Therapeutic response to bronchial thermoplasty: toward feasibility of patient selection based on modeling predictions.

Bronchial thermoplasty (BT) is a treatment for moderate-to-severe asthma in which the airway smooth muscle layer is targeted directly using thermal ablation. Although it has been shown to be safe and effective in long-term follow-up, questions remain about its mechanism of action, patient selection, and optimization of protocol based on structural phenotype. Using a cohort of 20 subjects who underwent thermoplasty and assessment by computed tomography (CT), we demonstrate that response to BT can be feasibly predicted based on pretreatment airway dimensions that inform a subject-specific computational model. Analysis revealed the need for CT assessment at total lung capacity, rather than functional residual capacity, which was less sensitive to the effects of BT. Final model predictions compared favorably with observed outcomes in terms of airway caliber and asthma control, suggesting that this approach could form the basis of improved clinical practice.NEW & NOTEWORTHY Bronchial thermoplasty is a treatment for asthma that targets the airway smooth muscle directly. We demonstrate the feasibility and constraints of predicting patient-specific response to thermoplasty using a computational model informed by pretreatment CT scans at different lung volumes. Predictions are compared with functional outcomes and posttreatment CT scans. This has the potential to form the basis for improved clinical practice.

Pharmacological bronchodilation is partially mediated by reduced airway wall stiffness.

BACKGROUND AND PURPOSE: In asthmatic patients, airflow limitation is at least partly reversed by administration of pharmacological bronchodilators, typically ß2 -adrenoceptor agonists. In addition to receptor-mediated bronchodilation, the dynamic mechanical environment of the lung itself can reverse bronchoconstriction. We have now explored the possibility that bronchodilators exert a synergistic effect with oscillatory loads by virtue of reducing airway wall stiffness, and therefore, enhancing the bronchodilatory response to breathing manoeuvres. EXPERIMENTAL APPROACH: Whole porcine bronchial segments in vitro were contracted to carbachol and relaxed to the non-specific ß-adrenoceptor agonist, isoprenaline, under static conditions or during simulated breathing manoeuvres. KEY RESULTS: The bronchodilatory response to isoprenaline was greater during breathing manoeuvres compared with the response under static conditions. As the bronchodilatory response to breathing manoeuvres is dependent upon airway smooth muscle (ASM) strain, and therefore, airway wall stiffness, our findings are likely to be explained by the effect of isoprenaline on reducing airway wall stiffness, which increased ASM strain, producing greater bronchodilation. CONCLUSIONS AND IMPLICATIONS: A contribution of reduced airway stiffness and increased ASM strain to the bronchodilator action of isoprenaline is shown, suggesting that oscillatory loads act synergistically with pharmacologically mediated bronchodilation. The implications for the treatment of asthma are that reducing airway wall stiffness represents a potential target for novel pharmacological agents.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb.

RATIONALE: The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. METHODS: Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. RESULTS: Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. CONCLUSION: These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.

Measuring airway dimensions during bronchoscopy using anatomical optical coherence tomography.

Airway dimensions are difficult to quantify bronchoscopically because of optical distortion and a limited ability to gauge depth. Anatomical optical coherence tomography (aOCT), a novel imaging technique, may overcome these limitations. This study evaluated the accuracy of aOCT against existing techniques in phantom, excised pig and in vivo human airways. Three comparative studies were performed: 1) micrometer-derived area measurements in 10 plastic tubes were compared with aOCT-derived area; 2) aOCT-derived airway compliance curves from excised pig airways were compared with curves derived using an endoscopic technique; and 3) airway dimensions from the trachea to subsegmental bronchi were measured using aOCT in four anaesthetised patients during bronchoscopy and compared with computed tomography (CT) measurements. Measurements in plastic tubes revealed aOCT to be accurate and reliable. In pig airways, aOCT-derived compliance measurements compared closely with endoscopic data. In human airways, dimensions measured with aOCT and CT correlated closely. Bland-Altman plots showed that aOCT diameter and area measurements were higher than CT measurements by 7.6% and 15.1%, respectively. Airway measurements using aOCT are accurate, reliable and compare favourably with existing imaging techniques. Using aOCT with conventional bronchoscopy allows real-time measurement of airway dimensions and could be useful clinically in settings where knowledge of airway calibre is required.

