Studies of human airways in vitro: a review of the methodology.

The pathophysiology of human airway narrowing is only partly understood. In order to gain more insight in the mechanisms of human lung diseases and potential beneficial therapeutic agents, adequate models are needed. Animal airway models are of limited value since lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are unique to humans and because the mechanisms of airway narrowing differ between species. Therefore, it is important to perform studies on human isolated airways. We describe the models that have been developed to study airway function in vitro, emphasizing human airway preparations. The easily prepared airway strip and ring preparations are described first. The potential damage during preparation and the interference with airway structure are important drawbacks in these preparations. Lung parenchymal strips, described next, were designed in order to study responsiveness of small airways. However, parenchymal strips are anatomically complex, and responsiveness is determined by the relative amounts of airway and vascular smooth muscle. The lack of reproducibility between species and even within one animal limits their usefulness. Airway tube preparations, in which luminal and serosal stimulation can be separated, enable us to study the modulatory role of the airways epithelium in vitro. Furthermore, airway compliance can be measured. In the isolated perfused lung preparation, relationships between the airways and the vascular system are preserved and the interaction between these two systems can be studied. Weight gain due to fluid extravasation is a problem in this model which has not been used yet to study human lungs in vitro. Next, methodological aspects such as tissue handling and storage, recording of responses, removal of the epithelium, and electrical field stimulation are discussed in some detail. Although animal airways tissue can be studied immediately after removal, human tissue is often obtained with some delay. However, this seems tenable since electron microscopy of lung tissue obtained at autopsy showed that recovery of the preparation occurs during incubation of carbogenated Krebs-Henseleit (K-H) buffer. Dissected airways can be stored overnight in cooled K-H buffer until up to 55 hr after resection without losing viability. Commonly used physiological salt solutions which bath the tissue contain osmotic molecules, ions important for contractility, glucose as a substrate, and a bicarbonate-carbon dioxide buffer.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrical field stimulation causes oxidation of exogenous histamine in Krebs-Henseleit buffer: a potential source of error in studies of isolated airways.

Electric field stimulation (EFS) relaxes human histamine-precontracted airways in vitro. This relaxation is only partly neurally mediated. Nonneural relaxation has been also shown in blood vessels and is due to the generation of oxygen radicals by EFS. In isolated airways the origin of the nonneural component of the relaxation is not clear. Because exogenous catecholamines are oxidized during EFS of carbogenated Krebs-Henseleit (K-H) buffer, we questioned whether this is also the case for exogenous histamine. Human airways precontracted with histamine or methacholine were exposed to either EFS-stimulated carbogenated K-H buffer that also contained histamine or methacholine or unstimulated buffer. Airways exposed to EFS-stimulated buffer that contained histamine relaxed, whereas airways exposed to buffer containing methacholine or exposed to unstimulated buffer did not. It appeared that the histamine concentrations in the organ baths decreased during 30 min of EFS. This decrease was significantly reduced in the presence of ascorbic acid. We conclude that EFS causes oxidation of histamine in carbogenated K-H buffer, and this may at least partly explain the nonneural component of EFS-induced relaxations of precontracted human isolated airways. Therefore, histamine should not be used to induce precontraction in EFS experiments.

The perfused human bronchiolar tube characteristics of a new model.

Strips or rings of airway tissue are often used to study contractile responses of human airways in vitro. These preparations have the disadvantage that it is impossible to deliver stimuli selectively to the mucosal or serosal surface. Hence, they allow only for a limited evaluation of the modulatory role of the airway epithelium. We developed an in vitro model that allows independent stimulation from either the serosal or the mucosal side of human peripheral airways. Segments of human peripheral airways were perfused with a Krebs solution at a constant pressure, and responsiveness was measured as a change in flow rate. Pressure/flow relationships indicated laminar flow over a wide pressure range, and a working pressure of 6 cm H2O was chosen because this is a physiological transpulmonary pressure. When stepwise stretching the airway to 180% of its length, we noted an increase in baseline flow and a decrease in flow reduction after methacholine 10(-5) M. At 140% of the length, accurate and reproducible measurements of the sensitivity (EC50) to methacholine were obtained, and airway closure did not occur. A one-way analysis of variance (ANOVA) revealed that the between-patients differences accounted for 91% of the total variability for -log EC50. We conclude that this in vitro model offers interesting possibilities for evaluating the modulatory effects of the human airway epithelium. In addition, the model provides the opportunity to study human small-airway mechanical properties and secretory functions.

Evolution and natural history of chronic lung disease of prematurity.

