"What needs to be seen": An exploration into the visual anticipation behaviour of different skill-level football referees while observing long passes on-field.

It is well established that elite football referees possess superior anticipatory skills in specific game scenarios such as when assessing foul situations. Referees might also have better anticipatory skills in other important scenarios such as when observing a long pass. In these often-occurring situations, a referee has to use visual information to anticipate the outcome of the pass, in particular to foresee any potential infringements that might occur when players battle for ball possession. However, little is known about if and how football referees might anticipate outcomes in these scenarios. The aim of the current study was therefore to analyse the visual anticipatory behaviour of football referees when long passes occur during actual football matches. Elite (N = 4) and sub-elite referees (N = 12) officiated an actual football match while wearing a mobile eye-tracker to analyse their gaze behaviour when long passes occurred (N = 196). The results revealed differences in the way that the elite and sub-elite referees tracked the ball and anticipated the outcome of the ball trajectories. The elite referees used a lower search rate (1.3 vs 1.8 fix/s; p < .05) and were more likely to direct their gaze towards the ball during the moment of kick (77 vs 52%; p < .05) and the early flight-phase of the pass (68 vs 45%; p < .05), and subsequently produced earlier anticipatory eye movements to the player(s) receiving the ball (at 50% vs 60% of the ball flight; p < .05). This earlier anticipation may help the elite referees to better pick-up relevant information about the receivers that could be vital in making adjudications about any potential infringement when the ball does arrive. Referee education programs can use the current study to highlight the importance of visual search behaviour and help referees to adapt a strategy that is beneficial for long-pass situations.

CYP3A4 genotype is associated with sildenafil concentrations in patients with heart failure with preserved ejection fraction.

Despite its established inter-individual variability, sildenafil has been the subject of only a few pharmacogenetic investigations, with limited data regarding the genetic modulators of its pharmacokinetics. We conducted a pharmacogenetic sub-study of patients randomized to sildenafil (n=85) in the RELAX trial, which investigated the impact of high-dose sildenafil in patients with heart failure with preserved left ventricular ejection fraction (HFpEF). In the overall population, the CYP3A4 inferred phenotype appeared associated with the dose-adjusted peak concentrations of sildenafil at week 12 and week 24 (adjusted P=0.045 for repeated measures analysis), although this P-value did not meet our corrected significance threshold of 0.0167. In the more homogeneous Caucasian subgroup, this association was significant (adjusted P=0.0165 for repeated measures). Hence, CYP3A4 inferred phenotype is associated with peak sildenafil dose-adjusted concentrations in patients with HFpEF receiving high doses of sildenafil. The clinical impact of this association requires further investigation.

A pharmacogenetic investigation of intravenous furosemide in decompensated heart failure: a meta-analysis of three clinical trials.

We conducted a meta-analysis of pharmacogenomic substudies of three randomized trials conducted in patients with decompensated heart failure (HF) that were led by National Heart Lung and Blood Institute (NHLBI)-funded HF Network to test the hypothesis that candidate genes modulate net fluid loss and weight change in patients with decompensated HF treated with a furosemide-based diuretic regimen. Although none of the genetic variants previously shown to modulate the effects of loop diuretics in healthy individuals were associated with net fluid loss after 72 h of treatment, a set of rare variants in the APOL1 gene, which codes for apolipoprotein L1 (P=0.0005 in the random effects model), was associated with this end point. Moreover, a common variant in the multidrug resistance protein-4 coding gene (ABCC4, rs17268282) was associated with weight loss with furosemide use (P=0.0001). Our results suggest that both common and rare genetic variants modulate the response to a furosemide-based diuretic regimen in patients with decompensated HF.

Expert consensus statement to guide the evidence-based classification of Paralympic athletes with vision impairment: a Delphi study.

BACKGROUND: Paralympic sports are required to develop evidence-based systems that allocate athletes into 'classes' on the basis of the impact of their impairment on sport performance. However, sports for athletes with vision impairment (VI) classify athletes solely based on the WHO criteria for low vision and blindness. One key barrier to evidence-based classification is the absence of guidance on how to address classification issues unique to VI sport. The aim of this study was to reach expert consensus on how issues specific to VI sport should be addressed in evidence-based classification. METHOD: A four-round Delphi study was conducted with 25 participants who had expertise as a coach, athlete, classifier and/or administrator in Paralympic sport for VI athletes. RESULTS: The experts agreed that the current method of classification does not fulfil the requirements of Paralympic classification, and that the system should be different for each sport to account for the sports' unique visual demands. Instead of relying only on tests of visual acuity and visual field, the panel agreed that additional tests are required to better account for the impact of impairment on sport performance. There was strong agreement that all athletes should not be required to wear a blindfold as a means of equalising the impairment during competition. CONCLUSIONS: There is strong support within the Paralympic movement to change the way that VI athletes are classified. This consensus statement provides clear guidance on how the most important issues specific to VI should be addressed, removing key barriers to the development of evidence-based classification.

