An optimized non-destructive protocol for testing mechanical properties in decellularized rabbit trachea.

Successful tissue-engineered tracheal transplantation relies on the use of non-immunogenic constructs, which can vascularize rapidly, support epithelial growth, and retain mechanical properties to that of native trachea. Current strategies to assess mechanical properties fail to evaluate the trachea to its physiological limits, and lead to irreversible destruction of the construct. Our aim was to develop and evaluate a novel non-destructive method for biomechanical testing of tracheae in a rabbit decellularization model. To validate the performance of this method, we simultaneously analyzed quantitative and qualitative graft changes in response to decellularization, as well as in vivo biocompatibility of implanted scaffolds. Rabbit tracheae underwent two, four and eight cycles of detergent-enzymatic decellularization. Biomechanical properties were analyzed by calculating luminal volume of progressively inflated and deflated tracheae with microCT. DNA, glycosaminoglycan and collagen contents were compared to native trachea. Scaffolds were prelaminated in vivo. Native, two- and four-cycle tracheae showed equal mechanical properties. Collapsibility of eight-cycle tracheae was significantly increased from -40cm H2O (-3.9kPa). Implantation of two- and four-cycle decellularized scaffolds resulted in favorable flap-ingrowth; eight-cycle tracheae showed inadequate integration. We showed a more limited detergent-enzymatic decellularization successfully removing non-cartilaginous immunogenic matter without compromising extracellular matrix content or mechanical stability. With progressive cycles of decellularization, important loss of functional integrity was detected upon mechanical testing and in vivo implantation. This instability was not revealed by conventional quantitative nor qualitative architectural analyses. These experiments suggest that non-destructive, functional evaluation, e.g. by microCT, may serve as an important tool for mechanical screening of scaffolds before clinical implementation. STATEMENT OF SIGNIFICANCE: Decellularization is a front-running strategy to generate scaffolds for tracheal tissue-engineering. Preservation of biomechanical properties of the trachea during this process is paramount to successful clinical transplantation. In this paper, we evaluated a novel method for biomechanical testing of decellularized trachea. We detected important loss of functional integrity with progressive cycles of decellularization. This instability was not revealed by our quantitative nor qualitative analyses. These experiments suggest that the technique might serve as a performant, non-destructive tool for mechanical screening of scaffolds before clinical implementation.

The role of recipient derived interleukin-17A in a murine orthotopic lung transplant model of restrictive chronic lung allograft dysfunction.

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model. Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist. After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice. The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD.

Combined or Serial Liver and Lung Transplantation for Epithelioid Hemangioendothelioma: A Case Series.

Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor with variable biological and clinical behavior. There is increasing experience with liver transplantation (LiTx) for hepatic EHE, even in cases of extrahepatic disease localization. Until now, no cases of lung transplantation (LuTx) had been reported for pulmonary EHE. This report describes three cases of EHE with multifocal disease in patients who underwent either serial or combined LiTx and LuTx.

Azithromycin and the treatment of lymphocytic airway inflammation after lung transplantation.

Lymphocytic airway inflammation is a major risk factor for chronic lung allograft dysfunction, for which there is no established treatment. We investigated whether azithromycin could control lymphocytic airway inflammation and improve allograft function. Fifteen lung transplant recipients demonstrating acute allograft dysfunction due to isolated lymphocytic airway inflammation were prospectively treated with azithromycin for at least 6 months (NCT01109160). Spirometry (FVC, FEV1 , FEF25-75 , Tiffeneau index) and FeNO were assessed before and up to 12 months after initiation of azithromycin. Radiologic features, local inflammation assessed on airway biopsy (rejection score, IL-17(+) cells/mm(2) lamina propria) and broncho-alveolar lavage fluid (total and differential cell counts, chemokine and cytokine levels); as well as systemic C-reactive protein levels were compared between baseline and after 3 months of treatment. Airflow improved and FeNO decreased to baseline levels after 1 month of azithromycin and were sustained thereafter. After 3 months of treatment, radiologic abnormalities, submucosal cellular inflammation, lavage protein levels of IL-1ß, IL-8/CXCL-8, IP-10/CXCL-10, RANTES/CCL5, MIP1-α/CCL3, MIP-1ß/CCL4, Eotaxin, PDGF-BB, total cell count, neutrophils and eosinophils, as well as plasma C-reactive protein levels all significantly decreased compared to baseline (p < 0.05). Administration of azithromycin was associated with suppression of posttransplant lymphocytic airway inflammation and clinical improvement in lung allograft function.

Lymphocytic bronchiolitis after lung transplantation is associated with daily changes in air pollution.

