Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis

© 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM and RC were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young Investigator Award

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15–16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at −140 mV; −39.0 ± 4.5 and −42.3 ± 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V1/2 was −61.0 ± 1.7 and −64.5 ± 2.8 mV; k was 20.5 ± 0.8 and 22.8 ± 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 ± 36 to 312 ± 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an ∼40 and 60% reduction in membrane capacitance in FDB fibers from 15–16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90° out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein–tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle

Ishockeypublikens påverkan : "När det är bra tryck i arenan så är de ju som en sjätte spelare!"

Sammanfattning Titel: Ishockeypublikens påverkan – ”När det är bra tryck i arenan så är de ju som en sjätte spelare!” (Citatet i titeln är hämtat från intervjun med respondent 6) Nyckelord: Motivation, Självförtroende, Nervositet, Ängslan, Ishockeyspelare, Publikpåverkan. Till varje lag i elitserien finns det publik som vill stödja sitt lag. De använder sig utav olika metoder för att hjälpa sitt lag till framgång. Stödet från publiken påverkar naturligtvis spelarna men det finns en begränsad tidigare forskning som kan visa på vilket sätt de reagerar psykologiskt. Syftet med studien var att undersöka hur eltishockeyspelares motivation, självförtroende och ängslan/nervositet påverkas av publiken under matchdagar. Metoden som användes för att uppfylla detta syfte var en kvalitativ intervju. De som deltog i studien var sju stycken (ålder 19-34 år) manliga elitseriespelare i ishockey från två olika lag. Lagen som valdes ut är båda framgångsrika och har starka traditioner inom svensk ishockey. För att analysera intervjuerna så har dessa transkriberats. Resultatdelen presenterades i tre olika delar utifrån de tre teman som intervjuerna utgick ifrån. Det visade sig att alla de tillfrågade spelarna tycker att det är som roligast med ishockey när det är fullsatt i arenan. Då blir det en extra laddning i luften och spelarnas motivation höjs. Samtidigt bildas en form av nervositet hos spelarna eftersom de då förstår att matchen betyder något extra. En viss nervositet innan match gör oftast att spelarna presterar bättre. I diskussionen kopplades tidigare forskning ihop med studiens resultat och flera av dem styrktes då med hjälp av de tidigare studierna. Vi analyserade och använde egna reflektioner för att verkligen fördjupa vår förståelse för hur elitishockeyspelare påverkas av publiken

Effective construction of data aggregation services in Java

Stora mängder data genereras dagligen av slutanvändare hos olika tjänster. Denna data tenderar att tillhandahållas av olika aktörer, vilket skapar en fragmenterad marknad där slutanvändare måste nyttja flera programvaror för att ta del av all sin data. Detta kan motverkas genom utvecklandet av aggregeringstjänster vilka samlar data från flera tjänster på en enskild ändpunkt. Utveckling av denna typ av tjänster riskerar dock att bli kostsamt och tidskrävande, då ny kod skrivs för flera projekt trots att stora delar av funktionaliteten är snarlik. För att undvika detta kan etablerade tekniker och ramverk användas för att på så vis återanvända mer generella komponenter. Vilka av dessa tekniker som är bäst lämpade och således kan anses vara mest effektiva ur ett utvecklingsperspektiv, kan dock vara svårt att avgöra. Därför baseras denna uppsats på vad som genom analys av akademisk litteratur kan utläsas som ett akademiskt konsensus. Innan denna uppsats påbörjades utvecklades en Java-baserad dataaggeringstjänst baserad på krav från ÅF i Malmö. Denna experimentella implementation har som syfte att samla in data från två separata tjänster, och tillgängliggöra denna på en enskild ändpunkt. Efter att implementationen färdigställts påbörjades arbetet på uppsatsen. Denna består av en litteraturstudie för att undersöka vilka tekniker och ramverk som akademisk forskning funnit bäst lämpad för användningsområdet. Vidare används resultaten från studien även för att analysera i vilken grad dessa korrelerar med de krav som ÅF presenterade inför den experimentella implementationen. Litteraturstudien visar på att de teknikmässiga val som gjordes av företaget i stor utsträckning korrelerar med de tekniker som akademisk forskning funnit bäst lämpade för användningsområdet. Detta innefattar bland annat OAuth 2.0 för autentisering, JSON som serialiseringsformat samt REST som kommunikationsarkitektur. Vidare visar denna litteraturstudie på en eventuell lucka inom den tillgängliga litteraturen, då sökningar kring specifika programvaror relaterade till området endast resulterar i en mindre mängd artiklar.Large quantities of data are generated daily by the end users of various services. This data is often provided by different providers, which creates a fragmented market where the end users have to utilize multiple applications in order to access all of their data. This can be counteracted by the development of aggregation services that gather data from multiple services to a combined endpoint. The development of these kinds of services does however run the risk of becoming costly and time-consuming since new code is written for several projects even though large portions of the functionality is similar. To avoid this, established technologies and frameworks can be utilized, thereby reusing the more general components. Which of the technologies are the best suited, and thereby can be considered the most effective from a development perspective, can however be difficult to determine. This essay is therefore based on what can be considered an academic consensus through analysis of literature regarding earlier reasearch on the subject. Before the writing of the essay began a Java-based data aggregation service was developed, based on requirements from the company ÅF in Malmö. The purpose of this experimental implementation is to gather data from two separate services, and make them accessible on a unified endpoint. After the implementation was finished, work on the essay began. This consists of a literature review to investigate what technologies and frameworks that has been found best suited for this area of application by academic research. The results from this study are also used to analyze the extent of the correlation between the results and the requirements presented by ÅF regarding the experimental implementation. The literature review shows that the choices made by the company largely correlates with the technologies that the academic research has found best suited for this area of application. This includes OAuth 2.0 for authentication, JSON as a serialization format and REST for communications architecture. The literature review also indicates a possible gap within the available academic literature since searches regarding specific pieces of software related to the subject only results in a small amount of articles