Th17/Tc17 infiltration and associated cytokine gene expression in elicitation phase of allergic contact dermatitis.

BACKGROUND: Allergic contact dermatitis (ACD) is a typical delayed-type hypersensitivity to sensitizing haptens mediated by T cells. Th1/Tc1 cells are currently considered to be the primary effectors in ACD. There is little information concerning the role played in ACD in humans by Th17/Tc17 cells, a recently defined subpopulation of effector T cells. OBJECTIVES: In the present report we attempted to characterize Th17/Tc17 cells in the infiltrates of the skin in the elicitation phase of ACD. METHODS: Th17 as well as Th1/Th2 cytokine gene expression was examined by semiquantitative real-time polymerase chain reaction in paired samples of positive patch test biopsies and normal skin from 11 patients allergic to nine different allergens. The in situ characterization of interleukin (IL)-17-producing cells was carried out using anti-RORC and anti-T-cell subset antibodies by double immunofluorescence. RESULTS: Compared with normal paired skin samples, gene expression of transcription factor for human Th17 cells, RORC, and Th17-related cytokines IL-17A, IL-17F and IL-23 was significantly increased in positive patch test biopsies. The mRNA for interferon-gamma and IL-4 was also increased. In the dermal infiltrates, about 20% of the infiltrating cells were IL-17-producing cells as they expressed RORC, and such RORC-expressing cells were detected in both CD4+ (approximately 30%) and CD8+ (approximately 20%) subsets. CONCLUSIONS: This is the first demonstration of Th17/Tc17 cells in the elicitation phase of human ACD, showing that they are a regular participant in the immunopathology of this common allergic reaction regardless of the nature of the triggering allergen.

Differentiation between African populations is evidenced by the diversity of alleles and haplotypes of HLA class I loci.

The allelic and haplotypic diversity of the HLA-A, HLA-B, and HLA-C loci was investigated in 852 subjects from five sub-Saharan populations from Kenya (Nandi and Luo), Mali (Dogon), Uganda, and Zambia. Distributions of genotypes at all loci and in all populations fit Hardy-Weinberg equilibrium expectations. There was not a single allele predominant at any of the loci in these populations, with the exception of A*3002 [allele frequency (AF) = 0.233] in Zambians and Cw*1601 (AF = 0.283) in Malians. This distribution was consistent with balancing selection for all class I loci in all populations, which was evidenced by the homozygosity F statistic that was less than that expected under neutrality. Only in the A locus in Zambians and the C locus in Malians, the AF distribution was very close to neutrality expectations. There were six instances in which there were significant deviations of allele distributions from neutrality in the direction of balancing selection. All allelic lineages from each of the class I loci were found in all the African populations. Several alleles of these loci have intermediate frequencies (AF = 0.020-0.150) and seem to appear only in the African populations. Most of these alleles are widely distributed in the African continent and their origin may predate the separation of linguistic groups. In contrast to native American and other populations, the African populations do not seem to show extensive allelic diversification within lineages, with the exception of the groups of alleles A*02, A*30, B*57, and B*58. The alleles of human leukocyte antigen (HLA)-B are in strong linkage disequilibrium (LD) with alleles of the C locus, and the sets of B/C haplotypes are found in several populations. The associations between A alleles with C-blocks are weaker, and only a few A/B/C haplotypes (A*0201-B*4501-Cw*1601; A*2301-B*1503-Cw*0202; A*7401-B* 1503-Cw*0202; A*2902-B*4201-Cw*1701; A*3001-B*4201-Cw*1701; and A*3601-B*5301-Cw*0401) are found in multiple populations with intermediate frequencies [haplotype frequency (HF) = 0.010-0.100]. The strength of the LD associations between alleles of HLA-A and HLA-B loci and those of HLA-B and HLA-C loci was on average of the same or higher magnitude as those observed in other non-African populations for the same pairs of loci. Comparison of the genetic distances measured by the distribution of alleles at the HLA class I loci in the sub-Saharan populations included in this and other studies indicate that the Luo population from western Kenya has the closest distance with virtually all sub-Saharan population so far studied for HLA-A, a finding consistent with the putative origin of modern humans in East Africa. In all African populations, the genetic distances between each other are greater than those observed between European populations. The remarkable current allelic and haplotypic diversity in the HLA system as well as their variable distribution in different sub-Saharan populations is probably the result of evolutionary forces and environments that have acted on each individual population or in their ancestors. In this regard, the genetic diversity of the HLA system in African populations poses practical challenges for the design of T-cell vaccines and for the transplantation medical community to find HLA-matched unrelated donors for patients in need of an allogeneic transplant.

