Rheumatoid arthritis synovial microenvironment induces metabolic and functional adaptations in dendritic cells.

Rheumatoid arthritis (RA) is a chronic autoimmune disease which causes degradation of cartilage and bone. It is well appreciated that the pathogenic hallmark of RA is the mass influx of inflammatory cells into the joint. However, the role that dendritic cells (DC) may play in this inflammatory milieu is still relatively unexplored. Moreover, the contribution this unique synovial microenvironment has on DC maturation is still unknown. Using monocyte-derived DC (MoDC), we established an in-vitro model to recapitulate the synovial microenvironment to explore DC maturation. MoDC treated with conditioned media from ex-vivo synovial tissue biopsy cultures [explant-conditioned media (ECM)] have increased expression of proinflammatory cytokines, chemokines and adhesion molecules. ECM DC have increased expression of CD83 and CC-chemokine receptor (CCR)7 and decreased expression of CCR5 and phagocytic capacity, suggestive of heightened DC maturation. ECM-induced maturation is concomitant with altered cellular bioenergetics, whereby increased expression of glycolytic genes and increased glucose uptake are observed in ECM DC. Collectively, this results in a metabolic shift in DC metabolism in favour of glycolysis. These adaptations are in-part mediated via signal transducer and activator of transcription-3 (STAT-3), as demonstrated by decreased expression of proinflammatory cytokines and glycolytic genes in ECM DC in response to STAT-3 inhibition. Finally, to translate these data to a more in-vivo clinically relevant setting, RNA-seq was performed on RA synovial fluid and peripheral blood. We identified enhanced expression of a number of glycolytic genes in synovial CD1c+ DC compared to CD1c+ DC in circulation. Collectively, our data suggest that the synovial microenvironment in RA contributes to DC maturation and metabolic reprogramming.

Dysregulated bioenergetics: a key regulator of joint inflammation.

OBJECTIVES: This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation. METHODS: Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO2 mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). RESULTS: DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO2 levels <20 mm Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO2 in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions. CONCLUSIONS: Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.

Tofacitinib regulates synovial inflammation in psoriatic arthritis, inhibiting STAT activation and induction of negative feedback inhibitors.

BACKGROUND: Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway is implicated in the pathogenesis of PsA. OBJECTIVES: To examine the effect of tofacitinib (JAK inhibitor) on proinflammatory mechanisms in PsA. METHODS: Primary PsA synovial fibroblasts (PsAFLS) and ex vivo PsA synovial explants were cultured with tofacitinib (1 µM). PhosphoSTAT3 (pSTAT3), phosphoSTAT1 (pSTAT1), suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3) and nuclear factor kappa B cells (NFκBp65) were quantified by western blot. The effect of tofacitinib on PsAFLS migration, invasion, Matrigel network formation and matrix metallopeptidase (MMP)2/9 was quantified by invasion/migration assays and zymography. Interleukin (IL)-6, IL-8, IFN-gamma-inducible protein 10 (IP-10) monocyte chemoattractant protein (MCP)-1, IL-17, IL-10, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were assessed by ELISA. RESULTS: Tofacitinib significantly decreased pSTAT3, pSTAT1, NFκBp65 and induced SOCS3 and PIAS3 expression in PsAFLS and synovial explant cultures (p<0.05). Functionally, PsAFLS invasion, network formation and migration were inhibited by tofacitinib (all p<0.05). In PsA explant, tofacitinib significantly decreased spontaneous secretion of IL-6, IL-8, MCP-1, MMP9/MMP2, MMP3 (all p<0.05) and decreased the MMP3/TIMP3 ratio (p<0.05), with no effect observed for IP-10 or IL-10. CONCLUSIONS: This study further supports JAK-STAT inhibition as a therapeutic target for the treatment of PsA.

Dysregulated cytokine expression in lesional and nonlesional skin in hidradenitis suppurativa.

