A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 µM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

Free secretory component from cystic fibrosis sputa displays the cystic fibrosis glycosylation phenotype

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.

Safety and use of sputum induction in children with cystic fibrosis

We assessed the safety and use of induced sputum (IS) in children with cystic fibrosis (CF). Forty-eight children (19 males) with CF, mean age 12.6 (range, 7.3-17.0) years and median forced expired volume in 1 sec (FEV1) 48% (range, 14-77%) predicted were recruited. Patients spontaneously expectorated sputum and then performed sputum induction by inhalation of nebulized 7% hypertonic saline. Samples were sent for bacteriological culture, and for measurement of the following inflammatory mediators: interleukin-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase activity. FEV1 was performed before and after inhalation of hypertonic saline. There was no increase in mediator levels in IS compared to expectorated sputum (ES) samples. Only 3 patients demonstrated significant bronchoconstriction following inhalation of hypertonic saline, by the method used. From the ES samples, Pseudomonas aeruginosa was isolated in 13 patients, Staphylococcus aureus in 7 patients, Stenotrophomonas maltophilia in 1 patient, and both Pseudomonas aeruginosa and Staphylococcus aureus in 5 patients. All these organisms were found in the IS samples. However, in 2 patients whose ES grew no organisms, one patient's IS grew Pseudomonas aeruginosa, and the other patient's IS grew Staphylococcus aureus. In our study, sputum induction was safe, with no proinflammatory effect.

Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis

Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC

Trademark law and theory:a handbook of contemporary research

Publication Review: Trademark Law and Theory: A Handbook of Contemporary Research, Edited by Graeme B. Dinwoodie and Mark D. Janis

Analysis of foveation sequences in congenital nystagmus

Congenital nystagmus (CN) is an ocular-motor disorder that appears at birth or during the first few months of life; it is characterised by involuntary, conjugated, bilateral to and fro ocular oscillations. Pathogenesis of congenital nystagmus is still unknown. Eye movement recording allow to extract and analyse nystagmus main features such as shape, amplitude and frequency; depending on the morphology of the oscillations nystagmus can be classified in different categories (pendular, jerk, horizontal unidirectional, bidirectional). In general, CN patient show a considerable decrease of the visual acuity: image fixation on the retina is disturbed by nystagmus continuous oscillations; however, image stabilisation is still achieved during the short foveation periods in which eye velocity slows down while the target image is placed onto the fovea. Visual acuity was found to be mainly dependent on foveation periods duration, but cycle-to-cycle foveation repeatability and reduction of retinal image velocities also contribute in increasing visual acuity. This study concentrate on cycle-to-cycle image position variation onto fovea, trying to characterise the sequences of foveation positions. Eye-movement (infrared oculographic or electro oculographic) recordings, relative to different gaze positions and belonging to more than 30 CN patients, were analysed. Preliminary results suggest that sequences of foveations show a cyclic pattern with a dominant frequency (around 0.3 Hz on average) much lower than that of the nystagmus (about 3.3 Hz on average). Sequences of foveations reveals an horizontal ocular swing of more than 2 degree on average, which can explain the low visual acuity of the CN patient. Current CN therapies, pharmacological treatment or surgery of the ocular muscles, mainly aim to increase the patient's visual acuity. Hence, it is fundamental to have an objective parameter (expected visual acuity) for therapy planning. The information about sequences of foveations can improve estimation of patient visual acuity. © 2008 Springer-Verlag.

Is pupil diameter influenced by refractive error?

