Single Molecule In Vivo Analysis of Toll-Like Receptor 9 and CpG DNA Interaction

Toll-like receptor 9 (TLR9) activates the innate immune system in response to oligonucleotides rich in CpG whereas DNA lacking CpG could inhibit its activation. However, the mechanism of how TLR9 interacts with nucleic acid and becomes activated in live cells is not well understood. Here, we report on the successful implementation of single molecule tools, constituting fluorescence correlation/cross-correlation spectroscopy (FCS and FCCS) and photon count histogram (PCH) with fluorescence lifetime imaging (FLIM) to study the interaction of TLR9-GFP with Cy5 labeled oligonucleotide containing CpG or lacking CpG in live HEK 293 cells. Our findings show that i) TLR9 predominantly forms homodimers (80%) before binding to a ligand and further addition of CpG or non CpG DNA does not necessarily increase the proportion of TLR9 dimers, ii) CpG DNA has a lower dissociation constant (62 nM±9 nM) compared to non CpG DNA (153 nM±26 nM) upon binding to TLR9, suggesting that a motif specific binding affinity of TLR9 could be an important factor in instituting a conformational change-dependant activation, and iii) both CpG and non CpG DNA binds to TLR9 with a 1∶2 stoichiometry in vivo. Collectively, through our findings we establish an in vivo model of TLR9 binding and activation by CpG DNA using single molecule fluorescence techniques for single cell studies

Influence of Caloric Restriction on Constitutive Expression of NF-κB in an Experimental Mouse Astrocytoma

Many of the current standard therapies employed for the management of primary malignant brain cancers are largely viewed as palliative, ultimately because these conventional strategies have been shown, in many instances, to decrease patient quality of life while only offering a modest increase in the length of survival. We propose that caloric restriction (CR) is an alternative metabolic therapy for brain cancer management that will not only improve survival but also reduce the morbidity associated with disease. Although we have shown that CR manages tumor growth and improves survival through multiple molecular and biochemical mechanisms, little information is known about the role that CR plays in modulating inflammation in brain tumor tissue.Phosphorylation and activation of nuclear factor κB (NF-κB) results in the transactivation of many genes including those encoding cycloxygenase-2 (COX-2) and allograft inflammatory factor-1 (AIF-1), both of which are proteins that are primarily expressed by inflammatory and malignant cancer cells. COX-2 has been shown to enhance inflammation and promote tumor cell survival in both in vitro and in vivo studies. In the current report, we demonstrate that the p65 subunit of NF-κB was expressed constitutively in the CT-2A tumor compared with contra-lateral normal brain tissue, and we also show that CR reduces (i) the phosphorylation and degree of transcriptional activation of the NF-κB-dependent genes COX-2 and AIF-1 in tumor tissue, as well as (ii) the expression of proinflammatory markers lying downstream of NF-κB in the CT-2A malignant mouse astrocytoma, [e.g. macrophage inflammatory protein-2 (MIP-2)]. On the whole, our date indicate that the NF-κB inflammatory pathway is constitutively activated in the CT-2A astrocytoma and that CR targets this pathway and inflammation.CR could be effective in reducing malignant brain tumor growth in part by inhibiting inflammation in the primary brain tumor

The regulation of IL-10 expression

Interleukin (IL)-10 is an important immunoregulatory cytokine and an understanding of how IL-10 expression is controlled is critical in the design of immune intervention strategies. IL-10 is produced by almost all cell types within the innate (including macrophages, monocytes, dendritic cells (DCs), mast cells, neutrophils, eosinophils and natural killer cells) and adaptive (including CD4(+) T cells, CD8(+) T cells and B cells) immune systems. The mechanisms of IL-10 regulation operate at several stages including chromatin remodelling at the Il10 locus, transcriptional regulation of Il10 expression and post-transcriptional regulation of Il10 mRNA. In addition, whereas some aspects of Il10 gene regulation are conserved between different immune cell types, several are cell type- or stimulus-specific. Here, we outline the complexity of IL-10 production by discussing what is known about its regulation in macrophages, monocytes, DCs and CD4(+) T helper cells

The emerging modern face of mood disorders: a didactic editorial with a detailed presentation of data and definitions