Acute rejection represents a major problem after organ transplantation, being a recognized risk for chronic rejection and mortality. Recently, it became clear that lymphocytic bronchiolitis (LB, B-grade acute rejection) is more important than previously thought, as it predisposes to chronic rejection. We aimed to verify whether daily fluctuations of air pollution, measured as particulate matter (PM) are related to histologically proven A-grade rejection and/or LB and bronchoalveolar lavage (BAL) fluid cellularity after lung transplantation. We fitted a mixed model to examine the association between daily variations in PM(10) and A-grade rejection/LB on 1276 bronchoscopic biopsies (397 patients, 416 transplantations) taken between 2001 and 2011. A difference of 10 µg/m(3) in PM(10) 3 days before diagnosis of LB was associated with an OR of 1.15 (95% CI 1.04-1.27; p = 0.0044) but not with A-grade rejection (OR = 1.05; 95% CI 0.95-1.15; p = 0.32). Variations in PM(10) at lag day 3 correlated with neutrophils (p = 0.013), lymphocytes (p = 0.0031) and total cell count (p = 0.024) in BAL. Importantly, we only found an effect of PM10 on LB in patients not taking azithromycin. LB predisposed to chronic rejection (p < 0.0001). The risk for LB after lung transplantation increased with temporal changes in particulate air pollution, and this was associated with BAL neutrophilia and lymphocytosis. Azithromycin was protective against this PM effect.

Azithromycin reduces pulmonary fibrosis in a bleomycin mouse model.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease without proper treatment. Despite intensive research, the exact underlying pathogenesis remains elusive. It is regarded as a continuous injury, resulting in inflammation, infiltration, and proliferation of fibroblasts and extracellular matrix deposition, leading to an irreversible restrictive lung function deterioration and death. In this study the effect of azithromycin, a macrolide antibiotic on bleomycin-induced pulmonary fibrosis was investigated. C57BL/6 mice were intratracheally instilled with bleomycin (0.5 mg/kg) or saline. In the bleomycin group, half of the animals received azithromycin every other day from day 1 on. Bronchoalveolar lavage and histology were performed at days 7 and 35, and pulmonary function tests on day 35. At day 35, fibrotic lesions (spindle cell proliferation/collagen I deposition) were paralleled by a restrictive lung function pattern. Alterations were found in neutrophils and macrophages (innate immunity) and in T(H)2, T(H)17, and Treg cytokines (adaptive immunity). Azithromycin significantly reduced both fibrosis and the restrictive lung function pattern. This study demonstrated a beneficial effect of azithromycin on bleomycin-induced pulmonary fibrosis. A possible mechanism could be a modulation of both innate immunity and adaptive immunity. These findings might suggest a potential role for azithromycin in the treatment of IPF.

Follicular bronchiolitis: a rare cause of bronchiolitis obliterans syndrome after lung transplantation: a case report.

This case report is the first confirmed case of follicular bronchiolitis (FB), a rare bronchiolar disorder characterized by peribronchiolar lymphoid follicles, in a series of over 400 lung transplantations performed in our center. It is to our knowledge, the first publication describing FB after lung transplantation (LTx), presenting as chronic allograft dysfunction or bronchiolitis obliterans syndrome (BOS).

A dichotomy in bronchiolitis obliterans syndrome after lung transplantation revealed by azithromycin therapy.

Bronchiolitis obliterans syndrome (BOS) is the most important cause of late mortality following lung transplantation, resulting in major morbidity and a huge burden on healthcare resources. Treatment options are limited, resulting in a mere stabilisation of the lung function decline. Recent introduction of the macrolide antibiotic azithromycin raised new hope after demonstrating lung function improvement in subsets of patients. The present study aimed to provide an overview of the clinical effects on azithromycin in the setting of BOS after lung transplantation, with special emphasis on the anti-inflammatory actions. Moreover, the authors proposed a new frame of thinking centred on a dichotomy in the pathogenesis and clinical phenotype of BOS. Subsets of BOS patients were identified who do or do not respond to azithromycin (regarding forced expiratory volume in one second (FEV(1)), bronchoalveolar lavage (BAL) neutrophilia/interleukin-8). These observations have shed new light on the current belief that BOS represents a homogenous clinical entity in which the neutrophil is the main culprit. Recent clinical observations, supported by research findings, have revealed a dichotomy in the clinical spectrum of BOS with neutrophilic (partially) reversible allograft dysfunction (responding to azithromycin) and fibroproliferative BOS (not responding to azithromycin). This concept is reinforced by unique data obtained in BOS patients, consisting of histology specimens, physical and radiological examination, FEV(1 )and BAL examination. The acceptance of this dichotomy can improve understanding of the heterogeneous pathological condition that constitutes bronchiolitis obliterans syndrome, thus encouraging a more accurate diagnosis and, ultimately, better tailored treatment for each bronchiolitis obliterans syndrome patient.

Multidisciplinary interobserver agreement in the diagnosis of idiopathic pulmonary fibrosis.

