overall association between any of the tested LEPR variants and overweight. However, the choice of a BMI cut-off value to distinguish cases from controls was crucial to explain heterogeneity in Q223R. Differences in allele frequencies across ethnic groups are compatible with natural selection of derived alleles in Q223R and K109R and of the ancient allele in K656N in Asians. In CoLaus, the rs10128072, rs3790438 and rs3790437 variants showed interaction with sex for their association with overweight, waist circumference and fat mass in linear regressions. Conclusions Our systematic review and analysis of primary data from the CoLaus study did not show an overall association between LEPR SNPs and overweight. Most studies were underpowered to detect small effect sizes. A potential effect modification by sex, population stratification, as well as the role of natural selection should be addressed in future genetic association studies.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD + BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD + BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD + BCP did not show any advantage over the use of EMD alone.

Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 mumol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 mumol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.

[(99m)Tc]Demomedin C, a radioligand based on human gastrin releasing peptide(18-27): synthesis and preclinical evaluation in gastrin releasing peptide receptor-expressing models

The synthesis and preclinical evaluation of [(99m)Tc]Demomedin C in GRPR-expressing models are reported. Demomedin C resulted by coupling a Boc-protected N(4)-chelator to neuromedin C (human GRP(18-27)), which, after (99m)Tc-labeling, afforded [(99m)Tc]Demomedin C. Demomedin C showed high affinity and selectivity for the GRPR during receptor autoradiography on human cancer samples (IC(50) in nM: GRPR, 1.4 ± 0.2; NMBR, 106 ± 18; and BB(3)R, >1000). It triggered GRPR internalization in HEK-GRPR cells and Ca(2+) release in PC-3 cells (EC(50) = 1.3 nM). [(99m)Tc]Demomedin C rapidly and specifically internalized at 37 °C in PC-3 cells and was stable in mouse plasma. [(99m)Tc]Demomedin C efficiently and specifically localized in human PC-3 implants in mice (9.84 ± 0.81%ID/g at 1 h pi; 6.36 ± 0.85%ID/g at 4 h pi, and 0.41 ± 0.07%ID/g at 4 h pi block). Thus, human GRP-based radioligands, such as [(99m)Tc]Demomedin C, can successfully target GRPR-expressing human tumors in vivo while displaying attractive biological features--e.g. higher GRPR-selectivity--vs their frog-homologues.

Influence of GABA(A) receptor α subunit isoforms on the benzodiazepine binding site

Classical benzodiazepines, such as diazepam, interact with α(x)β(2)γ(2) GABA(A) receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and with receptors containing the homologous mutations in α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2). The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and homologous positions in α(2), α(3), α(5) and α(6) with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA(A) receptor isoform α(1)β(2)γ(2), α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2).

Marked global reduction in mGluR5 receptor binding in smokers and ex-smokers determined by [11C]ABP688 positron emission tomography

Nicotine addiction is a major public health problem, resulting in primary glutamatergic dysfunction. We measured the glutamate receptor binding in the human brain and provided direct evidence for the abnormal glutamate system in smokers. Because antagonism of the metabotropic glutamate receptor 5 (mGluR5) reduced nicotine self-administration in rats and mice, mGluR5 is suggested to be involved in nicotine addiction. mGluR5 receptor binding specifically to an allosteric site was observed by using positron emission tomography with [(11)C]ABP688. We found a marked global reduction (20.6%; P < 0.0001) in the mGluR5 distribution volume ratio (DVR) in the gray matter of 14 smokers. The most prominent reductions were found in the bilateral medial orbitofrontal cortex. Compared with 14 nonsmokers, 14 ex-smokers had global reductions in the average gray matter mGluR5 DVR (11.5%; P < 0.005), and there was a significant difference in average gray matter mGluR5 DVR between smokers and ex-smokers (9.2%; P < 0.01). Clinical variables reflecting current nicotine consumption, dependence and abstinence were not correlated with mGluR5 DVR. This decrease in mGluR5 receptor binding may be an adaptation to chronic increases in glutamate induced by chronic nicotine administration, and the decreased down-regulation seen in the ex-smokers could be due to incomplete recovery of the receptors, especially because the ex-smokers were abstinent for only 25 wk on average. These results encourage the development and testing of drugs against addiction that directly target the glutamatergic system.

