Impact of Azithromycin on the Quorum Sensing-Controlled Proteome of Pseudomonas aeruginosa.

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and 1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12-13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.

Central administration of nicotine suppresses tracheobronchial cough in anesthetized cats.

We tested the hypothesis that nicotine, which acts peripherally to promote coughing, might inhibit reflex cough at a central site. Nicotine was administered via the vertebral artery [intra-arterial (ia)] to the brain stem circulation and by microinjections into a restricted area of the caudal ventral respiratory column in 33 pentobarbital anesthetized, spontaneously breathing cats. The number of coughs induced by mechanical stimulation of the tracheobronchial airways; amplitudes of the diaphragm, abdominal muscle, and laryngeal muscles EMGs; and several temporal characteristics of cough were analyzed after administration of nicotine and compared with those during control and recovery period. (-)Nicotine (ia) reduced cough number, cough expiratory efforts, blood pressure, and heart rate in a dose-dependent manner. (-)Nicotine did not alter temporal characteristics of the cough motor pattern. Pretreatment with mecamylamine prevented the effect of (-)nicotine on blood pressure and heart rate, but did not block the antitussive action of this drug. (+)Nicotine was less potent than (-)nicotine for inhibition of cough. Microinjections of (-)nicotine into the caudal ventral respiratory column produced similar inhibitory effects on cough as administration of this isomer by the ia route. Mecamylamine microinjected in the region just before nicotine did not significantly reduce the cough suppressant effect of nicotine. Nicotinic acetylcholine receptors significantly modulate functions of brain stem and in particular caudal ventral respiratory column neurons involved in expression of the tracheobronchial cough reflex by a mecamylamine-insensitive mechanism.

Tracheal occlusion conditioning causes stress, anxiety and neural state changes in conscious rats.

Evidence from human and animal studies indicates that mechanical loads to breathing are stressful stimuli and evoke compensatory behaviours. Conditioning of stressful stimuli is known to cause changes in basal stress levels and behaviour. Individuals with respiratory obstructive diseases repeatedly experience bouts of airway obstruction, which may act as a form of conditioning, and often have affective disorders, such as anxiety and depression. It is unknown whether the development of affective disorders in these individuals results from the unexpected recurring respiratory perturbations. To investigate this possibility, we developed a model to elicit tracheal occlusion (TO) in conscious rats and exposed them to 10 days of TO conditioning. We hypothesized that healthy, conscious animals exposed to TO conditioning would develop stress and anxiety and would have modulated neural activity in respiratory, stress, discriminative and affective neural regions. Following TO conditioning, rats had increased basal corticosterone levels, greater adrenal weights and elevated anxiety levels compared with animals not receiving TO. Significant increases in cytochrome oxidase staining were found in brainstem respiratory nuclei, periaqueductal grey, dorsal raphe, thalamus and insular cortex. These results suggest that healthy animals develop stress and anxiety responses to respiratory load conditioning via inescapable tracheal occlusions, which may be mediated through state changes in specific brain nuclei.

Urge to cough with voluntary suppression following mechanical pharyngeal stimulation.

OBJECTIVE: The goal of this project was to determine if mechanical stimulation to the posterolateral oropharynx would elicit the urge-to-cough and/or cough. BACKGROUND: Inhaled agents, such as capsaicin and citric acid, readily produce coughing and the sensation of urge-to-cough. Areas below the glottis are thought to be the primary sensory mediators of these responses, however it is unknown if there are specific areas in the oropharynx or laryngopharynx that are important for the sensation and production of coughing. METHODS: Paired-pulse air puffs were delivered to the posterolateral oropharyngeal walls of 11 healthy adults (5 men, 6 women) between the ages of 18 and 30 years. Air puffs were delivered via custom mouthpiece in 4 trials, 50 sets per trial. Instances of cough were recorded, and a modified Borg scale was used to gauge urge-to-cough throughout each trial. RESULTS: Instances of cough were recorded in 12/37 trials, and the sensation of an urge-to-cough was present in 25/37 trials. No motor cough response was elicited with an urge-to-cough rating less than 2.4 on the modified Borg scale. A trend towards higher urge-to-cough was noted for later (3rd and 4th) trials. CONCLUSIONS: Oropharyngeal mechanical stimulation elicits urge-to-cough and cough in healthy young adults. Like other methods to elicit coughing, the motor and sensory thresholds are different using the oropharyngeal air-puff stimuli. Further, it appears there is a sensitization to the air puff stimuli with later trials associated with stronger urge-to-cough and higher likelihood of coughing versus the first and second trial (Tab. 1, Fig. 5, Ref. 21).

