Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways.

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea, the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular, this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging, we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at deliver into mouse trachea via immediate retrograde fluid motion and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways. These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small-animal models.

Selective recording and stimulation of individual identified neurons in freely behaving Aplysia.

A neuroethological technique is described for selective recording and stimulation of an individual neuron in freely behaving Aplysia by means of a fine wire glued into the connective tissue sheath above the identified cell body. A whole-nerve cuff electrode simultaneously monitored functionally related multiunit axon activity. For biophysical analysis the soma was impaled with a microelectrode when the ganglion was subsequently exposed. The technique is illustrated for several identified neurons involved in different behaviors.

Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent.

Effective adenoviral gene therapy requires efficient viral vector entry into epithelial cells. Injured airway epithelia display enhanced gene transfer, reflecting in part increased vector access to protected cell populations and/or protected basolateral membranes. We tested whether adenoviral gene transfer is enhanced by modification of the epithelial barrier in mouse nasal airways with a nonionic detergent (polidocanol, PDOC). In C57BL/6 mice, 1.6 x 10(9) PFU of Ad5CMV LacZ (AdLacZ) instilled into the right nostril produced negligible gene transfer to the nasal epithelium 2 days after dosing, but significant, dose-dependent increases in gene transfer were achieved by pretreatment with PDOC. Permeation of the electron-dense tracer lanthanum into the intercellular junctions of PDOC (0.1%)-treated murine nasal epithelium, but not into intercellular junctions of vehicle controls, is consistent with PDOC-mediated increases in tight junctional permeability. In CF(-/-) mice, significant gene expression was not detectable after exposure to Ad5CBCFTR alone (1.4 x 10(9) PFU in 20 microl; AdCFTR), but PDOC pretreatment prior to AdCFTR instillation produced functional expression of CFTR (measured as deltaPD) 5 days after instillation. Because the development and testing of lung gene therapy will principally occur in children and adults with airway disease, AdLacZ gene transfer with and without PDOC pretreatment was examined in infected nasal airways. Gene expression was significantly reduced in infected as compared with uninfected airways. We conclude that the use of adjuvant surface-active and/or membrane-perturbing agents, synthetic or naturally derived, may provide a novel approach to enhancing the efficiency of adenoviral gene transfer.

Reduced neurocognition in children who snore.

Obstructive sleep apnea syndrome (OSAS) has been associated with reduced neurocognitive performance in children, but the underlying etiology is unclear. The aim of this study was to evaluate the relationship between hypoxemia, respiratory arousals, and neurocognitive performance in snoring children referred for adenotonsillectomy. Thirteen snoring children who were referred for evaluation regarding the need for adenotonsillectomy to a children's hospital otolaryngology/respiratory department underwent detailed neurocognitive and polysomnographic (PSG) evaluation. PSGs were evaluated for respiratory abnormalities and compared with 13 nonsnoring control children of similar age who were studied in the same manner. The snoring children had an obstructive respiratory disturbance index within normal range (mean obstructive apnea/hypopnea index, 0.6/hr). Despite this, several domains of neurocognitive function were reduced in the snoring group. These included mean verbal IQ scores (snorers 92.6 vs. nonsnorers 110.2, P < 0.001), mean global IQ scores (snorers 96.7 vs. nonsnorers 110.2, P < 0.005), mean selective attention scores (snorers 46.4 vs. nonsnorers 11.8, P < 0.001), mean sustained attention scores (snorers 8.0 vs. nonsnorers 2.2, P = 0.001), and mean memory index (snorers 95.2 vs. nonsnorers 112.1, P = 0.001). There was a direct relationship between number of mild oxygen desaturations of > or = 3%, obstructive hypopneas with > or = 3% oxygen desaturations, and respiratory arousals and severity of neurocognitive deficits, with the greatest effect being on memory scores. The disruption of sleep in snoring children produced by relatively mild changes in oxygen saturation or by increases in respiratory arousals may have a greater effect on neurocognitive function than hitherto appreciated. A possible explanation for these neurocognitive deficits may be the combination of the chronicity of sleep disruption secondary to snoring which is occurring at a time of rapid neurological development in the first decade of life. Future studies need to confirm the reversal of these relatively mild neurocognitive decrements post adenotonsillectomy.