Chronic lung disease (CLD) of prematurity is a common disorder in preterm infants who were ventilated for respiratory distress syndrome (RDS) at birth. Premature birth, mechanical ventilation and supplemental oxygen are the major risk factors for the development of CLD. Although the exact pathophysiology is unclear, recent evidence suggests that pulmonary inflammation may play a pivotal role in the development of CLD. Histologically, the evolution of CLD can be divided into an early inflammatory phase followed by a subacute and chronic fibroproliferative phase. The early, inflammatory phase of CLD is clinically indistinguishable from RDS. In bronchoalveolar lavage fluid an influx of inflammatory cells and increased levels of cytokines can be found. Pathological examination of the lungs reveals persisting hyaline membranes, necrosis of airway and alveolar epithelium and an influx of inflammatory cells in the lung. In the subacute fibroproliferative or reparative phase of CLD, persistent respiratory distress and hypercapnia are seen and patients require oxygen with or without ventilatory support. Histologically, this phase is characterized by hyperplasia of type II pneumocytes, hypertrophy of bronchial and bronchiolar smooth muscle and interstitial and perialveolar fibrosis. In the chronic fibroproliferative phase (up to 1 yr), airway remodelling occurs. Respiratory distress continues and many patients remain oxygen dependent. Cyanotic spells are frequently seen and chronic hypoxia may lead to pulmonary hypertension and right heart failure. Many patients have severe feeding problems and somatic growth is poor. In surviving patients, persisting lung function abnormalities are found. Airway resistance and airway responsiveness are increased and residual volume (RV) and RV/total lung capacity ratios remain elevated, indicating air trapping. Although lung function improves during childhood, residual abnormalities are still found in young adults, raising concerns about the evolution of pulmonary function in old age.

[Turkish children with recurrent abdominal pain and fever: familial Mediterranean fever]. / Turkse kinderen met recidiverende buikpijn en koorts: familiale mediterrane koorts.

Four children of Turkish origin, three boys aged 12, 8 and 7 years, and a girl aged 5 years, presented with clinical symptoms of familial Mediterranean fever. They had the characteristic episodes of fever combined with abdominal pain, thoracic pain, general malaise or arthralgia. Familial Mediterranean fever is an autosomal recessive genetic disorder restricted to people originating from the Middle East. The causative gene (MEFV) and many missense mutations have been identified. The clinical syndrome is characterised by self-limiting febrile episodes accompanied by inflammation of the serous membranes, resulting in peritonitis, pleuritis or synovitis. In untreated patients systemic amyloidosis may develop, which manifests as renal insufficiency. The diagnosis is based on the characteristic medical history and is confirmed by DNA analysis. Meanwhile, treatment with colchicine can be started. This is effective in 90% of affected patients. Being aware of the prevalence of familial Mediterranean fever in immigrant populations can improve the quality of life and prevent long-term complications.

[Bronchopulmonary dysplasia]. / Bronchopulmonale dysplasie.

Bronchopulmonary dysplasia (BPD) is a serious lung disease which is increasingly recognised in prematurely born infants in neonatal intensive care units. Little is known about the cause of the disease, the treatment is mainly empirical. Although oxygen is well known to be one of the causal agents for BPD, it is, at the same time, part of its treatment. The neurodevelopmental outcome of infants with BPD is likely to be disturbed, while the lasting treatment often causes problems in the family. Because of the tendency to treat premature infants born after still younger pregnancies, neonatologists are facing a growing number of patients with BPD. In this article the presentation, pathogenesis, diagnostical approach, therapy and the course as well as the follow-up of our patients is discussed.

Autonomic innervation of human airways: structure, function, and pathophysiology in asthma.