Workshop: tuning the 'cough center'.

The Workshop considered the mechanisms whereby the 'cough center' could be tuned by various afferent inputs. There were particular presentations on the effects of inputs from the nose, mouth, respiratory tract and lungs, cerebral cortex, somatic tissues and the pharynx. From all these sites cough induced from the lungs could be increased or decreased in its strength or modified in its pattern. Thus 'tuning' of cough could be due to the interaction of afferent inputs, or to the sensitization or desensitization of brainstem neural pathways. The pattern of response depended on the 'type' of cough being studied and, in some instances, on the timing of the sensory input into the brainstem. Cough inputs could also affect various 'non-cough' motor outputs from the brain, although this was not the main theme of the Workshop. The main conclusion was that cough is not a stereotyped output from the medullary 'cough center', but that its pattern and strength depend on many afferent inputs acting on the 'cough center'.

The role of nicotine on respiratory sensory gating measured by respiratory-related evoked potentials.

Respiratory perception can be altered by changes in emotional or psychological states. This may be due to affective (i.e., anxiety) modulation of respiratory sensory gating. Nicotine withdrawal induces elevated anxiety and decreased somatosensory gating. Respiratory sensory gating is evidenced by decreased amplitude of the respiratory-related evoked potentials (RREP) N(1) peak for the second occlusion (S2) when two 150-ms occlusions are presented with a 500-ms interval during an inspiration. The N(1) peak amplitude ratio of the S2 and first occlusion (S1) (S2/S1) is <0.5 and due to central neural sensory gating. We hypothesized that withdrawal from nicotine is anxiogenic and reduces respiratory gating in smokers. The RREP was recorded in smokers with 12-h withdrawal from nicotine and nonsmokers using a paired occlusion protocol. In smokers, the RREP was measured after nicotine withdrawal, then with either nicotine or placebo gum, followed by the second RREP trial. Nonsmokers received only placebo gum. After nicotine withdrawal, the smokers had a higher state anxiety compared with nonsmokers. There was a significant interaction between groups (nonsmokers vs. smokers with nicotine vs. smokers with placebo) and test (pre- vs. posttreatment) in RREP N(1) peak amplitude S2/S1. The S2/S1 in the smokers were larger than in nonsmokers before treatment. After gum treatment, the smoker-with-placebo group had a significantly larger S2/S1 than the other two groups. The S2/S1 was significantly decreased after the administration of nicotine gum in smokers due to significantly decreased S2 amplitudes. The RREP N(f) and P(1) peaks were unaffected. These results demonstrated that respiratory sensory gating was decreased in smokers after nicotine withdrawal. Nicotine increased respiratory sensory gating in smokers with a S2/S1 similar to that of the nonsmokers. Nicotine did not change respiratory sensory information arrival, but secondary information processing in respiratory sensation.

Clinical cough I: the urge-to-cough: a respiratory sensation.