Effect of immediate versus slow intrauterine reduction of congenital diaphragmatic hernia on lung development in the sheep: a morphometric analysis of term pulmonary structure and maturity.

The incidence of congenital diaphragmatic hernia (CDH) is 1:1,200-5, 000, and the condition is associated with high mortality and morbidity attributed principally to associated pulmonary hypoplasia. One treatment approach has been for intrauterine intervention to induce lung growth to a sufficient level to allow survival at birth. Repair of the hernia in utero has been attempted, using a method of immediate reduction and repair of the hernia (patch) compared to a slow reduction method using a silastic "silo" sewn over the diaphragm defect to contain the hernial contents. In animal studies, this second method has been associated with lower fetal morbidity and mortality. This study, utilizing the sheep model of CDH, focuses on analysis of lung structural development and maturation, comparing the efficacy of the immediate vs. slow methods of hernial repair in preventing/reversing pulmonary hypoplasia. We hypothesized that: a) Both the immediate (patch) and slow (silo) methods of hernia repair performed in the lamb model of CDH will stimulate lung growth and structural development and restore lung structure and maturity towards normal levels by term gestation; b) Effects will be detectable by morphometric measurement of the following parameters: lung volume; parenchyma to nonparenchyma tissue ratio; volume density of connective tissue in nonparenchyma; gas exchange tissue to airspace ratio; gas exchange surface area; capillary loading; alveolar/airspace density; and alveolar perimeter; c) Effects will be seen in all lobes of the lung; and d) There will be no significant difference in lung size or structural parameters between the two groups. Forty-four pregnant ewes were allocated randomly to one of four groups. Fetal lambs in three groups (n = 36) underwent CDH creation at days 72-74 of gestation. Of surviving lambs showing an adequate hernia, 9 were not operated on further, 11 underwent "repair" using a silastic chimney around the hernial contents (slow reduction), and 11 underwent "repair" by a silastic patch over the diaphragmatic defect (immediate reduction). The fourth group were normal controls. All surviving lambs (n = 8 in each group) were delivered by Cesarian section at 141-143 days (term = 145-149 days). Lungs were obtained at autopsy, inflation-fixed, divided into lobes, and sampled, and morphometric analysis was performed. Comparisons were made between these groups and with matched normal controls and CDH untreated animals prepared in conjunction and previously reported. The lungs from the CDH animals treated by both methods of fetal hernia repair showed significant lung growth and structural development and maturation, although they remained significantly hypoplastic compared to normal. There were minor differences in the lung parameters between these two groups, with a tendency for the slow method to provide more normal parameter values. An exception was the increase in lung volume that was greater for the immediate (patch) method, particularly in the left lower lobe. In conclusion, intrauterine hernia repair by both methods is capable of partially reversing total lung and lobar structural hypoplasia and immaturity. The slow reduction method, with reduced potential for mortality and morbidity, is at least as good at reversing pulmonary hypoplasia as the immediate method. Alternative intrauterine interventions to prevent or reverse pulmonary hypoplasia are discussed and compared with the hernia repair methods used in this study.

Efficacy of once-daily tobramycin monotherapy for acute pulmonary exacerbations of cystic fibrosis: a preliminary study.