BACKGROUND: There is a dearth of information on the precise pathogenesis of hidradenitis suppurativa (HS), but immune dysregulation is implicated. OBJECTIVES: To determine the nature of the immune response in HS. METHODS: Skin biopsies - lesional, perilesional (2 cm away) and uninvolved (10 cm away) - were obtained from patients with HS and healthy controls. The expression of various cytokines was determined by enzyme-linked immunosorbent assay, flow cytometry and real-time polymerase chain reaction. RESULTS: The expression of the inflammatory cytokines interleukin (IL)-17, IL-1ß and tumour necrosis factor-α was enhanced in lesional skin of patients with HS. In addition, IL17A and IL1B mRNA were enhanced in clinically normal perilesional skin. CD4(+) T cells produced IL-17 in HS, while CD11c(+) CD1a(-) CD14(+) cells were sources of IL-1ß. Activated caspase-1 was detected in HS skin and was associated with enhanced expression of NLRP3 and IL18. Inhibition of caspase-1 decreased IL-1ß and IL-18 production, suggesting that the caspase-1 pathway participates in IL-1ß and IL-18 expression in HS. Abnormal cytokine expression was detected in perilesional and uninvolved skin, which may suggest that subclinical inflammation is present in HS skin prior to the formation of an active lesion. CONCLUSIONS: This study demonstrates that CD4(+) T cells produce IL-17 in HS and that the IL-17 pathway may be important in HS pathogenesis. CD11c(+) CD1a(-) CD14(+) cells are a source of IL-1ß in HS, the production of which was shown to be mediated, in part, via a caspase-1-dependent pathway. These results suggest that IL-17 and the caspase-1-associated cytokines IL-1ß and IL-18 may play a role in the pathogenesis of HS.

Spatiotemporal coordination behaviors in futsal (indoor football) are guided by informational game constraints.

This report investigated the behavioral dynamics of teams in futsal game practice when the goalkeeper of the attacking team is substituted for an extra outfield player. To this end, the lateral and longitudinal displacements of the ball and both teams, as well as their kinematics expressed in angles and radial distances from the goal center, were obtained and subjected to relative phase analysis. The results demonstrated (a) stronger phase relations with the ball for the defending team than the attacking team for both coordinate systems, (b) phase relations between each team and ball, and, to a lesser extent, between teams themselves, produced greater stabilities in the lateral (side-to-side) direction than the longitudinal (forward-backward) direction, and (c) phase attractions were most pronounced for the defending team and ball when using angles as a measure of association, indicating ball position and goal location as key informational constraints for futsal game behavior. These findings advance understanding of self-organizing sports game dynamics with implications for sports practice.

Interpersonal coordination and ball dynamics in futsal (indoor football).

Here, we report an investigation of the patterned movement behavior of players for a specific sub-phase of the game of futsal, namely when the goalkeeper for the attacking team is substituted with an extra outfield player. The movement trajectories of the ball and players were recorded in both lateral and longitudinal directions and investigated using relative phase analysis. Some differences in phase relations between different playing dyads were noted, indicating specificity of phase attractions, or otherwise, for certain players. In general terms, the defenders demonstrated strong in-phase attractions with the ball and with each other, whereas weaker phase attractions, indicated by increased relative phase variability, were observed for the attackers and ball, as well as between attackers themselves. These results demonstrate different coordination dynamics for the defending and attacking dyads, from which we interpret evidence for different playing sub-systems consistent with different team objectives linked together in an overarching game structure. In keeping with dynamical systems theory for complex systems, we view this sub-phase of futsal as being characterized by coordinated behavior patterns that emerge as a result of self-organizing processes. These dynamic patterns are generated within functional constraints, with players and teams exerting mutual influence on each other.

Lymphangitic metastasis of recurrent renal cell carcinoma to the contralateral lung causing lymphangitic carcinomatosis and respiratory symptoms.

Renal cell carcinoma comprises 80%-85% of kidney malignancies. For early presentations, nephrectomy provides a high cure rate, but patients usually present at advanced stages, leading to poor outcomes. Even for patients without metastatic spread who undergo nephrectomy, metastatic recurrence is frequent. We report the case of a patient who underwent nephrectomy for stage iii renal cell carcinoma and who presented 20 months later with respiratory symptoms consistent with pneumonia, influenza, or (less likely) congestive heart failure or a cardiac event. Persistent right pleural effusion on serial chest radiographs despite treatment prompted computed tomography evaluation, which revealed lymphangitic carcinomatosis, a very rare form of renal cell carcinoma metastasis to the lung. This preliminary finding was confirmed by right middle lobe tissue biopsy through bronchoscopy and cytopathology examination.

Evaluation of the effect of shape on a contemporary CAD system.

The TracerCAD system is one of the leading prosthetic CAD systems in the world and is increasingly used in clinics to replace traditional methods of residual limb shape capture. Accurate dimensional capture of the residuum is arguably the most important process in the production of a prosthetic socket. TracerCAD system accuracy has previously been tested on a cylindrical model but not a trans-tibial shape. Residual limbs are irregular in shape therefore it is important to assess if shape has an effect on the accuracy of data collected when using TracerCAD. The objective of this study is to investigate the accuracy of the TracerCAD system in measuring a model of a trans-tibial stump of known dimensions and volume. A model of a trans-tibial stump was produced and filled with plaster and measured using a data acquisition system with an accuracy of five micron (0.005 mm). The model was repeatedly traced using the TracerCAD system by an individual user. The mean value of measures taken by the dynamic indicator was calculated and compared to individual and mean values of TracerCAD measurement. Results showed that the TracerCAD measurement was not as consistent on the more complex trans-tibial model as for the cylindrical model.