Potent bronchodilation and reduced stiffness by relaxant stimuli under dynamic conditions.

Airway relaxation in response to isoprenaline, sodium nitroprusside (SNP) and electrical field stimulation (EFS) was compared under static and dynamic conditions. The capacity of relaxants to reduce airway stiffness and, thus, potentially contribute to bronchodilation was also investigated. Relaxation responses were recorded in fluid filled bronchial segments from pigs under static conditions and during volume oscillations simulating tidal and twice tidal manoeuvres. Bronchodilation was assessed from the reduction in carbachol-induced lumen pressure, at isovolume points in pressure cycles produced by volume oscillation, and stiffness was assessed from cycle amplitudes. Under static conditions, all three inhibitory stimuli produced partial relaxation of the carbachol-induced contraction. Volume oscillation alone also reduced the contraction in an amplitude-dependent manner. However, maximum relaxation was observed when isoprenaline or SNP were combined with volume oscillation, virtually abolishing contraction at the highest drug concentrations. The proportional effects of isoprenaline and EFS were not different under static or oscillating conditions, whereas relaxation to SNP was slightly greater in oscillating airways. All three inhibitory stimuli also strongly reduced carbachol-induced airway stiffening. The current authors conclude that bronchoconstriction is strongly suppressed by combining the inhibitory stimulation of airway smooth muscle with cyclical mechanical strains. The capacity of airway smooth muscle relaxants to also reduce stiffness may further contribute to bronchodilation.

Airway mechanics and methods used to visualize smooth muscle dynamics in vitro.

Contraction of airway smooth muscle (ASM) is regulated by the physiological, structural and mechanical environment in the lung. We review two in vitro techniques, lung slices and airway segment preparations, that enable in situ ASM contraction and airway narrowing to be visualized. Lung slices and airway segment approaches bridge a gap between cell culture and isolated ASM, and whole animal studies. Imaging techniques enable key upstream events involved in airway narrowing, such as ASM cell signalling and structural and mechanical events impinging on ASM, to be investigated.

Airway narrowing in porcine bronchi with and without lung parenchyma.

During bronchoconstriction elastic after-loads arise due to distortion of lung parenchyma by the narrowing airway. In the present study, the functional effect of parenchymal elastic after-load on airway narrowing was determined. Airway narrowing was measured in vivo over a range of transpulmonary pressures and compared with in vitro narrowing measured at corresponding transmural pressures. Bronchi were generation 10 with internal diameters of approximately 4 mm. In vivo luminal narrowing was measured by videobronchoscopy in anaesthetised and ventilated pigs. In vitro luminal narrowing was measured by videoendoscopy in isolated bronchial segments. Airways were activated by maximum vagal nerve stimulation and maximum electrical field stimulation in vivo and in vitro, respectively. At 5 cmH2O, stimulation produced a 35.9+/-3.2% (n = 6) and a 36.5+/-2.4% (n = 11) decrease in lumen diameter in vivo and in vitro, respectively. At 30 cmH2O, luminal narrowing fell to 23.7+/-2.0% in vivo and 23.4+/-2.5% in vitro. There was no difference between luminal narrowing in vivo and in vitro at any pressure. In conclusion, these findings suggest that in mid-sized, cartilaginous bronchi, parenchymal elastic after-loads do not restrict airway narrowing.

Elastic properties of the bronchial mucosa: epithelial unfolding and stretch in response to airway inflation.