Generation of CD4(+) and CD8(+) T lymphocyte responses by dendritic cells armed with PSA/anti-PSA (antigen/antibody) complexes.

Dendritic cells (DC) acquire antigens through a number of cell surface structures including receptors for the Fc portion of immunoglobulins and mannose. Little is known about the effects of antigen uptake via these receptors on antigen processing and presentation. We compared the capacity of DC to generate CD4(+) and CD8(+) T cell responses after exposure to prostate-specific antigen (PSA) alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47). Autologous CD3(+) T cells were added to monocyte-derived immature DC that had been cultured with GM-CSF/IL-4 for 4 days, exposed to antigen, and matured with CD40L or TNFalpha/IFN-alpha. After several rounds of stimulation, T cell responses were assessed by intracellular IFN-gamma production using flow cytometry. Both CD4(+) and CD8(+) T cell responses were observed after stimulation with DC exposed to the PSA/anti-PSA complexes, whereas CD4(+) predominated over CD8(+) T cell responses after stimulation with PSA-armed DC or PSA-m. These CD8(+) T cells responded when rechallenged with DC pulsed with HLA allele-restricted PSA peptides. These results indicate that PSA and PSA-m are processed primarily through pathways that favor HLA Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived DC. These findings have potential applications in designing more effective cancer vaccines for prostate cancer.

Brief murine myocardial I/R induces chemokines in a TNF-alpha-independent manner: role of oxygen radicals.

Early chemokine induction in the area at risk of an ischemic-reperfused (I/R) myocardium is first seen in the venular endothelium. Reperfusion is associated with several induction mechanisms including increased extracellular tumor necrosis factor (TNF)-alpha, reactive oxygen intermediate (ROI) species formation, and adhesion of leukocytes to the venular endothelium. To test the hypothesis that chemokine induction in cardiac venules can occur by ROIs in a TNF-alpha-independent manner, and in the absence of leukocyte accumulation, we utilized wild-type (WT) and TNF-alpha double-receptor knockout mice (DKO) in a closed-chest mouse model of myocardial ischemia (15 min) and reperfusion (3 h), in which there is no infarction. We demonstrate that a single brief period of I/R induces significant upregulation of the chemokines macrophage inflammatory protein (MIP) -1 alpha, -1 beta, and -2 at both the mRNA and protein levels. This induction was independent of TNF-alpha, whereas levels of these chemokines were increased in both WT and DKO mice. Chemokine induction was seen predominantly in the endothelium of small veins and was accompanied by nuclear translocation of nuclear factor-kappa B and c-Jun (AP-1) in venular endothelium. Intravenous infusion of the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) initiated 15 min before ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun had no effect. The results suggest that ROI generation in the reperfused myocardium rapidly induces C-C and C-X-C chemokines in the venular endothelium in the absence of infarction or irreversible cellular injury.

Differential regulation of mitogen-activated protein kinases in the failing human heart in response to mechanical unloading.

Background- Mechanical unloading of the heart with a left ventricular assist device (LVAD) leads to favorable changes in the biology of the failing cardiac myocyte. To determine a potential mechanism for these improvements, we examined the regulation of mitogen-activated protein kinases (MAPKs) in the failing heart in the presence and absence of LVAD support. Methods and Results- We examined the degree of activation (ie, phosphorylation) of p44/42 extracellularly regulated kinase, p38 kinase, and c-Jun N-terminal kinase (JNK1/2), and the corresponding activity levels of these MAPKs in myocardial samples obtained from 11 patients with LVAD support and in 11 patients without LVAD support. MAPK activity was also examined in an additional 6 patients from whom paired samples were obtained before and after LVAD support. The activity of p44/42 and JNK1/2 were reduced significantly, whereas p38 activity levels were significantly increased after LVAD support. We examined functional parameters that are linked to MAPK activation, namely cardiac myocyte hypertrophy and apoptosis. Both cardiac myocyte cell size and the incidence of cardiac myocyte apoptosis were significantly reduced after LVAD support. Conclusions- Mechanical unloading of the failing heart leads to differential regulation of MAPKs. These changes in MAPK activity are associated with changes in myocyte hypertrophy and viability, suggesting a potential mechanistic basis for some of the observed salutary changes after LVAD support.

Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules.