Comparison of the results of four users of a contemporary CAD/CAM system.

The objective of this study was to investigate the variation of measurements recorded when four different users of the TracerCAD system trace a model of known dimensions and volume. This complements a previous study where the accuracy and consistency of a single user was measured. Landmarks were added to indicate proximal, distal, anterior, medial and lateral regions of a specially manufactured cylindrical nylon 6.6 model. Four circumferential lines were added at regular intervals along the length of the cylinder with a view to calculating diameters and volumes relative to these landmarks. The model was measured using a comparator with guaranteed accuracy to one hundredth of a millimetre, and was traced using the TracerCAD system by four different users. The difference in mean volume between measured results and TracerCAD scans of differing users ranged to -3%. Individual trace volumes varied by up to -7.85%. In all volumes measured, 11 out of 12 maximum volume percentage differences measured greater than 2%, and of these, seven results showed maximum volume percentage difference to measure greater than 4%.

Evaluation of a contemporary CAD/CAM system.

The objective of this study is to investigate the accuracy of the TracerCAD system in measuring a model of known dimensions and volumes. A cylindrical nylon 6.6 model was prepared. Landmarks were added to indicate proximal, distal, anterior, medial, and lateral. Four additional landmarks were added at regular intervals along the length of the cylinder with a view to calculating diameters and volumes relative to these landmarks. The model was measured using a comparator with a guaranteed accuracy of 0.01 mm and was traced using the TracerCAD system (Test 1). The test was repeated with the model rotated by 90 degrees (Test 2), to determine whether there were any effects related to the orientation of the model in relation to the transmitter. The difference in average volume between measured results and TracerCAD scans was between 0.20% and -1.96%. Individual trace volumes varied between -0.0085% and -4.50%. In all volumes measured in Tests 1 and 2, all maximum volume percentage differences measured greater than 3%.

Patients' preferences for characteristics associated with treatments for osteoarthritis.

OBJECTIVE: The purpose of this study was to investigate patient preferences for attributes associated with the efficacy and side-effects of treatment for osteoarthritis. METHODS: A stated preference design questionnaire was administered to a sample of 412 individuals diagnosed with osteoarthritis (OA). RESULTS: Statistically significant attributes in influencing treatment preferences were the level of joint aches, the level of physical mobility and the risk of experiencing serious side-effects from treatment. Respondents were relatively more concerned about the risk of serious side-effects (even with a very low probability) than mild to moderate side-effects (at a much higher probability). Data segmentation revealed some variations in preferences according to respondent characteristics. The importance of joint aches increased according to the severity of the symptoms of osteoarthritis, indicating that this attribute is more troublesome to those respondents with more severe symptoms. Older respondents were more willing than younger respondents to accept an increased risk of experiencing serious side-effects for an improvement in the symptoms of OA. Individuals in lower income brackets appeared to attach greater importance to joint aches and the level of mobility experienced than those in higher income brackets. Respondents who had previously experienced gastrointestinal side-effects from treatment were, as expected, more tolerant of them than those who had not. CONCLUSION: The use of conjoint analysis to assess patient preferences provides a useful insight to the likely attitudes of patients to novel treatments for osteoarthritis.

The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis.

We have identified a Drosophila homolog of the DNA replication initiation inhibitor Geminin (Dm geminin) and show that it has all of the properties of Xenopus and human Geminin. During Drosophila development, Dm Geminin is present in cycling cells; protein accumulates during S phase and is degraded at the metaphase to anaphase transition. Overexpression of Dm geminin in embryos inhibits DNA replication, but cells enter mitosis arresting in metaphase, as in dup (cdt1) mutants, and undergo apoptosis. Overexpression of Dm Geminin also induces ectopic neural differentiation. Dm geminin mutant embryos exhibit anaphase defects at cycle 16 and increased numbers of S phase cells later in embryogenesis. In a partially female-sterile Dm geminin mutant, excessive DNA amplification in the ovarian follicle cells is observed. Our data suggest roles for Dm Geminin in limiting DNA replication, in anaphase and in neural differentiation.

Against a final ballistic process in the control of voluntary action: evidence using the Hoffmann reflex.