The human airways are innervated via efferent and afferent autonomic nerves, which regulate many aspects of airway function. It has been suggested that neural control of the airways may be abnormal in asthmatic patients, and that neurogenic mechanisms may contribute to the pathogenesis and pathophysiology of asthma. In this review, the autonomic innervation of the human airways and possible abnormalities in asthma are discussed. The parasympathetic nervous system is the dominant neuronal pathway in the control of airway smooth muscle tone. Stimulation of cholinergic nerves causes bronchoconstriction, mucus secretion, and bronchial vasodilation. Although abnormalities of the cholinergic innervation have been suggested in asthma, thus far the evidence for cholinergic dysfunction in asthmatic subjects is not convincing. Sympathetic nerves may control tracheobronchial blood vessels, but no innervation of human airway smooth muscle has been demonstrated. beta-Adrenergic receptors, however, are abundantly expressed on human airway smooth muscle and activation of these receptors causes bronchodilation. The physiological role of beta-adrenergic receptors is unclear and their function seems normal in asthmatic patients. Inhibitory nonadrenergic noncholinergic (NANC) nerves, containing vasoactive intestinal peptide and nitric oxide, may be the only neural bronchodilator pathways in human airways. Although a dysfunction of inhibitory NANC nerves has been proposed in asthma, thus far no differences in inhibitory NANC responses have been found between asthmatics and healthy subjects. Excitatory NANC nerves, extensively studied in animal airways, have also been detected in human airways. In animal studies, stimulation of excitatory NANC nerves causes bronchoconstriction, mucus secretion, vascular hyperpermeability, cough, and vasodilation, a process called 'neurogenic inflammation'. Recent studies have demonstrated an interaction between the excitatory NANC nervous system and inflammatory cells. Neuropeptides may influence the recruitment, proliferation, and activation of leukocytes. On the other hand, inflammatory cells may modulate the neuronal phenotype and function. The functional relevance of the excitatory NANC nervous system and its interaction with the immune system in asthma still remains to be elucidated.

Acute idiopathic scrotal oedema: four cases and a short review.

We report four cases of acute idiopathic scrotal oedema, an underreported cause of acute painless or moderately painfull swelling and erythema of the scrotum in young boys. It is a self-limiting disease and to prevent unnecessary surgical exploration or antibiotic therapy, it is very important to distinguish acute idiopathic scrotal oedema from more serious diseases.

Negative effect of insufflation on cardiac output and pulmonary blood volume.

In 14 anaesthetized young pigs the changes in pulmonary blood flow and pulmonary blood volume (Qp) during mechanical ventilation were quantified. Ventilation was performed at 10 cycles per min and tidal volume (VT) was adjusted to an arterial PCO2 of about 40 mmHg (5.3 kPa). In 4 animals, 7 ventilatory cycles with an inspiratory pause (IP) of 7.2 s but different tidal volumes were inserted at intervals of 5 min to determine the decrease in Qp (delta Qp) from the differences between right ventricular (Qs,rv) and left ventricular (Qs,lv) stroke volume, and to relate delta Qp to VT. We measured pressure in the aorta (Pao), central veins (Pcv), right and left ventricles (Prv, Plv) pericardium (Pit), and trachea (PT). Blood flow was measured electromagnetically (EM) in the pulmonary artery (Q'pa) and aorta (Q'ao). Stroke volumes were derived from the EM-flow curves. In the other 10 experiments, Qs,lv was derived from the aortic pulse contour. Beat-to-beat analyses of Qs,rv and Qs,lv and blood pressures during the normal ventilatory cycles and those with an IP revealed the following: 1) The end-expiratory RV output and LV output were constant and were defined as baseline values. 2) The accumulated decrease in Qs,rv during insufflation caused a mean deficit in cardiac output of 10.3 +/- 3.2% (s.d.), n = 135; the same was found for Qs,lv, indicating the pulse contour as a useful method to estimate the variations in cardiac output during a ventilatory cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin-induced contraction of human peripheral airways mediated by both bradykinin beta 2 and thromboxane prostanoid receptors.

Bradykinin (BK) induces bronchoconstriction in asthmatic but not in normal individuals. Studies in vivo in the human suggest that BK causes cholinergic nerve activation, release of prostanoids, and local axon reflexes with release of tachykinins in the airways. To determine the mechanisms of BK-induced airway narrowing, we investigated the effects of epithelium removal, inhibition of the enzymes neutral endopeptidase (NEP) and cyclooxygenase, and blockade of neural conductance with tetrodotoxin (TTX) on BK-induced responses of human isolated peripheral airways. Responses to BK were recorded from airways with spontaneous intrinsic tone and from airways precontracted with methacholine. Furthermore, we measured the BK-induced release of the prostanoids PGE2, PGI2, and TXA2 from airways with and without epithelium in the absence and presence of indomethacin by radioimmunoassay. Finally, we examined the effect of the bradykinin beta 2 receptor antagonist Hoe 140 and the thromboxane prostanoid (TP) receptor blocking drug GR32191 on BK-induced responses. BK contracted intact and epithelium-denuded airways with spontaneous intrinsic tone, whereas precontracted airways either relaxed or contracted to BK. Removal of the epithelium increased the sensitivity to BK sevenfold without changing the direction of the response. The NEP inhibitor phosphoramidon tended to increase the sensitivity to BK (NS) and did not change the direction of the response. Both contractile and relaxation responses to BK and the release of the prostanoids PGE2, PGI2, and TXA2 by the airway tissues were largely inhibited by indomethacin, whereas TTX had no effect. PGE2, PGI2, and TXA2 were released by both intact and epithelium-denuded airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative epithelial damage produces hyperresponsiveness of human peripheral airways.