Cough is generated by a brainstem neural network. Chemical and mechanical stimulation of the airway can elicit a reflex cough and can elicit a cognitive sensation, the urge-to-cough. The sensation of an urge-to-cough is a respiratory-related sensation. The role of the respiratory sensation of an urge-to-cough is to engage behavioral modulation of cough motor action. Respiratory sensations are elicited by a combination of modalities: central neural, chemical, and mechanical. Stimulation of respiratory afferents or changes in respiratory pattern resulting in a cognitive awareness of breathing are mediated by central neural processes that are the cognitive neural basis for respiratory sensations, including the urge-to-cough. It is proposed that the urge-to-cough is a component of the cough motivation-to-action system. The urge-to-cough is induced by stimuli that motivate subjects to protect their airway by coughing. Cough receptor stimulation is gated into suprapontine brain systems. In the proposed cough motivation system, the cough stimulus would produce an urge-to-cough which then matches with the cognitive desire for a response to the urge. If a cough is produced by the motor action system, the descending cognitive drive modulates the brainstem cough neural network. Receptors within the respiratory system provide sensory feedback indicating if the cough occurred, the motor pattern, and the magnitude. The limbic system uses that information to determine if the coughing behavior satisfied the urge. Cough is stopped if the urge-to-cough is satisfied; if the urge has not been satisfied then the urge-to-cough will continue to motivate the central nervous system. The central component within this cough motivation system is the intrinsic brain mechanism which can be activated to start the cycle for motivating a cough, the urge-to-cough. Eliciting a cognitive urge-to-cough is dependent on the integration of respiratory afferent activity, respiratory motor drive, affective state, attention, experience, and learning.

Effects of systemic administration of mirtazapine on respiratory muscle activity in sleeping rats.

Mirtazapine (MIRT) is an antidepressant with mixed noradrenergic and serotonergic effects in central nervous system. The present study was undertaken to assess whether MIRT can stimulate genioglossus muscle (GG) activity in the conscious, behaving rat. Nine male rats were chronically instrumented with GG and neck muscle EMG electrodes. EEG electrodes were implanted to acquire sleep stage. Results demonstrated a dose-dependent effect of MIRT on GG activity during sleep, although no changes reached statistical significance. Low dose MIRT (0.1 mg/kg) showed a slight increase in GG phasic activity. In contrast, higher doses of MIRT (0.5-1.0 mg/kg) tended to decrease GG activity relative to vehicle, in addition to decreasing total sleep time.

Disinhibition of the dorsomedial hypothalamus increases the frequency of augmented breaths in the anesthetized rat.

The present study was undertaken to identify if activation of the dorsomedial hypothalamus (DMH) elicits augmented breaths (ABs). DMH disinhibition in urethane anesthetized rats produced both an increase in baseline respiratory rate (RR) and an increase in the number of ABs. The increase in RR was associated with a decrease in both the time of inspiration (T(i)) and expiration (T(e)) and the peak change in RR was observed 5 min post DMH activation. In contrast, the increase in ABs was greatest during the first 1.25 min, and both T(i)s of the ABs did not change significantly from pre-injection values. The T(e) of the ABs did decrease but remained significantly greater than the T(e) of the normal breath during DMH disinhibition. Our results support the hypothesis that the central neural pathway involved in the maintenance of normal respiratory pattern may be distinct from pathways involved in the generation of ABs.

A pilot study of high-adsorption hemofiltration in human septic shock.

BACKGROUND: To compare the hemodynamic and biological effects of high-adsorption continuous veno-venous hemofiltration (CVVH) with standard CVVH in septic shock. METHODS: In a randomized cross-over clinical trial twelve patients with septic shock and multiple organ failure were enrolled at a tertiary intensive care unit. Patients were allocated to either 9 hours of high-adsorption hemofiltration (CVVH with 3 hourly filter change using AN69 hemofilters - 3FCVVH) or 9 hours of standard hemofiltration (CVVH without filter change - 1F-CVVH). RESULTS: Changes in hemodynamic variables, dose of noradrenaline required to maintain a mean arterial pressure greater than 75 mmHg and plasma concentrations of cytokines (IL-6, IL-8, IL-10 and IL-18) were measured. A 9-hour period of 3F-CVVH was associated with greater reduction in noradrenaline dose than a similar period of 1F-CVVH (median reduction: 16 vs. 3.5 microg/min, p=0.036; median percentage reduction: 48.1% vs. 17.5%, p=0.028). Unlike 1F-CVVH, 3F-CVVH was associated with a reduction in the plasma concentration of IL-6, IL-10 and IL-18 at 9 hours and a significant decrease 30 minutes after additional filter changes (IL-6: p<0.01, p<0.01; IL-10: p=0.03, p=0.016 and IL-18: p=0.016, p<0.01, respectively). Both, 3F-CVVH and 1F-CVVH were associated with decreased plasma concentrations of IL-8 at 9 hours (p<0.01, p<0.01, respectively). In a confirmatory ex-vivo experiment IL-6 concentrations substantially decreased during 3F-CVVH (at baseline 511 pg/mL and at end: 21 pg/mL) whereas IL-6 concentrations increased in control blood (at baseline 511 pg/mL and at end: 932 pg/mL). CONCLUSIONS: High-adsorption CVVH appears more effective than standard CVVH in decreasing noradrenaline requirements and plasma concentrations of cytokines in septic shock patients.