The bronchial mucosa contributes to elastic properties of the airway wall and may influence the degree of airway expansion during lung inflation. In the deflated lung, folds in the epithelium and associated basement membrane progressively unfold on inflation. Whether the epithelium and basement membrane also distend on lung inflation at physiological pressures is uncertain. We assessed mucosal distensibility from strain-stress curves in mucosal strips and related this to epithelial length and folding. Mucosal strips were prepared from pig bronchi and cycled stepwise from a strain of 0 (their in situ length at 0 transmural pressure) to a strain of 0.5 (50% increase in length). Mucosal stress and epithelial length in situ were calculated from morphometric data in bronchial segments fixed at 5 and 25 cmH(2)O luminal pressure. Mucosal strips showed nonlinear strain-stress properties, but regions at high and low stress were close to linear. Stresses calculated in bronchial segments at 5 and 25 cmH(2)O fell in the low-stress region of the strain-stress curve. The epithelium of mucosal strips was deeply folded at low strains (0-0.15), which in bronchial segments equated to < or =10 cmH(2)O transmural pressure. Morphometric measurements in mucosal strips at greater strains (0.3-0.4) indicated that epithelial length increased by approximately 10%. Measurements in bronchial segments indicated that epithelial length increased approximately 25% between 5 and 25 cmH(2)O. Our findings suggest that, at airway pressures <10 cmH(2)O, airway expansion is due primarily to epithelial unfolding but at higher pressures the epithelium also distends.

Cyclical elongation regulates contractile responses of isolated airways.

Bronchoconstrictor responses are quantitatively different when they are evoked under static conditions and during or after periods of deep inspiration. In vivo, deep inspirations produce bronchodilation and protect the lung from subsequent bronchoconstriction (termed bronchoprotection). These effects may be due in part to dynamic stretch on airways produced by cyclical expansion of airway diameter. However, airways also lengthen cyclically during breathing. The effects of cyclical airway elongation on evoked bronchoconstriction have not been examined. This study recorded evoked contractions of pig bronchial segments 1) at different airway lengths, 2) after a period of cyclical lengthening in relaxed airways, and 3) during cyclical lengthening in pretoned airways. Airway segments were mounted in organ baths and bathed in Krebs solution luminally and on the adventitia. Airways were cyclically lengthened by 5-30% of their deflated length at 0.5-2 Hz for 5 min. Contractions were evoked by electrical field stimulation or carbachol and were recorded under isovolumic conditions. Under static conditions, there was a blunt relationship between length and response to electrical field stimulation. After a period of airway length cycling, electrical field stimulation-induced contractions were increased. In airways pretoned with carbachol, cyclical lengthening produced a transient bronchodilation and a sustained increase in contraction. Contractile responses were not blocked by indomethacin. The results show that isolated airways respond actively to dynamic changes in length. Our results indicate that cyclical lengthening of airways could contribute to lung function in vivo but does not appear to account for the phenomenon of bronchoprotection.

Cholinergic responsiveness of the individual airway after allergen instillation in sensitised pigs.

Allergen exposure of sensitised lungs produces bronchial hyperresponsiveness in vivo associated with airway inflammation and remodelling. It is unclear if hyperresponsiveness is also present in airways in vitro under similar conditions of drug provocation as carried out in vivo, and at different times after allergen-challenge. This study records responsiveness of individual airway segments to acetylcholine (ACh) in sensitised bronchi after instillation of allergen (ovalbumin, OA). Airway histology and sensitivity and maximum effects to ACh were recorded 1, 24 and 72 h and 1 week after OA. OA-instilled airways exhibited eosinophilia and epithelial proliferation. Physiological recordings showed no change in maximum contractions of airway segments to acetylcholine placed in the airway lumen except at 24 h where they were reduced. In contrast maximum contractions to ACh to the airway adventitia were reduced at all times except 1 week, with the greatest change occurring at 24 h. There were no changes in airway sensitivity to either route of ACh in OA-instilled airways but the difference in sensitivity to adventitial and lumenal ACh was reduced. Results show that allergen does not produce hyperresponsiveness at the airway wall but it may alter an interaction between airway smooth muscle and other structural components of the airway.