The epithelium probably modulates airway smooth muscle responsiveness by producing relaxing factors, by inactivating agonists, or by acting as a physical barrier. In isolated airway strips, however, only a limited modulatory role of the epithelium has been found, and this may well be due to shortcomings of this airway model. The present study compares the modulatory role of the airway epithelium in human airway tubes and strips. In addition, since oxygen radicals may contribute to epithelial damage in asthma, oxidative damage to the airway epithelium was induced with luminally applied hydrogen peroxide (H2O2), and changes in responsiveness to the agonists histamine, methacholine, and salbutamol were measured. To examine whether intact epithelium acts as a barrier to histamine, the histamine concentration in the organ bath was measured in tubes with intact and damaged epithelium stimulated from the mucosal side. In airway strips, no differences in responsiveness were found between intact and epithelium-denuded airways for any of the three agonists. In contrast, the sensitivity of airway tubes to both histamine and methacholine was significantly lower with mucosal stimulation than with serosal stimulation (-log EC50: 4.87 and 4.92 versus 5.87 and 5.45 for histamine and methacholine, respectively, p < 0.001). No difference was found between the sensitivity to salbutamol of mucosally and serosally stimulated airways (-log EC50: 6.19 and 6.20, respectively). The modulation of the sensitivity to contractile agonists by the epithelium increased with increasing airway size, and was abolished after treatment with H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability of human isolated airways increases after hydrogen peroxide and poly-L-arginine.

In asthma, the airway epithelium may be damaged by oxidants and cationic proteins produced by eosinophils. The degree of epithelial damage correlates with the level of bronchial hyperreactivity. An increase in airway permeability to bronchoactive agonists may be one of the factors responsible for airway hyperresponsiveness in asthma. We investigated the effects of the oxidant hydrogen peroxide and the major basic protein (MPB) analogue poly-L-arginine on the permeability and morphology of nonasthmatic human peripheral airways. In addition, we examined whether airway permeability depends on airway size. Human airway tubes (generation 7 to 12) mounted in an organ bath were luminally perfused with Krebs-Henseleit buffer containing the hydrophilic tracer 111In-diethyletriamine pentaacetic acid (111In-DTPA) or the lipophilic tracer 14C-antipyrine (14C-AP). Permeability of the airways was calculated from the fluxes of the tracer molecules across the airway wall. After experiments, light- and electromicroscopic examination of the airway epithelium was performed. Baseline permeability to 14C-AP was five times greater than to 111In-DTPA. Luminal exposure of the airways to 100 mmol/L H2O2 produced a significant, sixfold increase in permeability to 111In-DTPA but not to 14C-AP, indicating the opening of paracellular pathways. The H2O2-induced increase in permeability was partly reversible. Luminal exposure to polyarginine for 3 h and 16 h produced a significant 4.5- and 7-fold increase in permeability to 111In-DTPA, respectively. Histologic examination of epithelium exposed to H2O2 of poly-L-arginine showed focal loss of superficial cells with preservation of basal cells. Baseline airway permeability increased with decreasing airway size (rs = -0.75, p < 0.01). These results suggest that epithelial damage due to eosinophil products may increase airway permeability, and that this may at least partly explain the increased responsiveness to inhaled stimuli in asthma.

Epithelium removal and peptidase inhibition enhance relaxation of human airways to vasoactive intestinal peptide.

In this study we evaluated the role of epithelial versus subepithelial peptidases in the responses of isolated peripheral and central human airways to VIP. Human airways were obtained at thoracotomies (n = 8) and studied in organ baths. Intact or epithelium-denuded strips of central and peripheral airways were incubated with or without a cocktail of peptidase inhibitors containing phosphoramidon (2.5 micrograms/ml), leupeptin, aprotinin, captopril, soybean trypsin inhibitor (all 20 micrograms/ml), and bestatin (2.8 micrograms/ml). After precontraction with histamine (5 x 10(-6) M), cumulative concentration-response curves to VIP (10(-10) to 10(-7) M) were obtained. Both intact central and peripheral airways showed only minor relaxations to VIP irrespective of the precontraction level. Removal of the epithelium and addition of peptidase inhibitors additively increased the sensitivity (> 20-fold) and maximal response to VIP in both central and peripheral airways. We conclude that (1) VIP relaxes both central and peripheral human airways but only in the absence of epithelium and/or the presence of peptidase inhibitors, and (2) both epithelial and subepithelial peptidases are important in the inactivation of VIP in human airways.