Orexin receptor antagonism, a new sleep-enabling paradigm: a proof-of-concept clinical trial

The orexin system is a key regulator of sleep and wakefulness. In a multicenter, double-blind, randomized, placebo-controlled, two-way crossover study, 161 primary insomnia patients received either the dual orexin receptor antagonist almorexant, at 400, 200, 100, or 50 mg in consecutive stages, or placebo on treatment nights at 1-week intervals. The primary end point was sleep efficiency (SE) measured by polysomnography; secondary end points were objective latency to persistent sleep (LPS), wake after sleep onset (WASO), safety, and tolerability. Dose-dependent almorexant effects were observed on SE , LPS , and WASO . SE improved significantly after almorexant 400 mg vs. placebo (mean treatment effect 14.4%; P < 0.001). LPS (–18 min (P = 0.02)) and WASO (–54 min (P < 0.001)) decreased significantly at 400 mg vs. placebo. Adverse-event incidence was dose-related. Almorexant consistently and dose-dependently improved sleep variables. The orexin system may offer a new treatment approach for primary insomnia.

Induction of the Neurokinin 1 Receptor by TNFα in Endometriotic Tissue Provides the Potential for Neurogenic Control Over Endometriotic Lesion Growth

Context: Endometriosis is characterized by the growth of ectopic endometrial tissue. Nerve fibers are frequently associated with ectopic lesions, and neurogenic inflammation may play a role in endometriosis. Objective: The purpose of this study was to determine the presence of tachykinin receptors in endometriotic lesions and the role of TNFα on their expression. Design: This study was an assessment of matching eutopic and ectopic endometrial tissue and peritoneal fluid from patients with endometriosis and an in vitro analysis of primary endometrial cells. Setting: The setting was a university hospital. Patients: Participants were premenopausal women undergoing laparoscopy. Interventions: Endometriotic lesions were removed surgically. Main Outcome Measures: Tachykinin mRNA (TACR1/2) and protein (neurokinin 1 receptor [NK1R]) expression in both eutopic and ectopic endometrial tissue from patients with endometriosis and the correlation to peritoneal fluid TNFα were measured. Primary endometrial epithelial and stromal cells were assessed in vitro to determine the induction of TACR1/2 and NK1R expression after TNFα treatment. Cell viability of endometrial stromal cells after substance P exposure was also assessed. Results: Expression of both TACR1 and TACR2 mRNA was significantly higher in the ectopic than in the eutopic tissue. Both TACR1 mRNA and NK1R protein expression was significantly correlated with peritoneal fluid TNFα, and in vitro studies confirmed that TNFα treatment induced both TACR1 mRNA and NK1R protein expression in endometrial stromal cells. In endometrial stromal cells, substance P treatment enhanced cell viability, which was inhibited by a specific NK1R antagonist. Conclusions: NK1R expression is induced in ectopic endometrial tissue by peritoneal TNFα. Induction of NK1R expression may permit endometriotic lesion maintenance via exposure to substance P.

First continuous phosphate record from Greenland ice cores

A continuous and highly sensitive absorption method for detection of soluble phosphate in ice cores has been developed using a molybdate reagent and a 2m liquid waveg- uide (LWCC). The method is optimized to meet the low concentrations of phosphate in Greenland ice, it has a detection limit of around 0.1ppb and a depth resolution of approximately 2cm. The new method has been applied to obtain phosphate concen- trations from segments of two Northern Greenland ice cores: from a shallow firn core covering the most recent 120yr and from the recently obtained deep NEEM ice core in which sections from the late glacial period have been analysed. Phosphate con- centrations in 20th century ice are around 0.32ppb with no indication of anthropogenic influence in the most recent ice. In the glacial part of the NEEM ice core concentra- tions in the cold stadial periods are significantly higher, in the range of 6–24ppb, while interstadial ice concentrations are around 2ppb. In the shallow firn core, a strong cor- relation between concentrations of phosphate and insoluble dust suggests a similar deposition pattern for phosphate and dust. In the glacial ice, phosphate and dust also correlate quite strongly, however it is most likely that this correlation originates from the phosphate binding to dust during transport, with only a fraction coming directly from dust. Additionally a constant ratio between phosphate and potassium concentrations shows evidence of a possible biogenic land source.

Expression of FGFRL1, a novel fibroblast growth factor receptor, during embryonic development

FGFRL1 is a novel member of the fibroblast growth factor receptor (FGFR) family. To investigate its expression during mammalian embryonic development, we have used the mouse system. Expression of Fgfrl1 is very low in mouse embryos of day 6 but steadily increases until birth. As demonstrated by in situ hybridization of 16-day-old embryos, the Fgfrl1 mRNA occurs in cartilaginous structures such as the primordia of bones and the permanent cartilage of the trachea, the ribs and the nose. In addition, some muscle types, including the muscles of the tongue and the diaphragm, express Fgfrl1 at relatively high level. In contrast, the heart and the skeletal muscles of the limbs, as well as many other organs (brain, lung, liver, kidney, gut) express Fgfrl1 only at basal level. It is conceivable that Fgfrl1 interacts with other Fgfrs, which are expressed in cartilage and muscle, to modulate FGF signaling.