X-ray Analysis of Structural Changes Induced by Reduced Nicotinamide Adenine Dinucleotide When Bound to Cysteine-46-Carboxymethylated Liver Alcohol Dehydrogenase

The structure of the complex between Cys-46-carboxymethylated horse liver alcohol dehydrogenase (CM-LADH) and reduced nicotinamide adenine dinucleotide (NADH) has been determined by X-ray analysis. The complex represents NADH binding to the orthorhombic, "open" conformation of the enzyme. Coenzyme binding here induces a local structural change in the peptide loop 293-297, but there is no domain rotation, as observed for the "closed" conformation of the protein. This local movement of a few residues in the loop is sufficient to trap the nicotinamide ring of NADH within the active-site area close to a productive binding position. The carboxymethyl group on the zinc ligand cysteine-46 is oriented between the pyrophosphate bridge of NADH and the guanidinium group of arginine-369 and can occupy this position because the coenzyme binding cleft remains open and unchanged upon coenzyme binding. The zinc coordination sphere is distorted, and the position of the metal atom is shifted 1 A compared to native unliganded LADH. The distance between the zinc ion and the sulfur of the alkylated cysteine residue is of the order of 3 A. Alkylation experiments were performed at 0.15 and 10 mM iodoacetate, and peptide maps were examined. Gentle treatment with reagent yields an enzyme product which is substituted at only one of the two zinc binding sites per subunit of LADH (Cys-46). This enzyme species maintains its structural integrity; it binds coenzyme which induces conformational changes resolved into two steps. Thus, in addition to the orthorhombic complex, a crystalline NADH complex in the closed conformation of CM-LADH was obtained. These crystals showed enzymic activity, and single crystals were analyzed with microspectro-photometric methods. Formation of the stable crystalline abortive complex between CM-LADH-NAD"1" and 4-fran5-(7V,A-dimethylamino)cinnamaldehyde (DACA) could be observed upon addition of excess aldehyde to the closed complex of CM-LADH-NADH. The CM-LADH-NAD+-DACA complex is characterized by an intense absorption band with a at 456 nm which corresponds to a shift in the spectrum of free DACA of approximately 60 nm. At the higher concentration of iodoacetate, three of the cysteine ligands to the second zinc atom (Cys-100, -103, and -111) are alkylated in addition to Cys-46. This enzyme product rapidly denatures and cannot be crystallized under our conditions. This is an experimental indication that the intact noncatalytic zinc binding site contributes to the structural stability of the protein. © 1985, American Chemical Society. All rights reserved