Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism

Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder

Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance.This research has been funded through projects FEDER MINECO INNPACTO IPT-2011-1132-010000, CTQ/2013/45875R, and PrometeoII/2014/040 (GVA).Tortajada-Genaro, LA.; Mena-Mollá, S.; Niñoles Rodenes, R.; Puigmule, M.; Viladevall, L.; Maquieira Catala, Á. (2016). Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Analytical and Bioanalytical Chemistry. 408(9):2339-2345. https://doi.org/10.1007/s00216-016-9332-3S233923454089Cortese S. The neurobiology and genetics of Attention-Deficit/Hyperactivity Disorder (ADHD): what every clinician should know. Eur J Paediatr Neurol. 2012;16:422–33.Contini V, Rovaris DL, Victor MM, Grevet EH, Rohde LA, Bau CH. Pharmacogenetics of response to methylphenidate in adult patients with attention-deficit/hyperactivity disorder (ADHD): a systematic review. Eur Neuropsychopharmacol. 2013;23:555–60.Gardiner SJ, Begg EJ. Pharmacogenetics, drug-metabolizing enzymes, and clinical practice. Pharmacol Rev. 2006;58(3):521–90.Abul-Husn NS, Obeng AO, Sanderson SC, Gottesman O, Scott SA. Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med. 2014;7:227.Altman RB, Flockhart D, Goldstein DB, editors. Principles of pharmacogenetics and pharmacogenomics. Cambridge: Cambridge University Press; 2012.Hawi Z, Cummins TDR, Tong J, Johnson B, Lau R, Samarrai W, et al. The molecular genetic architecture of attention deficit hyperactivity disorder. Mol Psychiatry. 2015;20:289–97.Limaye N. Pharmacogenomics, Theranostics and Personalized Medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics. 2013;2:17–21.Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R, et al. Implementing genomic medicine in the clinic: the future is here. Genet Med. 2013;15:258–67.Kim S, Misra A. PharmGKB: the Pharmacogenomics Knowledge Base. Annu Rev Biomed Eng. 2007;9:289–320.Lucarelli F, Tombelli S, Minunni M, Marrazza G, Mascini M. Electrochemical and piezoelectric DNA biosensors for hybridisation detection. Anal Chim Acta. 2008;609:139–59.Knez K, Spasic D, Janssen KP, Lammertyn J. Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst. 2014;139:353–70.Choi JY, Kim YT, Byun JY, Ahn J, Chung S, Gweon DG, et al. Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping. Lab Chip. 2012;12:5146–54.Ragoussis J. Genotyping Technologies for Genetic Research. Annu Rev Genomics Hum Genet. 2009;10:117–33.Sethi D, Gandhi RP, Kuma P, Gupta KC. Chemical strategies for immobilization of oligonucleotides. Biotechnol J. 2009;4:1513–29.Bañuls MJ, Morais SB, Tortajada-Genaro LA, Maquieira A. Microarray Developed on Plastic Substrates. Microarray Technology: Methods and Applications, 2016; 37-51.Tortajada-Genaro LA, Rodrigo A, Hevia E, Mena S, Niñoles R, Maquieira A. Microarray on digital versatile disc for identification and genotyping of Salmonella and Campylobacter in meat products. Anal Bioanal Chem. 2015;407:7285–94.Kieling C, Genro JP, Hutz MH, Rohde LA. A current update on ADHD pharmacogenomics. Pharmacogenomics. 2010;11:407–19.Kim BN, Kim JW, Cummins TD, Bellgrove MA, Hawi Z, Hong SB, et al. Norepinephrine genes predict response time variability and methylphenidate-induced changes in neuropsychological function in attention deficit hyperactivity disorder. J Clin Psychopharmacol. 2013;33:356–62.Carpentier PJ, Arias Vasquez A, Hoogman M, Onnink M, Kan CC, Kooij JJS, et al. Shared and unique genetic contributions to attention deficit/hyperactivity disorder and substance use disorders: A pilot study of six candidate genes. Eur Neuropsychopharmacol. 2013;23:448–57.Zhang Y, Haraksingh R, Grubert F, Abyzov A, Gerstein M, Weissman S, et al. Child development and structural variation in the human genome. Child Dev. 2013;84:34–48.Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K. Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem. 2009;386:85–90.Choi JY, Kim YT, Ahn J, Kim KS, Gweon DG, Seo TS. Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping. Biosens Bioelectron. 2012;35:327–34.Konstantou JK, Ioannou PC, Christopoulos TK. Dual-allele dipstick assay for genotyping single nucleotide polymorphisms by primer extension reaction. Eur J Hum Genet. 2009;17:105–11.Sebastian T, Cooney CG, Parker J, Qu P, Perov A, Golova JB, et al. Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva. Clin Chim Acta. 2014;429:198–205