Intraluminal pressure oscillation enhances subsequent airway contraction in isolated bronchial segments.

A period of deep inspiration in humans has been shown to attenuate subsequent bronchoconstriction, a phenomenon termed bronchoprotection. The bronchoprotective effect of deep inspiration may be caused though a depression in the force production of airway smooth muscle (ASM). We determined the response of whole airway segments and isolated ASM to a period of cyclic stretches. Isovolumetric contraction to electrical field stimulation (EFS) was assessed in porcine bronchial segments before and after intraluminal pressure oscillation from 5 to 25 cmH(2)O for 10 min at 0.5 Hz. Morphometry showed that this pressure oscillation stretched ASM length by 21%. After pressure oscillation, the response to EFS was not reduced but instead was modestly enhanced (P < 0.01). Airway responses to EFS returned to preoscillation levels 10 min after the end of oscillation. The increase in EFS response after pressure oscillation was not altered by the addition of indomethacin. In a separate experiment, we assessed isometric force in isolated ASM strips before and after length oscillation. The amplitude, frequency, and duration of length oscillation were similar to those induced in bronchial segments. In contrast to bronchial segments, length oscillation of ASM produced a significant depression in isometric force induced by EFS (P < 0.01). These results suggest that the response of ASM to length oscillation is modified by the airway wall. They also suggest that the phenomenon of bronchoprotection reported in some in vivo studies may not be an intrinsic property of the airway.

Relationship of airway narrowing, compliance, and cartilage in isolated bronchial segments.

Structural components of the airway wall may act to load airway smooth muscle and restrict airway narrowing. In this study, the effect of load on airway narrowing was investigated in pig isolated bronchial segments. In some bronchi, pieces of cartilage were removed by careful dissection. Airway narrowing was produced by maximum electrical field stimulation. An endoscope was used to record lumen narrowing. The compliance of the bronchial segments was determined from the cross-sectional area of the lumen and the transmural pressure. Airway narrowing and the velocity of airway narrowing were increased in cartilage-removed airways compared with intact control bronchi. Morphometric assessment of smooth muscle length showed greater muscle shortening to acetylcholine in cartilage-removed airways than in controls. Airway narrowing was positively correlated with airway compliance. Compliance and area of cartilage were negatively correlated. These results show that airway narrowing is increased in compliant airways and that cartilage significantly loads airway smooth muscle in whole bronchi.

A novel fluorochrome for the microscopic observation of microbial morphology in wet mounts.

A family of novel nontoxic fluorochromes, including trans-4-(p-N,N-dimethylaminostyryl)-N-butoxycarbonyl-methylpyridin ium bromide and other dimethylaminostyrylpyridinium salts, has been used for the examination of microorganisms in the living state. Equipment required is similar to that for immunofluorescence, but the technique does not necessitate the utilization of specific antibodies. Procaryotic vegetative cells and endospores were readily visualized by their fluorescence with no observable quenching. Other bacteria observed included capsule-forming species, acid-fast mycobacteria, and spirochetes. In addition, the technique is also applicable to the examination of eucaryotic microorganisms. The technique as described is simple and convenient, and serves as an alternate means for the microscopic study of viable microorganisms.

Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro.

The invasion and migration occurring in primary neoplastic tissue explants were studied by using a three-dimensional collagen matrix model, subsequent time-lapse videomicroscopy, and computer-assisted cell tracking. We show that not only single cells but groups of clustered cells comprising 5 to more than 100 cells detach from the primary tumor lesion and migrate within the adjacent extracellular matrix. These clusters were highly polarized, resulting in a high directional persistence of migration. Locomoting cell clusters were observed in primary cultures from invasive oral squamous cell carcinomas (6 of 9), ductal breast carcinomas (2 of 3), and rhabdomyosarcoma (1 of 1), whereas normal oral mucosa (0 of 4) was cell cluster negative. Thus, locomoting cell clusters could be a novel and potentially important mechanism of cancer cell invasion and metastasis.