Our objective was to compare the efficacy, safety, and microbiology of once-daily intravenous (IV) tobramycin with conventional 8-hourly tobramycin/ceftazidime IV therapy for acute Pseudomonas aeruginosa (PA) pulmonary exacerbations in cystic fibrosis (CF). CF patients with PA-induced pulmonary exacerbations were allocated to receive either once-daily tobramycin (Mono) or conventional therapy with tobramycin/ceftazidime given 8-hourly (Conv). The two longitudinal groups received therapy in a double-blind, randomized manner over a period of 2 years. Tobramycin doses were adjusted to achieve a daily area under the time-concentration curve of 100 mg x hr/L in both groups. Results were assessed for both short-term changes (efficacy and safety after 10 days of IV antibiotics during acute exacerbations) and long-term changes (efficacy, safety, and sputum microbiology between study entry and exit). Pulmonary function tests (PFTs) on admission were similar in both groups. After 10 days of IV antibiotics, absolute mean improvements in percent of predicted PFTs were 12.8, 12.1, and 13.7 for forced expiratory volume in 1 sec (FEV(1)), forced vital capacity (FVC), and forced expired flow between 25--75% of FVC (FEF(25--75%)) in the Conv group (n = 51 admissions) compared to 10.6, 9.9, and 10.6 in the Mono group (n = 47)(P<0.05 for all). Sixteen percent in the Conv group and 15% of patients in the Mono group did not respond to therapy by day 10. Long-term PFT patterns were similar for the Conv and Mono groups. The time between admissions did not differ. The Mono group showed a significant increase in tobramycin minimum inhibitory concentrations (MICs) against PA from study entry to study exit (P = 0.02, n = 27 strains); this failed to reach significance in the Conv group (P = 0.08, n = 25). There was no significant increase in the number of isolates, with MIC> or =8 mg/L in both groups. No short- or long-term changes in audiology or serum creatinine were found in either group. After 10 days of IV therapy, the urinary enzyme N-acetyl-beta-d-glucosaminidase/creatinine ratios increased in both groups (P0.05). This increase was greater in the Conv compared to the Mono group (P < 0.05). We conclude that this pilot study indicates once-daily tobramycin therapy to be as effective and safe as conventional 8-hourly tobramycin/ceftazidime therapy. Combination antibacterial therapy appears to offer no clinical advantage over once-daily tobramycin monotherapy. Tobramycin once-daily monotherapy is a potential alternative to conventional IV antibacterial therapy which deserves further investigation, including the impact on susceptibility of PA to tobramycin.

Intrathoracic silo for fetal diaphragmatic hernia: lung growth and slow reduction of abdominal viscera.

Correction of a left congenital diaphragmatic hernia in a human fetus with a large volume of liver in the chest requires reduction of liver and viscera into the abdomen. This can kink the ductus venosus and cause the death of the fetus. Therefore, we have repaired surgically created diaphragmatic hernias in fetal lambs by leaving viscera in the chest wrapped in a silastic chimney. With fetal growth there is a relative reduction of hernia volume over weeks, potentially avoiding kinking the ductus venosus. In four groups of lambs lung size and static respiratory system compliance at birth were compared. Lambs treated by this new technique (silo, n = 7) were compared with lambs that had undergone immediate complete correction with a flat silastic patch in the diaphragm plus an abdominal patch (patch, n = 8), with lambs with uncorrected hernias (n = 6), and with normals (n = 8). There was no significant difference between total lung weights (131 +/- 6 g v 157 +/- 13 g, mean +/- SEM, silo v patch) and lung displacement volumes (142 +/- 7 mL v 162 +/- 14 mL) in either surgically corrected group. Lungs from those corrected by silo were significantly heavier than those with uncorrected herniae (131 +/- 6 g v 56 +/- 5 g, P < .01), but were not as heavy as normal lungs (131 +/- 6 g v 257 +/- 16 g, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute respiratory infection in patients with cystic fibrosis with mild pulmonary impairment: comparison of two physiotherapy regimens.

Chest physiotherapy is an essential part of the management of cystic fibrosis, yet comparatively few studies have investigated the commonly used forms of chest physiotherapy during acute respiratory exacerbations. Fifteen subjects with cystic fibrosis and predominantly mild pulmonary impairment completed a randomised cross-over trial with 24 hours between treatments. The active cycle of breathing techniques (ACBT) assisted by a physiotherapist was compared with the ACBT performed independently by the patient. Measurement outcomes included pulmonary function tests, indirect calorimetry and oximetry parameters. Energy expenditure was not significantly different between the two treatment regimens, though significant improvements in pulmonary function were apparent 24 hours following the therapist-assisted ACBT. In this group of subjects, neither form of treatment proved superior in terms of energy consumption, but a reduction in airways obstruction was observed as a carry-over effect following the therapist-assisted ACBT.

Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.

Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials.

Phase contrast X-ray imaging for the non-invasive detection of airway surfaces and lumen characteristics in mouse models of airway disease.