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction: In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation. Methods: Two specific n-3 compounds were tested, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), each at 0.1, 1 and 10 μM. Full thickness bovine cartilage explants, 5 mm in diameter, were cultured for 5 days with or without IL-1β and in the presence or absence of each n-3 compound. The media were replaced every 24 hours and assayed for sGAG content using the 1,9-dimethylmethylene blue (DMB) method. Chondrocyte viability was determined at the end of the culture period using fluorescence microscopy to visualise cells labelled with calcein AM and ethidium homodimer. Results: Treatment with IL-1β (10 ng.ml-1) produced a large increase in sGAG release compared to untreated controls, but with no effect on cell viability, which was maintained above 80% for all treatments. In the absence of IL-1β, both n-3 compounds induced a mild catabolic response with increased loss of sGAG, particularly at 10 μM. By contrast, in the presence of IL-1β, both EPA and DHA at 0.1 and 1 μM significantly reduced IL-1β-mediated sGAG loss. The efficacy of the EPA treatment was maintained at approximately 75% throughout the 5-day period. However, at the same concentrations, the efficacy of DHA, although initially greater, reduced to approximately half that of EPA after 5 days. For both EPA and DHA, the highest dose of 10 μM was less effective. Conclusions: The results support the hypothesis that n-3 compounds are anti-inflammatory through competitive inhibition of the arachidonic acid oxidation pathway. The efficacy of these compounds is likely to be even greater at more physiological levels of IL-1β. Thus we suggest that n-3 PUFAs, particularly EPA, have exciting therapeutic potential for preventing cartilage degradation associated with chronic inflammatory joint disease

Divergent Pro-Inflammatory Profile of Human Dendritic Cells in Response to Commensal and Pathogenic Bacteria Associated with the Airway Microbiota

Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3–5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota

Functionalized Positive Nanoparticles Reduce Mucin Swelling and Dispersion

Multi-functionalized nanoparticles (NPs) have been extensively investigated for their potential in household and commercial products, and biomedical applications. Previous reports have confirmed the cellular nanotoxicity and adverse inflammatory effects on pulmonary systems induced by NPs. However, possible health hazards resulting from mucus rheological disturbances induced by NPs are underexplored. Accumulation of viscous, poorly dispersed, and less transportable mucus leading to improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF), and COPD (Chronic Obstructive Pulmonary Disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms have not been resolved. Herein, we report that positively charged functionalized NPs can hinder mucin gel hydration and effectively induce mucin aggregation. The positively charged NPs can significantly reduce the rate of mucin matrix swelling by a maximum of 7.5 folds. These NPs significantly increase the size of aggregated mucin by approximately 30 times within 24 hrs. EGTA chelation of indigenous mucin crosslinkers (Ca2+ ions) was unable to effectively disperse NP-induced aggregated mucins. Our results have demonstrated that positively charged functionalized NPs can impede mucin gel swelling by crosslinking the matrix. This report also highlights the unexpected health risk of NP-induced change in mucus rheological properties resulting in possible mucociliary transport impairment on epithelial mucosa and related health problems. In addition, our data can serve as a prospective guideline for designing nanocarriers for airway drug delivery applications

Effects of Inhibition of Interleukin-6 Signalling on Insulin Sensitivity and Lipoprotein (A) Levels in Human Subjects with Rheumatoid Diseases