The effect of codeine on the Urge-to-Cough response to inhaled capsaicin.

We have shown previously in normal subjects that a sensory measure, the Urge-to-Cough rating, increases at concentrations of inhaled capsaicin that are lower than those necessary to elicit reflex cough. This finding suggests that the Urge-to-Cough may represent an index of the cough response. Research on cough in the human has most often employed challenge with inhaled capsaicin to induce reflex cough. Current measures of cough sensitivity in the human provide no information regarding the intensity of cough. The influence of codeine on cough perceptual sensitivity and the relationship to cough intensity with capsaicin-induced cough in normal subjects has not been evaluated. This study determined the effect of codeine on capsaicin-induced cough perceptual sensitivity and motor response in normal subjects in a double-blind, placebo-controlled, crossover study. This approach investigated the relevance of cough sensitivity, intensity, and sensory modalities in the assessment of cough suppression in humans. This study consisted of three experimental trials: administration of placebo, 30 mg codeine and 60 mg codeine. The study was double-blinded. The order of the three trials was randomized. Respiratory motor pattern was recorded with EMGs from the rectus abdominis, lateral abdominal muscles and eighth intercostal space. The subjects leaned into a fume hood to inspire deeply for 2 s once through a mouthpiece connected to the nebulizer. A modified Borg scale was used to estimate their Urge-to-Cough. The experimental trial consisted of eight test solutions of 0-200 microM capsaicin. Each solution was presented three times in a randomized block order for a total of 24 presentations. The lowest capsaicin concentration to elicit a cough was determined. The lowest capsaicin concentration to elicit an Urge-to-Cough greater than zero was identified. The Urge-to-Cough sensitivity was determined from the log-log slope. For placebo, the Urge-to-Cough was zero with inhalation of the vehicle and no coughs were observed. The threshold capsaicin concentration for subjects to report an Urge-to-Cough was 15.6 microM (+/-2.6 SEM). The capsaicin concentration threshold for eliciting a cough was significantly greater, 39.3 microM (+/-5.6 SEM). As the capsaicin concentration increased, the magnitude estimation of the Urge to-Cough increased. The slope of the log-log relationship for the Urge-to-Cough was 0.94 (+/-0.07 SEM). As the capsaicin concentration increased, the number and intensity of the coughs increased. The administration of 30 and 60 mg codeine had no significant effect on the threshold capsaicin concentration for the Urge-to-Cough. There was also no significant codeine effect on the slope of the log-log Urge-to-Cough relationship. Thirty and sixty milligram codeine had no significant effect on the relationship between the capsaicin concentration and the number and intensity of the coughs. The results of this study demonstrate that the threshold for a subject to perceive an Urge-to-Cough was less than the capsaicin concentration that elicits the cough motor response. There was a direct relationship between the sensory intensity (magnitude estimation of the Urge-to-Cough) and the cough number and intensity. Thus, as the sense of an Urge-to-Cough increased the cough motor response increased. Neither the 30 nor 60 mg codeine affected the perceptual or motor sensitivity to capsaicin-induced cough. These results showed that the initial threshold for responding to capsaicin-induced cough is the perception of an Urge-to-Cough, followed by a motor cough response if the capsaicin is increased above the perceptual threshold. As the capsaicin concentration increases, both the perceptual need to cough and the cough motor response increase. The response of subjects to inhalation of capsaicin consisted of both a sensory component leading to perception of an Urge-to-Cough and motor cough behavior.