Reduced surface electromyogram (EMG) onsets, observed in some cases when trying to stop an earlier intended action before it begins, were taken to suggest a control process that remains open to stopping right up to the point of motor discharge. This interpretation argues against a final ballistic (involuntary) process in the control of voluntary action. That a final ballistic process may receive reduced input shortly before its manifestation as reduced output (i.e., motor discharge), however, provides for an alternative interpretation of these same data. In this study we used the Hoffmann (H) reflex to further investigate for stopping effects in the brief interval before voluntary motor discharge. Late stopping effects on the facilitated H-reflex within the time window that a final ballistic process would otherwise be expected (i.e, shortly before EMG onset) were observed in some instances. We conclude from these data good evidence against a final ballistic process in the control of voluntary action.

On winning the penalty shoot-out in soccer.

The penalty shoot-out is used to break tied games in the knock-out stages of soccer competition. The shoot-out, which consists of an alternating series of penalty kicks, is won by the team with the highest goal tally after n kicks per team (n = 5). In the event of a tie after five penalty kicks each, the shoot-out progresses to 'sudden death' by increasing n in iterative fashion (i.e. n = n + 1) until one team obtains a higher goal tally than the other after an equal number of kicks per team. The team to strike first is determined at the end of extra time by the toss of a coin. As each on-field player can be awarded only a single penalty kick, the line-up order in which the penalty kicks are taken allows for the possibility of tactical influence on the final outcome. Consequently, we report a probability analysis of the penalty shoot-out in soccer from which we identify the following pre- and post-game strategies. The best five ranked penalty takers from the on-field players should be assigned to the first five penalty kicks in their reverse order of ability. That is, the fifth best penalty taker should take the first penalty kick, the fourth best penalty taker should take the second penalty kick, and so on. In the event of sudden death, the next highest ranked on-field player should be assigned to the next penalty kick until the shoot-out ends. For this tactic to be successful, players should be ranked a priori on their penalty-taking ability. Similarly, goalkeepers should be ranked a priori on their penalty-stopping ability. These findings indicate that the tactical substitution of on-field players for higher ranked penalty takers, including higher ranked penalty stoppers (i.e. goalkeepers), with a view to an impending penalty shoot-out should be given due consideration. These results are of practical importance in that they are shown to maximize the likelihood of winning the penalty shoot-out under certain initial conditions.

Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis.

A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore, expression of a stable Xenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle. The vSecurin proteins share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts. Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation.

Classification system for complete edentulism. The American College of Prosthodontics.

The American College of Prosthodontists has developed a classification system for complete edentulism based on diagnostic findings. These guidelines may help practitioners determine appropriate treatments for their patients. Four categories are defined, ranging from Class I to Class IV, with Class I representing an uncomplicated clinical situation and a Class IV patient representing the most complex and higher-risk situation. Each class is differentiated by specific diagnostic criteria. This system is designed for use by dental professionals who are involved in the diagnosis of patients requiring treatment for complete edentulism. Potential benefits of the system include: 1) better patient care, 2) improved professional communication, 3) more appropriate insurance reimbursement, 4) a better screening tool to assist dental school admission clinics, and 5) standardized criteria for outcomes assessment.

On the presence and absence of behavioural traits in sport: an example from championship squash match-play.

Here we report two experiments that analysed sport (squash) competition as a non-linear system that transits intermittently between different behavioural states. The first experiment involved a perceptual analysis of 60 rallies in which stable behaviour and unstable behaviour, delineated by behavioural transitions (i.e. shot perturbations), were reliably (kappa = 0.930) and validly (kappa = 0.844) identified by independent observers. In addition, experts were better than non-experts at identifying the type of system behaviour (P < 0.01). These results provide for three alternative descriptions: (a) the system is multi-stable and transits between states via the mechanism of instability; (b) the system is bi-stable and abruptly transits between two states, labelled stable and unstable; or (c) the system is mono-stable and displays variability, marked by transient instability, as a result of system perturbations. The second experiment analysed squash behaviour as expressed in the phase relation between the two players from time-motion analysis. The data, from four rallies, yielded evidence of a tight anti-phase coupling with transient phase shifts, or perturbations, that were quickly damped. These data suggest a mono-stable system with a single (anti-phase) attractor onto which system fluctuations are occasionally written. However, these fluctuations failed to correspond with the short perturbations that were identified from perceptual analysis. Together, these results affirm the presence of transient behaviours in squash match-play, although the information that forms these perceptual judgements has yet to be identified.

Geminin, an inhibitor of DNA replication, is degraded during mitosis.

We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of the metaphase-anaphase transition. Geminin inhibits DNA replication by preventing the incorporation of MCM complex into prereplication complex (pre-RC). We propose that geminin inhibits DNA replication during S, G2, and M phases and that geminin destruction at the metaphase-anaphase transition permits replication in the succeeding cell cycle.