We seek to establish non-invasive imaging able to detect and measure aspects of the biology and physiology of surface fluids present on airways, in order to develop novel outcome measures able to validate the success of proposed genetic or pharmaceutical therapies for cystic fibrosis (CF) airway disease. Reduction of the thin airway surface liquid (ASL) is thought to be a central pathophysiological process in CF, causing reduced mucociliary clearance that supports ongoing infection and destruction of lung and airways. Current outcome measures in animal models, or humans, are insensitive to the small changes in ASL depth that ought to accompany successful airway therapies. Using phase contrast X-ray imaging (PCXI), we have directly examined the airway surfaces in the nasal airways and tracheas of anaesthetised mice, currently to a resolution of approximately 2 microm. We have also achieved high resolution three-dimensional (3D) imaging of the small airways in mice using phase-contrast enhanced computed tomography (PC-CT) to elucidate the structure-function relationships produced by airway disease. As the resolution of these techniques improves they may permit non-invasive monitoring of changes in ASL depth with therapeutic intervention, and the use of 3D airway and imaging in monitoring of lung health and disease. Phase contrast imaging of airway surfaces has promise for diagnostic and monitoring options in animal models of CF, and the potential for future human airway imaging methodologies is also apparent.

Airway gene therapy and cystic fibrosis.

Airway disease in cystic fibrosis (CF) is the major cause of death and is presently inadequately treatable, but genetic therapies offer the hope that such life-long disease will be curable, or at least satisfactorily treated. Normal pathogen defences that have evolved on airway surfaces also prevent the various gene vectors now available from producing effective gene transfer. Nevertheless, findings from basic research and human clinical trials are revealing how these barriers might be overcome or circumvented, with benefits to therapeutic efficacy and patient safety. Though progress is slower than expected or desired, the therapeutic rewards will be great when safe and effective gene therapy for CF airway disease becomes a clinical reality.

Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production.

BACKGROUND: We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated. METHODS: A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production. RESULTS: We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant. CONCLUSIONS: By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.

The morphology and ultrastructure of tension receptors in the walking legs of the crab, Carcinus maenas.

The tension receptor system of the crab merus consists of two size classes of receptor cell body distributed along one face of the flexor muscle apodeme. The receptors show the general arthropod mechanoreceptor structure of cell body, connecting cilium, and sheathed sensory processes, but there are several differences. Many processes show convolutions, and the distal portion of the sensory process is embedded in the apodeme cuticle. The terminations of the sensory processes lack the usual structural specialisations for mechanotransduction. Tension transduction appears to occur by flexion of the cuticle-embedded sensory process.

Swimming in Aplysia brasiliana: behavioral and cellular effects of serotonin.

1. Aplysia brasiliana is a marine mollusk that swims by repeated metachronal flapping movements of its bilateral fleshy parapodia. Animals with bilateral cerebropedal connective (CPC) lesions do not swim when suspended above the substrate, although tonic CPC stimulation can elicit normal parapodial flapping. Although the parapodial opener-phase (POP) cells, a previously identified group of neurons, fire synchronous bursts of efferent spikes in-phase with parapodial opening movements in both intact animals and dissected preparations, they are not likely to be primary parapodial motoneurons. These cells receive one or more large, apparently monosynaptic excitatory postsynaptic potentials (EPSPs) during CPC stimulation that are effective in producing the swimming motor program (SMP). 2. In suspended CPC-lesioned animals, injections of serotonin (5-HT) that produce an average hemolymph concentration of 10(-5) M induced full-amplitude parapodial flapping. Selected episodes of flapping were similar in frequency to normal suspended swimming. 3. In suspended CPC-lesioned animals, 5-HT injections elicited an apparently normal swimming motor program that was associated with synchronous bursts of large-amplitude efferent spikes in the parapodial nerves. In many semi-intact preparations, exposing the circumoesophageal ganglia to 5-HT elicited a similar rhythmic motor program, but usually at a lower frequency than during normal swimming or during tonic CPC stimulation. 4. In isolated-ganglion preparations, bath application of 5-HT produced immediate depolarization and tonic firing of individual POP neurons, followed by smooth and regular bursting in the apparent absence of synaptic input. In such preparations, the motor program elicited by bath-applied 5-HT differed from the one elicited by tonic CPC stimulation in that the 5-HT-elicited rhythmic bursting usually was not synchronous in different POP neurons. Tonic CPC stimulation during bath applications of 5-HT produced immediate synchronization of bursts among the POP neurons. 5. Hyperpolarization (or depolarization) of a POP neuron during bath application of 5-HT increased (or decreased) the burst period, but membrane polarization did not change the burst period elicited during tonic CPC stimulation.