Capsaicin exposure elicits complex airway defensive motor patterns in normal humans in a concentration-dependent manner.

The airway defensive response to tussive agents, such as capsaicin, is frequently assessed by counting the number of cough sounds, or expulsive events. This method does not identify or differentiate important respiratory events that occur in the respiratory muscles and lungs, which are critical in assessing airway defensive responses. The purpose of this study was to characterize the airway defensive behaviours (cough and expiration reflex) to capsaicin exposure in humans. We observed complex motor behaviours in response to capsaicin exposure. These behaviours were defined as cough reacceleration (CRn) and expiration reflex (ERn), where n is the number of expulsive events with and without a preceding inspiratory phase, respectively. Airway defensive responses were defined in terms of frequency (number of expulsive events), strength (activation of abdominal muscles) and behaviour type (CRn vs. ERn). Thirty-six subjects (15 females, 24+/-4 yr) were instrumented with EMG electrodes placed over the rectus abdominis (RA), external abdominal oblique (EO) and the 8th intercostal space (IC8). A custom-designed mouth pneumotachograph was used to assess the airflow acceleration, plateau velocity and phase duration of the expulsive phase. Subjects inhaled seven concentrations of capsaicin (5-200 microM) in a randomized block order. The total number of expulsive events (frequency) and the sum of integrated EMG for the IC8, RA and EO (strength) increased in a curvilinear fashion. Differentiating the airway defense responses into type demonstrated predominately CR1 and CR2 (i.e. inspiration followed by one and two expulsive events, respectively) with very few ER's at <50 microM capsaicin. At higher concentrations (>50 microM) ER's with one or more expulsive events (ER1) appeared, and the number of CR's with three or more expulsive events (CR3) increased. The decrease in EMG activation and airflow measurements with each successive expulsive event suggests a decline in power and shear force as the number of expulsive events increased. Therefore, the airway defensive response to capsaicin is a complex motor pattern that functions to coordinate ER's and CR's with differing numbers of expulsive events possibly to prevent aspirations and keep air moving to promote clearance.

Respiratory-related evoked potential elicited in tracheostomised lung transplant patients.

The present study investigated the role of removal of upper airway and lung vagal afferents in the respiratory-related evoked potential (RREP) response to inspiratory occlusions in two patients with a tracheostomy, who had undergone double lung transplantation (DLT). The patients were 1.5 and 3 months post-DLT and surgical placement of the tracheostomy. RREP recordings in response to inspiratory occlusions were obtained under four conditions: mouth breathing ignore trial; mouth breathing attend trial; tracheostomy breathing attend trial; and tracheostomy breathing ignore trial. The RREP peak components, Nf, P1 and N1, were present in both mouth and tracheostomy ignore breathing trials. The P300 was present in both mouth and tracheostomy attend trials. RREP peak latencies were similar between conditions. The peak amplitudes were greater with mouth breathing due to greater occlusion-related inspiratory pressure. These results demonstrate that the respiratory-related evoked potential can be elicited with inspiratory occlusion in the absence of mouth, upper airway and lung vagal afferent input. This suggests that inspiratory occlusion can elicit cortical activity with activation of inspiratory pump mechanoreceptors.

Heterotopic nasopharyngeal brain tissue associated with cleft palate.

Heterotopic brain tissue in a cleft palate is a very rare developmental anomaly. We present the eighth case reported worldwide with a review of the literature and suggestions on the management of this unusual condition.

Cytokine removal during continuous renal replacement therapy: an ex vivo comparison of convection and diffusion.