The A*0201, A *0202, A*0203, A*0206, and A*6802 binding capacity of single amino acid substitution analogs of known A2-supertype binding peptides and of large nonredundant peptide libraries was measured. The results were utilized to rigorously define the peptide binding specificities of these A2-supertype molecules. Although each molecule was noted to have unique preferences, large overlaps in specificity were found. The presence of L, I, V, M, A, T, and Q residues in position 2, and L, I, V, M, A, and T residues at the C-terminus of peptide ligands were tolerated by all molecules. Likewise, whereas examination of secondary influences on peptide binding revealed allele specific preferences, shared features could also be identified. These shared features were utilized to define an A2-supermotif and were noted to correlate with crossreactivity. Over 70% of the peptides that bound A *0201 with high affinity were found to bind at least two other A2-supertype molecules. Because the A2-supertype molecules studied herein cover the variants most common in different major ethnicities, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula.

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.

A novel bile duct cannulation method with tail cuff exteriorization allowing continuous intravenous infusion and enterohepatic recirculation in the unrestrained rat.

A variety of bile duct cannulation methods have been used in the study of biliary excretion in the rat. We now report the validation and use of one such method. In this method, the common bile duct and duodenum were cannulated, the free ends of the cannulae tunnelled through the abdominal wall, passed through a trochar and exteriorized at the ventral aspect of the tail. A purpose-designed stainless steel tail cuff was then attached, to protect the cannulae from the rat. The cannulae were passed through the top of a metabolism cage and attached to a dual swivel that allows the rat freedom of movement within the metabolism cage. Where necessary an additional cannula could be placed in the femoral vein to allow infusion of test material or blood sampling. The results demonstrate that the method is robust and that its use allows a reliable correlation between surgically prepared and intact animals, as physiological parameters are allowed to return to normal prior to inclusion of the animals in the study. The technique allows the animals a great deal of freedom and, as such, is considered to minimize stress associated with the procedure. This fact is reflected in the reliability and reproducibility of the data obtained over the wide range of studies that have been conducted using this method. This method has been in use for over 4 years at Inveresk and this paper describes the authors' experience with the method to date.

Effects of vesnarinone on peripheral circulating levels of cytokines and cytokine receptors in patients with heart failure: a report from the Vesnarinone Trial.

STUDY OBJECTIVES: Proinflammatory cytokines may contribute to disease progression in heart failure by virtue of the direct toxic effects that these molecules exert on the heart and the circulation. Accordingly, there is interest in developing therapeutic agents with anticytokine properties that might be used as adjunctive therapy to modulate proinflammatory cytokine levels in patients with heart failure. Previous experimental studies suggested that vesnarinone has potent anticytokine properties in vitro. Therefore, we examined the effects of vesnarinone on circulating levels of cytokines and cytokine receptors in a large-scale, multicenter, clinical trial of patients with moderate-to-advanced heart failure: the Vesnarinone Trial (VEST). METHODS: Circulating levels of tumor necrosis factor (TNF)-alpha, soluble TNF-receptor type 1, soluble TNF-receptor type 2, as well as interleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) were measured on plasma samples by enzyme-linked immunosorbent assay at baseline and at 24 weeks in patients who were receiving placebo (n = 352), 30 mg of vesnarinone (n = 367), and 60 mg of vesnarinone (n = 327). RESULTS: Treatment with 30 mg and 60 mg of vesnarinone had no effect on circulating levels of cytokines or cytokine receptors in patients with advanced heart failure over a 24-week period. CONCLUSIONS: In contrast to the potent anticytokine effects observed with vesnarinone in experimental studies in vitro, the results of this clinical study suggest that vesnarinone does not have any measurable anticytokine effects in vivo in patients with moderate-to-advanced heart failure.

Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor.

BACKGROUND: The mechanisms responsible for tumor necrosis factor (TNF)-induced LV structural remodeling in the adult heart are not known. METHODS AND RESULTS: We generated a line of transgenic mice (MHCsTNF) with cardiac restricted overexpression of TNF that develop progressive LV dilation/remodeling from 4 to 12 weeks of age. During the early phases of LV structural remodeling, there was a significant increase in total matrix metalloproteinase (MMP) activity that corresponded to a decrease in total myocardial fibrillar collagen content. As the MHCsTNF mice aged, there was a significant decrease in total MMP zymographic activity that was accompanied by an increase in total fibrillar collagen content. The changes in total MMP activity and myocardial fibrillar collagen content were related to a time- dependent increase in myocardial tissue inhibitor of metalloproteinases (TIMP)-1 levels, resulting in a significant time-dependent decrease in the MMP activity/TIMP level ratio in the MHCsTNF mice. To determine a possible mechanism for the increase in myocardial fibrosis, we also measured levels of TGF-beta(1) and TGF-beta(2) protein levels, which were shown to be significantly elevated in the hearts of the MHCsTNF mice. CONCLUSIONS: Our results suggest that progressive time-dependent changes in the balance between MMP activity and TIMP activity are responsible, at least in part, for the spectrum of TNF-induced changes in the myofibrillar collagen content that occur during LV structural remodeling in the MHCsTNF mice.

In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4.

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.