The effect of carbon monoxide on the function of a crustacean sensory receptor is no different to the effect of hypoxia.

The crayfish Cherax was used as a model to determine whether there are behavioural and neural effects that can be attributed to a direct carbon monoxide toxicity rather than to the hypoxia it produces. A comparison was made by replacing the oxygen in the ambient liquid with carbon monoxide or with nitrogen. We found that Cherax is resistant to extreme hypoxia produced by either gas, and that there is therefore no meaningful behavioural carbon monoxide toxicity. We describe hypoxia-induced changes in the tonic and phasic activity recorded from in-vitro preparations of the muscle receptor organs that are the same for nitrogen and carbon monoxide substituted saline solutions. We conclude that in the single cells of the muscle receptor organs of Cherax, carbon monoxide has no toxic effects additional to those attributable to hypoxia.

Swimming in Aplysia brasiliana: identification of parapodial opener-phase and closer-phase neurons.

1. In freely behaving Aplysia brasiliana, spontaneous swimming in the laboratory occurred primarily in the dark hours of the day-night cycle. Suspending an intact animal above the substrate elicited continuous parapodial flapping with the same frequency and amplitude as spontaneous swimming. Parapodial flapping with decreased frequency and amplitude could still be elicited by suspending minimally dissected, but not more radically dissected, preparations. 2. In otherwise intact animals, severing the cerebropedal connective (CPC) bilaterally abolished suspended parapodial flapping, but normal flapping was elicited by tonic stimulation of the distal CPC. In minimally dissected preparations, tonic CPC stimulation elicited parapodial flapping, but with reduced frequency and amplitude. 3. During normal parapodial flapping, chronically implanted electrodes on parapodial nerves recorded the swimming motor program (SMP). The whole-nerve SMP consisted of rhythmic bursts of large-amplitude efferent units in phase with parapodial opening, with no observable activity during parapodial closing. By contrast, simultaneous electromyogram (EMG) recordings from antagonistic parapodial muscles showed antiphasic bursts of activity during opening and closing. The SMP was inhibited by touching food to the animals' lips. 4. Parapodial nerve backfills, using nickel chloride, labeled several cell clusters in the ipsilateral pedal ganglion. Two of these clusters were located caudally: one tightly clustered medial group had large cell bodies, and another, more distributed, lateral group had small cell bodies. The two clusters were identified in semi-intact preparations and isolated brains, using tonic CPC stimulation to elicit a fictive SMP recorded in parapodial nerves, and intracellular electrodes to characterize and stain individual cells. 5. The large parapodial opener-phase (POP) neurons were normally silent. At the onset of CPC stimulation, POP neurons depolarized and fired tonically, and then burst rhythmically in phase with each other, and one for one with large-amplitude axon spikes observed extracellularly in parapodial nerves during the fictive SMP. Intracellular firing of POP cells, singly or in pairs, never produced observable papapodial movements or one-for-one responses in parapodial muscles. Lucifer yellow-filled POP neurons showed a process (with a pronounced rostral loop) that gave off many short, fine neurites in the pedal neuropile before branching into two or three axons projecting into different parapodial nerves. 6. The smaller parapodial closer-phase (PCP) neurons normally discharged tonically at low frequencies.(ABSTRACT TRUNCATED AT 400 WORDS)

Temperature dependence of egg laying in Aplysia brasiliana and A. californica.

The temperature dependence of egg laying was examined in winter-caught Aplysia. Cold-water Aplysia californica and warm-water A. brasiliana were individually housed in the same large aquarium for 16 days at 15 degrees C, and then for 16 days at 20 degrees C. Initially, the majority of the A. californica were not reproductively mature (as determined by injections of atrial gland extracts) whereas all of the A. brasiliana were reproductively mature. When the temperature was increased from 15 to 20 degrees C, both species showed a marked increase in the frequency of egg laying. At both temperatures, A. brasiliana laid eggs more frequently but produced smaller egg masses than A. californica. We conclude that increased egg laying in A. californica was attributable both to facilitation of oogenesis in previously reproductively immature animals and to increased activity of the bag cells which release an egg-laying hormone. Increased egg laying in A. brasiliana was attributable primarily to increased bag cell activity.