BACKGROUND AND OBJECTIVES: It is unknown whether cytokine adsorption to the membrane during continuous renal replacement therapy is affected by the technique. Such knowledge might affect the choice of technique in vivo. Accordingly, we conducted an ex vivo study to test whether continuous veno-venous hemofiltration (CVVH) or continuous veno-venous hemodialysis (CVVHD) affect cytokine adsorption differently. SETTING: Laboratory attached to the Intensive Care Unit of a tertiary hospital. DESIGN: Six healthy human volunteers donated blood, which was incubated with endotoxin. Control blood was left at room temperature, and treatment blood was recirculated for eight hours through closed circuits using polyacrylonitrile membranes (AN69). The effect of CVVH and CVVHD on cytokine removal from the circuits was compared. MEASUREMENTS: The concentrations of interleukins (IL)-1beta, 6, 8, 10 and TNF were measured in the control samples, pre-and post-filter and in the effluent at baseline and hourly thereafter. The clearances by adsorption, and filtration were calculated. RESULTS: Control cytokine concentrations remained the same or increased slightly. Adsorption was the major mechanism of removal for all cytokines with the exception of IL-1beta, but the effect was short-lived. Peak adsorption generally occurred at baseline before the start of CVVH and CVVHD, with clearances ranging from 43.7 ml/min (for IL-8) to 7.6 ml/min (for IL-10). The time-weighted average total clearances during CVVH were 23.3, 4.3, 3.8, -2.0, and 15 ml/min for IL-8, IL-1beta, TNF, IL-6, and IL-10 respectively. The corresponding clearances during CVVHD were 19.0, 10.7, 2.7, 2.4, and 0.3 ml/min. IL-10 clearances were greater during CVVH than CVVHD (p=0.03). Non adsorptive CVVH clearance of IL-1beta was greater than CVVHD clearance, but this advantage was outweighed by an increased tendency of the membrane to release IL-1beta into the circuit during HF. CONCLUSIONS: The technique of solute removal had only a minor effect on the magnitude of cytokine adsorption, and neither technique had the advantage for all the measured cytokines.

Super high flux hemofiltration: a new technique for cytokine removal.

OBJECTIVE: To test whether hemofiltration using a hemofilter with large pores (super high flux hemofiltration) achieves effective cytokine removal. DESIGN: : Ex vivo study. SETTING: Laboratory of an intensive care unit in a tertiary hospital. PATIENTS AND PARTICIPANTS: Five healthy volunteers. INTERVENTIONS: Blood was spiked with 1 mg of endotoxin and then circulated through a closed hemofiltration circuit with a large pore polyamide super high flux hemofilter (nominal cut-off point: 100 kDa). Hemofiltration was conducted at 1 l/h or 6 l/h of ultrafiltrate flow. Samples were taken from the arterial, venous and ultrafiltration sampling ports. MEASUREMENTS AND RESULTS: Sieving coefficients (SC) above 0.6 were achieved for interleukin (IL)-1beta, IL-6 and IL-10 and SCs above 0.3 were achieved for IL-8 and TNF-alpha at 1 l/h. SCs of all cytokines (except IL-1) were reduced when the ultrafiltration rate was increased from 1 l/h to 6 l/h ( p<0.01), but cytokine clearances still increased ( p<0.01). The highest SC for albumin was 0.1 at 1 l/h and fell to 0.01 at 6 l/h. No adsorption of cytokines and albumin was observed. CONCLUSION: High volume ultrafiltration using a super high flux filter achieved cytokine clearances comparable to, or greater than, those currently achieved for urea during standard continuous renal replacement therapy.

Necrotising fasciitis in an infant with congenital insensitivity to pain syndrome.

We present a rare case of necrotising fasciitis in an infant with congenital insensitivity to pain syndrome. The aetiology, diagnosis and management of necrotising fasciitis in children are compared with those in adults. In contrast to adults, children affected by necrotising fasciitis are usually previously healthy and have no predisposing factors. Early diagnosis, intravenous antibiotics and aggressive surgical debridement are mandatory for an optimal outcome.