The present work represents a detailed description of our current understanding and knowledge of the epidemiology, etiopathogenesis and clinical manifestations of mood disorders, their comorbidity and overlap, and the effect of variables such as gender and age. This review article is largely based on the 'Mood disorders' chapter of the Wikibooks Textbook of Psychiatry http://en.wikibooks.org/wiki/Textbook_of_Psychiatry/Mood_Disorders

Prevention of overweight at Dutch secondary schools: the national and regional picture

With regard to the food and physical activity offered at secondary schools, improvements can be made that can contribute to the reduction of overweight among the youth. This was shown in the first national survey on the prevention of overweight at Dutch secondary schools. The survey cited canteens and/or soda vending machines in almost 90% of the schools, and candy dispensers in 77%. More than half the schools described the food assortment as being primarily rich in calories. In most cases the school board can influence the food supplied to the schools: 60% have an influence on food supplied in machines and 80% have an influence on what is offered in canteens. Only one-quarter of the locations expected a positive change in the following six months. The school environment can also be important: 75% of schools are located near a supermarket, snack bar or petrol station. Pupils at about half the schools have access to these food facilities, since they are allowed to leave the schoolyard during school hours. In the lower classes most schools offer 3-4 hours a week of physical education, but 25% of the capacity of the sport facilities is considered as being insufficient. The schoolyard itself offers possibilities for physical activity: pupils can walk in or around the schoolyard at 80% of the schools and 40% of the schools offer additional sport facilities on the schoolyard. Traffic circumstances around the school are considered to be average to very safe at 90% of the schools. There is still a lot of improvement possible. Only one-third of the schools focuses on overweight prevention outside the curriculum and 13% have specific guidelines for screening and follow-up monitoring of overweight pupils. Most schools expect to pay more attention to overweight prevention in the near future. Since prevalence of overweight among Dutch teenagers will probably rise further, RIVM recommends that schools put these good intentions into practice.In het voedings- en beweegaanbod op middelbare scholen zijn verbeteringen mogelijk die kunnen bijdragen aan een reductie van overgewicht bij de jeugd. Dit blijkt uit de eerste landelijke enquete naar de preventie van overgewicht op middelbare schoollocaties in Nederland. Bijna 90 % van de schoollocaties heeft een kantine en/of frisdrankautomaat en 77 % heeft een snoepautomaat. Ruim de helft omschrijft het aanbod van producten op school als overwegend calorierijk. In veel gevallen kan de directie het aanbod beinvloeden: 60 % kan de samenstelling van de automaten aanpassen en 80% heeft invloed op het kantineaanbod. Slechts een kwart verwacht echter een verandering van het aanbod in het komende half jaar. Ook de omgeving van de school is van belang. Driekwart van de schoollocaties bevindt zich op minder dan 1 km afstand van een supermarkt, snackbar of tankstation. Op ongeveer de helft van de schoollocaties hebben leerlingen toegang tot deze voorzieningen omdat ze in tussenuren en pauzes het schoolterrein mogen verlaten. Op de meeste schoollocaties wordt in de onderbouw 3 tot 4 uur lichamelijke opvoeding per week gegeven. De capaciteit van de sportzalen wordt door 25% als onvoldoende ervaren. Het schoolterrein zelf biedt vaak mogelijkheden om te bewegen. Op 80% van de schoollocaties kunnen jongeren op of rondom het terrein wandelen en 40% van de schoollocaties biedt extra sportfaciliteiten op het schoolterrein. De verkeersveiligheid in de buurt van de school wordt door 90% als gemiddeld tot zeer veilig beoordeeld. Er is nog veel verbetering mogelijk. Slechts eenderde van de schoollocaties geeft buiten het reguliere onderwijs aandacht aan het thema overgewicht. Op 13% van de schoollocaties zijn er richtlijnen voor signalering, advisering en hulpverlening voor kinderen met overgewicht. De meeste schoollocaties verwachten in de toekomst meer aandacht te geven aan overgewicht. Gezien de verwachte toename van overgewicht onder jongeren in Nederland adviseert RIVM dat scholen deze voornemens in de praktijk brengen