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Career situation of first and presenting authorYoung investigator.IntroductionWithin the rheumatoid joint, mesenchymal stromal cells (MSC) up-regulate podoplanin with unknown consequences for disease pathogenesis. The function of podoplanin has been linked to enhanced migratory potential and interactions with platelets. However, it is unclear how these two cell types interact with one another, given that MSC and platelets are usually located in in different anatomical compartments (tissue vs blood respectively) separated by the blood vascular endothelial cells (EC).ObjectivesHere, we examined the functional consequences of podoplanin expression on the migratory potential of MSC and their interactions with circulating platelets.MethodsHuman MSC were isolated from healthy controls Comparisons were made between podoplanin positive and negative MSC. MSC migration across 8 um pore filters following treatment with anti-siRNA podoplanin or Rho GTPases inhibitors was assessed. MSC-platelet interactions were assessed by culturing MSC on the basal surface of 3 um pore filters and perfusing fluorescently labelled platelets in whole blood over the apical surface. In some cases, the apical surface of the filter was pre-coated with EC, forming an EC-MSC co-culture, prior to platelet perfusion.ResultsExpression of podoplanin significantly enhanced the migration of MSC compared to MSC lacking podoplanin. Rac-1 inhibition altered the membrane localisation of podoplanin and in turn significantly reduced MSC migration. Blocking Rac-1 activity had no effect on the migration of MSC lacking podoplanin, indicating it was responsible for regulation of migration through podoplanin. When podoplanin-expressing MSC were seeded on the basal surface of a porous filter, they were able to capture platelets perfused over the uncoated apical surface and induce platelet aggregation. Similar microthrombi were observed when EC were co-cultured on the apical surface. Confocal imaging shows podoplanin-expressing MSC extending processes into the EC layer, which could interact with circulating platelets. In both models, platelet aggregation induced by podoplanin-expressing MSC was inhibited by recombinant soluble CLEC-2.ConclusionsPodoplanin enhances the migratory capacity of tissue-resident MSC enabling them to move more rapidly within the rheumatoid joint. Moreover, podoplanin allows MSC to interact with both circulating and tissue platelets to elicit either protective or pathogenic responses.AcknowledgementsThis work was funded by an Versus Arthritis Career Development Fellowship, a MRC-funded PhD studentship and BHF Grant.Disclosure of InterestNone declared.

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Reprogramming of stromal cell metabolism toward glycolysis in cancerous tissue provides a source of high-energy metabolic intermediates which support proliferation, invasion and metastasis of invading tumour cells; a phenomenon known as the ‘Reverse Warburg’ effect. We have explored whether a similar interplay may fuel pathogenic processes in autoinflammatory diseases such as rheumatoid arthritis (RA). In RA, fibroblasts are themselves transformed to an aggressive, tumour-like phenotype and also reside in close proximity to energetically demanding immune cells including resident macrophages, infiltrating neutrophils and autoaggressive lymphocytes.We used nuclear magnetic resonance spectroscopy and metabolic flux analysis to identify pathological changes to metabolism in both primary human RA synovial cells, and murine cells from TNF-ΔARE and collagen-induced models of arthritis.We have shown that the fibroblast metabotype correlates with C-reactive protein as a systemic marker of inflammation, and can predict resolution of acute synovitis or progression to chronic RA prior. We have confirmed the clinical prognostic potential of metabolic profiling in inflammatory diseases and demonstrated that synovial fibroblasts increase glycolysis but not mitochondrial respiration in response to inflammatory cues such as tumour necrosis factor-α. Furthermore, analysis of monocarboxylate transporters (MCT-4) supports the likelihood that increased glycolytic products such as lactate may be shuttled between fibroblasts and myeloid cells in situ.Our findings shed light on the metabolic relationships between cells at sites of autoinflammatory pathology and support the therapeutic targeting of the glycolytic pathway beyond oncology.European Union's FP7 Health Programme FP7-HEALTH-F2–2012–305549AR UK Rheumatoid Arthritis Pathogenesis Centre of Excellence 20298

Bericht: Chemische Analyse anorganischer KSrper. Benzin (oder Benzol) empfoblen. Die Benzinschieht nimmt bei Gegenwart yon Kupfer eine intensiv grane Farbe an. I n der w/~ssrigen L6sung liisst sich mit Ammoniak k'ein Kupfer mehi" nachweisen Die Reaktion, fiber deren Empfindlichkeit Angaben nicht gemacht werden, beruht t~brigens, wie C h a r i t s c h k o f f 1) sp~ter erkann~ hat, lediglich auf der Anwesenheit der einbasischen P e n t a m e t h y l e n s~u r e und ist for den Pentamenthylenring charakteristisch. Eine intensive Blauf~rbung zeigt bei Anwesenheit von Kupfer eine alkalischeLSsung yon 1. 2 D i a m i d o a n t h r a c h i n o n-3-s u l f o s~i u r e. Diese Reaktion. die R. U h l e n h u t h~) gefunden hat, ist iiusserst empfindlich; die blaue Ftirbung ist bei 0.0019 mg Ca in 1 c c m noch gut sichtbur, die Grenze der Empfindliehkeit liegt bei 1 : 5 0 0 0 0 0 0. Man bereitet das Reagens durch Li)sen yon 0.5 g der Sulfos~ure in 500 c c m Wasser unter Zusatz yon 4 0 c c m NaOg-L0sung yon 40 o B6. G. M a l a t e s t a and E. Di N o l a 3) verst~rken die Reaktion durch Ammoniak; sie schlagen ffir das Reagens folgende Zusammensetzung vor: 0~5 g der Sulfosfiure. 100 c c m konz. Ammoniak. 340 c c m Wasser, 40 c c m Na0H-L(isung yon 40 o B6. Wie Kupfer so rufen auch Kobalt und Nickel Blauf~rbung hervor. Zur Unterscheidung ftigt man Ammoniumchlorid zu; die Kupferfi~rbung schl~gt dann in Rot urn. wiihrend Kobaltand Nickelfttrbung unveriindert bleiben. D. S c h e n k 4) verwertet die F e h l i n g s c l l e R e a k t i o n. Er macht 10 c c m der zu prtlfenden LSsung mit der Mischung yon Seignettesalz und Natronlauge schwach alkalisch, gibt 4 5 KOrnchen Traubenzucker hinzu and stellt die Misehung nach dem Umschtitteln in ein siedendes Wasserbad. Ist Kupfer zugegen, so scheidet sich Kuprooxyd aus. Die Empfindlichkeit der Reaktion ist etwa 1 : 3 1 4 0 0 0. Ein sehr empfindliches Reagens auf Kupfer i s t nach den Angaben yon W. G. L y l e , L. J. C u r t m a n n und J. T. W. M a r s h a l l b) ferner eine w~ssrige LSsung yon a-A m i n on -c a p r o n s~u r e (CH~(CH.~)3CHNH2COOH). Diese ruft auch in ganz verdannten LOsungen yon Kupfer (1 : 333000) noch eine deutliche Reaktion hervor. Zur Ausfahrung der Prfifung versetzt man 1 c c m der zu untersuchenden L0sung mit 1 c c m einer 40°,oigen NatriumazetatlOsung und 1 c c m Reagens (0,67g Siiure in 100ccm Wasser). Der sieh ausscheidende graublaue Niederschlag setzt sich beim Umriihren an den Wandungen des Reagensglases ab. Ausser Kupfer geben nut Quecksilber und Zink eine Fiillung. Die Gegenwart yon Natriumchlorid verhindert die Aus