T lymphocyte locomotion in a three-dimensional collagen matrix. Expression and function of cell adhesion molecules.

T cell locomotion within the extracellular matrix may be mediated by cell adhesion molecules. We investigated the expression and function of beta 1- and beta 2-integrins and CD44 on human peripheral CD4+ and CD8+ lymphocytes locomoting in a 3-D type I collagen matrix. Paths of randomly selected T cells were digitized from time-lapse videorecordings and were quantitatively analyzed. After the blocking of CD49b with mAb Gi9, the locomotion of a defined locomotor subset (50% of spontaneously locomoting cells) was inhibited. Anti-CD49d mAb HP2/1 and an activating anti-CD44 mAb (J173), respectively, induced transient recruitment (< 1 h) of previously nonmotile cells (10 to 35%). In contrast to the J173-induced short-term locomotion, hyaluronan incorporated within the matrix promoted locomotion for > 2 h. No significant effects were present for anti-CD49f (GoH3) and -CD11a (25.3) mAbs. After the addition of IL-8 to the matrix, rapid induction of locomotion in 20 to 30% of the cells (control) was evident, which was virtually abolished by anti-alpha 2- and alpha 6-integrin, and -CD11a mAbs. Thus, the locomotion of nonactivated and IL-8-activated T cells may involve different sets of integrins. Using flow cytometry, the development of a CD49b+CD29highCD44lowL-selectinlow T cell phenotype independent of activation markers including CD25, CD27, CD28, VLA-4, and CD45RA- to CD45RO-transition was observed after 4 days in the matrix. The initial development of spontaneous locomotion in the collagen matrix, however, was not accompanied by alterations in CAM surface staining and, therefore, may involve functional CAM activation rather than involving an increase in surface expression.

Locomotor phenotypes of unstimulated CD45RAhigh and CD45ROhigh CD4+ and CD8+ lymphocytes in three-dimensional collagen lattices.

The spontaneous locomotion of immunomagnetically isolated resting CD4+ and CD8+ lymphocytes in three-dimensional collagen gels was recorded by time-lapse videomicroscopy. Two-dimensional projections of the paths of randomly selected individual cells were digitized, plotted and quantitatively analysed. Among five different donors 46 +/- 10% of CD4+ and CD8+ lymphocytes (n = 180) showed initial spontaneous locomotion (individual speed 5-25 microns/min; mean speed CD4+ 5.2 +/- 3.6 microns, CD8+ 3.23 +/- 2.72 microns). Active CD4+ cells were constantly migrating for more than 4 hr, whereas CD8+ lymphocytes significantly slowed down after 60-90 min in the collagen gel (P < 0.003). Quantitative analysis of the paths indicated at least three migratory phenotypes: (1) spontaneously locomoting cells exhibiting high speed and low frequency of stopping; (2) a major non-motile fraction without significant displacement; and (3) a subpopulation within CD4+ and CD8+ cells with intermediate activity of speed and stopping. Further subtype analysis of immunomagnetically isolated CD45RAhigh/ROlow or CD45RAlow/ROhigh lymphocytes showed that more than 90% of CD4+ CD45RAhigh/ROlow cells were actively locomoting. In contrast, only 20% of the CD4+ CD45ROhigh/RAlow phenotype showed spontaneous motility to a limited degree. The data indicate that resting CD4+ and CD8+ lymphocytes comprise further locomotory subpopulation related to the expression of different CD45 isoforms.

Lymphocyte locomotion in three-dimensional collagen gels. Comparison of three quantitative methods for analysing cell trajectories.