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that has been found to be increased in type 2 diabetic subjects. However, it still remains unclear if these elevated IL-6 levels are co-incidental or if this cytokine is causally related to the development of insulin resistance and type 2 diabetes in humans. Therefore, in the present study we examined insulin sensitivity, serum adipokine levels and lipid parameters in human subjects before and after treatment with the IL-6 receptor antibody Tocilizumab.11 non-diabetic patients with rheumatoid disease were included in the study. HOMA-IR was calculated and serum levels for leptin, adiponectin, triglycerides, LDL-cholesterol, HDL-cholesterol and lipoprotein (a) (Lp (a)) were measured before as well as one and three months after Tocilizumab treatment. The HOMA index for insulin resistance decreased significantly. While leptin concentrations were not altered by inhibition of IL-6 signalling, adiponectin concentrations significantly increased. Thus the leptin to adiponectin ratio, a novel marker for insulin resistance, exhibited a significant decrease. Serum triglycerides, LDL-cholesterol and HDL-cholesterol tended to be increased whereas Lp (a) levels significantly decreased.Inhibition of IL-6 signalling improves insulin sensitivity in humans with immunological disease suggesting that elevated IL-6 levels in type 2 diabetic subjects might be causally involved in the pathogenesis of insulin resistance. Furthermore, our data indicate that inhibition of IL-6 signalling decreases Lp (a) serum levels, which might reduce the cardiovascular risk of human subjects

Early acquisition and high nasopharyngeal co-colonisation by Streptococcus pneumoniae and three respiratory pathogens amongst Gambian new-borns and infants

BACKGROUND: Although Haemophilus influenzae type b (Hib), Staphylococcus aureus and Moraxella catarrhalis are important causes of invasive and mucosal bacterial disease among children, co-carriage with Streptococcus pneumoniae during infancy has not been determined in West Africa. METHODS: Species specific PCR was applied to detect each microbe using purified genomic DNA from 498 nasopharyngeal (NP) swabs collected from 30 Gambian neonates every two weeks from 0 to 6 months and bi-monthly up to 12 months. RESULTS: All infants carried S. pneumoniae, H. influenzae and M. catarrhalis at several time points during infancy. S.pneumoniae co-colonized the infant nasopharynx with at least one other pathogen nine out of ten times. There was early colonization of the newborns and neonates, the average times to first detection were 5, 7, 3 and 14 weeks for S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus respectively. The prevalence of S. pneumoniae, H. influenzae and M. catarrhalis increased among the neonates and exceeded 80% by 13, 15 and 23 weeks respectively. In contrast, the prevalence of S. aureus decreased from 50% among the newborns to 20% amongst nine-week old neonates. S. pneumoniae appeared to have a strong positive association with H. influenzae (OR 5.03; 95% CI 3.02, 8.39; p<0.01) and M. catarrhalis (OR 2.20; 95% CI 1.29; p<0.01) but it was negatively associated with S. aureus (OR 0.53; 95% CI 0.30, 0.94; p=0.03). CONCLUSION: This study shows early acquisition and high co-carriage of three important respiratory pathogens with S. pneumoniae in the nasopharyngeal mucosa among Gambian neonates and infants. This has important potential implications for the aetiology of respiratory polymicrobial infections, biofilm formation and vaccine strategies

The NAMPT inhibitor FK866 reverts the damage in spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Emerging data implicate nicotinamide phosphoribosyl transferase (NAMPT) in the pathogenesis of cancer and inflammation. NAMPT inhibitors have proven beneficial in inflammatory animal models of arthritis and endotoxic shock as well as in autoimmune encephalitis. Given the role of inflammatory responses in spinal cord injury (SCI), the effect of NAMPT inhibitors was examined in this setting.</p> <p>Methods</p> <p>We investigated the effects of the NAMPT inhibitor FK866 in an experimental compression model of SCI.</p> <p>Results</p> <p>Twenty-four hr following induction of SCI, a significant functional deficit accompanied widespread edema, demyelination, neuron loss and a substantial increase in TNF-α, IL-1β, PAR, NAMPT, Bax, MPO activity, NF-κB activation, astrogliosis and microglial activation was observed. Meanwhile, the expression of neurotrophins BDNF, GDNF, NT3 and anti-apoptotic Bcl-2 decreased significantly. Treatment with FK866 (10 mg/kg), the best known and characterized NAMPT inhibitor, at 1 h and 6 h after SCI rescued motor function, preserved perilesional gray and white matter, restored anti-apoptotic and neurotrophic factors, prevented the activation of neutrophils, microglia and astrocytes and inhibited the elevation of NAMPT, PAR, TNF-α, IL-1β, Bax expression and NF-κB activity.</p> <p>We show for the first time that FK866, a specific inhibitor of NAMPT, administered after SCI, is capable of reducing the secondary inflammatory injury and partly reduce permanent damage. We also show that NAMPT protein levels are increased upon SCI in the perilesional area which can be corrected by administration of FK866.</p> <p>Conclusions</p> <p>Our findings suggest that the inflammatory component associated to SCI is the primary target of these inhibitors.</p