Purpose: To investigate the relationship between pupil diameter and refractive error and how refractive correction, target luminance, and accommodation modulate this relationship. Methods: Sixty emmetropic, myopic, and hyperopic subjects (age range, 18 to 35 years) viewed an illuminated target (luminance: 10, 100, 200, 400, 1000, 2000, and 4100 cd/m2) within a Badal optical system, at 0 diopters (D) and −3 D vergence, with and without refractive correction. Refractive error was corrected using daily disposable contact lenses. Pupil diameter and accommodation were recorded continuously using a commercially available photorefractor. Results: No significant difference in pupil diameter was found between the refractive groups at 0 D or −3 D target vergence, in the corrected or uncorrected conditions. As expected, pupil diameter decreased with increasing luminance. Target vergence had no significant influence on pupil diameter. In the corrected condition, at 0 D target vergence, the accommodation response was similar in all refractive groups. At −3 D target vergence, the emmetropic and myopic groups accommodated significantly more than the hyperopic group at all luminance levels. There was no correlation between accommodation response and pupil diameter or refractive error in any refractive group. In the uncorrected condition, the accommodation response was significantly greater in the hyperopic group than in the myopic group at all luminance levels, particularly for near viewing. In the hyperopic group, the accommodation response was significantly correlated with refractive error but not pupil diameter. In the myopic group, accommodation response level was not correlated with refractive error or pupil diameter. Conclusions: Refractive error has no influence on pupil diameter, irrespective of refractive correction or accommodative demand. This suggests that the pupil is controlled by the pupillary light reflex and is not driven by retinal blur.

Muscle movement and electrodes motion artifact during vibration treatment

Vibration treatment by oscillating platforms is more and more employed in the fields of exercise physiology and bone research. The rationale of this treatment is based on the neuromuscular system response elicited by vibration loads. surface Electromyography (EMG) is largely utilized to assess muscular response elicited by vibrations and Root Mean Square of the electromyography signals is often used as a concise quantitative index of muscle activity; in general, EMG envelope or RMS is expected to increase during vibration. However, it is well known that during surface bio-potential recording, motion artifacts may arise from relative motion between electrodes and skin and between skin layers. Also the only skin stretch, modifying the internal charge distribution, results in a variation of electrode potential. The aim of this study is to highlight the movements of muscles, and the succeeding relevance of motion artifacts on electrodes, in subjects undergoing vibration treatments. EMGs from quadriceps of fifteen subjects were recorded during vibration at different frequencies (15-40 Hz); Triaxial accelerometers were placed onto quadriceps, as close as possible to muscle belly, to monitor motion. The computed muscle belly displacements showed a peculiar behavior reflecting the mechanical properties of the structures involved. Motion artifact related to the impressed vibration have been recognized and related to movement of the soft tissues. In fact large artifacts are visible on EMGs and patellar electrodes recordings during vibration. Signals spectra also revealed sharp peaks corresponding to vibration frequency and its harmonics, in accordance with accelerometers data. © 2008 Springer-Verlag.

Global trends in myopia management attitudes and strategies in clinical practice

PURPOSE: Myopia is a global public health issue; however, no information exists as to how potential myopia retardation strategies are being adopted globally. METHODS: A self-administrated, internet-based questionnaire was distributed in six languages, through professional bodies to eye care practitioners globally. The questions examined: awareness of increasing myopia prevalence, perceived efficacy and adoption of available strategies, and reasons for not adopting specific strategies. RESULTS: Of the 971 respondents, concern was higher (median 9/10) in Asia than in any other continent (7/10, p<0.001) and they considered themselves more active in implementing myopia control strategies (8/10) than Australasia and Europe (7/10), with North (4/10) and South America (5/10) being least proactive (p<0.001). Orthokeratology was perceived to be the most effective method of myopia control, followed by increased time outdoors and pharmaceutical approaches, with under-correction and single vision spectacles felt to be the least effective (p<0.05). Although significant intra-regional differences existed, overall most practitioners 67.5 (±37.8)% prescribed single vision spectacles or contact lenses as the primary mode of correction for myopic patients. The main justifications for their reluctance to prescribe alternatives to single vision refractive corrections were increased cost (35.6%), inadequate information (33.3%) and the unpredictability of outcomes (28.2%). CONCLUSIONS: Regardless of practitioners' awareness of the efficacy of myopia control techniques, the vast majority still prescribe single vision interventions to young myopes. In view of the increasing prevalence of myopia and existing evidence for interventions to slow myopia progression, clear guidelines for myopia management need to be established.

We are being myopic about myopia control

Editorial