We evaluated three different quantitative evaluation methods for lymphocyte locomotion in three-dimensional collagen gels: (1) the length of the two-dimensional migration path (distance migrated) was compared to (2) the resulting average displacement from the starting to the end point and (3) the displacement of the furthest migrating population (cells with high displacement). Locomotion of immunomagnetically isolated human CD4+ and CD8+ peripheral blood lymphocytes suspended in type I collagen gels was recorded using time-lapse videomicroscopy. Paths of randomly selected locomoting cells were digitized, reconstructed and quantitatively analysed. For spontaneously locomoting CD4+ and CD8+ lymphocytes (90 min observation period) the mean total distance migrated was 10.0 +/- 3.7 microns/min (CD4+; n = 114 cells) and 5.6 +/- 3.3 microns/min (CD8+; n = 90 cells). The mean displacement from the individual starting point amounted to 1.3 +/- 0.7 micron/min for CD4+ and 1.1 +/- 0.7 micron/min for CD8+ cells, thus representing only 5-25% of the total migration path (index range displacement/distance migrated: 0.13-50%). Incubation with interleukin-8 and/or receptor blocking by monoclonal antibodies against VLA-2 (Gi9) or VLA-4 (HP2/1) integrins significantly altered the mean length of the migration paths for six out of ten different experimental conditions. Average displacement or displacement of the most active cells detected significant changes in two and three out of ten samples. Whereas the interleukin-8 induced locomotory changes were correctly represented by end point determination, relatively slight but significant modulation in lymphocyte behaviour by anti-integrin antibodies was revealed solely by analysis of the complete cell trajectory. In conclusion, the cell trajectory may represent a sensitive method for evaluating induced subtle changes in lymphocyte locomotory characteristics.

Changes in cell three-dimensional locomotory characteristics upon expression of the EJ-ras oncogene.

Activation of the ras oncogene induces ruffling of membranes, enhances cell locomotion and has been implicated in the invasive process. We have investigated the locomotory changes of a 212 cell line due to ras activation using a novel system for determining the three-dimensional trajectories of cells migrating within collagen lattices. It was found that a small, yet statistically significant increase in cell speed resulted and that the duration of locomotory periods was prolonged in ras activated cells. These locomotory periods was prolonged in ras activated cells. These locomotory periods were cyclic in nature with a periodicity of approximately 30 min. These results are discussed in the relation to the current hypotheses concerning the molecular mechanisms involved in pseudopod kinetics.

Does a genetically determined aggressive locomotion favour the establishment of intracellular macrophage parasites?

A novel computerized methodology was used to quantify, for the first time, the 3-dimensional locomotory phenotypes of individual macrophages moving within an extracellular matrix-like hydrated collagen lattice/gel. Comparisons between two macrophage lines genetically susceptible to intracellular parasites (B10S4 and ANA-1) and two genetically resistant (B10R4 and CD2) demonstrated that Bcgs alleles consistently endowed macrophages with an outward searching and active locomotion, whereas Bcgr imparted a retrocessive phenotype. Several different macrophage pathogens may have exploited this genetically determined aggressive locomotory behavior of susceptible macrophages to allow them to rapidly enter a sanctuary.

Locomotory characteristics of fibroblasts within a three-dimensional collagen lattice: modulation by a helium/neon soft laser.

It has been postulated that low energy (soft) lasers can enhance wound healing. Considering the importance of cell locomotion in the wound healing process, we have studied the effects of a helium/neon laser upon the locomotory behaviour of a population of fibroblasts migrating within a three-dimensional hydrated collagen lattice. Statistical methods were used to quantify cell three-dimensional trajectories obtained using a computer-assisted tracking system. A two-minute exposure of embryonic fibroblasts to soft laser light decreased the speed and increased the frequency and duration of stops compared to controls. The locomotory phenotype induced in embryonic fibroblasts by the laser light resembled that of the developmentally older c20 fibroblasts, which are known to express a more mature locomotory phenotype. The relevance of these results to wound healing is discussed.