The purpose of the present study was to evaluate the accuracy of the diagnosis of idiopathic pulmonary fibrosis (IPF) by respiratory physicians in six European countries, and to calculate the interobserver agreement between high-resolution computed tomography reviewers and histology reviewers in IPF diagnosis. The diagnosis of usual interstitial pneumonia (UIP) was assessed by a local investigator, following the American Thoracic Society/European Respiratory Society consensus statement, and confirmed when a minimum of two out of three expert reviewers from each expert panel agreed with the diagnosis. The level of agreement between readers within each expert panel was calculated by weighted kappa. The diagnosis of UIP was confirmed by the expert panels in 87.2% of cases. A total of 179 thoracic high-resolution computed tomography scans were independently reviewed, and an interobserver agreement of 0.40 was found. Open or thoracoscopic lung biopsy was performed in 97 patients, 82 of whom could be reviewed by the expert committee. The weighted kappa between histology readers was 0.30. It is concluded that, although the level of agreement between the readers within each panel was only fair to moderate, the overall accuracy of a clinical diagnosis of idiopathic pulmonary fibrosis in expert centres is good (87.2%).

The role of interleukin-17 during acute rejection after lung transplantation.

Acute rejection (AR) is an important complication that can occur after lung transplantation and constitutes a risk factor for bronchiolitis obliterans syndrome, which is characterised by a neutrophilic airway inflammation. The specific aim of this study was to investigate the role of interleukin (IL)-17, which promotes chemotaxis of neutrophils by inducing IL-8 production, in AR. Cell differentials, mRNA and protein levels were quantified in bronchoalveolar lavages (BALs) taken from patients at 28 and 90 days after lung transplantation. The patient's rejection status was assessed by transbronchial biopsy. An AR was found in nine out of the 26 patients examined, 28 days after transplantation. The number of BAL neutrophils and lymphocytes were increased in these patients. IL-17 mRNA and protein levels in the BAL were increased in patients with AR. Analysis of BAL obtained at day 90 after transplantation, demonstrated that the increase in IL-17 had disappeared, whereas the increase in neutrophils and lymphocytes persisted. These data showed that interleukin-17 is temporarily upregulated in bronchoalveolar lavage during acute rejection. The number of lymphocytes and neutrophils are increased in bronchoalveolar lavage during acute rejection and may persist up to 2 months after acute rejection. These findings suggest that interleukin-17 is important in the pathophysiology of acute lung rejection.

Kinetics of an intratracheally administered chromium catalyst in rats.

Chromium-based catalysts are used for the synthesis of polyethylene, but little is known about the hazard and biomonitoring possibilities of this type of chromium for workers who may be occupationally exposed to such compounds. Therefore, the bioavailability and toxicokinetics of chromium were studied in male Wistar rats after a single intratracheal instillation (2 ml/kg body weight) of various doses (1, 5, or 25 mg/kg body weight) of the catalyst (approximately 1% chromium bound to an amorphous silica matrix), either before (CAT-Cr[III]) or after (CAT-Cr[VI]) heat treatment. The results were compared with those of equivalent amounts of two chromium salts (CrCl(3) and K(2) Cr(2) O (7). Each dose group was composed of three rats. The concentration of chromium was determined by atomic absorption spectrometry in urine (collected daily for 7 d) and in plasma, erythrocytes, lung, and liver tissue obtained 2 d (only highest concentration) and 7 d after dosing. On d 2, a significant increase in lung weight was found in the animals treated with the highest dose of the hexavalent Cr products. On d 7, on the basis of body weights, lung weights, and lung histology, there was no overt toxicity, except after the highest dose of CAT-Cr(VI). The elimination of all forms of chromium was apparently monoexponential, with calculated half-life elimination times in urine of 4-11 h for Cr(III) (CAT-Cr[III] and CrCl3 ) and 8-21 h for Cr(VI) (CAT-Cr[VI] and K(2) Cr(2) O(7). On d 2, the erythro-cytes Cr concentrations were significantly higher for the hexavalent Cr products than for the trivalent Cr products. After 7 d, the erythrocytes Cr concentrations were significantly increased above control values (3 microg/L) only in rats treated with the 2 highest doses of Cr( VI) compounds (12 and 64 microg/L for K(2) Cr(2) O(7), and 14 and 79 microg/L for CAT-Cr[VI]). The present study shows that intratracheally instilled Cr(VI) and Cr(III) have different toxicokinetic profiles and that the Cr(VI) catalyst has the same bioavailability and excretion kinetics as a water-soluble Cr(VI) salt. Exposure to chromium compounds could be monitored by measuring Cr concen-trations in urine (shortly after exposure) and in erythrocytes (also at later time points after high Cr[VI] exposure).

Microscopic haematuria in giant cell arteritis.

Giant cell arteritis (GCA) is traditionally considered to spare the kidney, although an uncontrolled study reported microscopic haematuria in 10 out of 30 patients with GCA. To study the frequency and the characteristics of microscopic haematuria in GCA, we retrospectively studied 42 patients with biopsy-proven GCA, 39 patients with polymyalgia rheumatica (PMR) and 62 control patients >or=60 years of age, admitted to the general internal medicine unit. Patients with pyuria, significant bacteriuria or a known haematuric disorder were excluded. Microscopic haematuria was defined as the presence of >5 red blood cells (RBC) per high-power field (sediment counts) or of >8 RBC/microl (direct counting). Microscopic haematuria was present at presentation in 47.6% of the GCA patients, versus 17.9% of the PMR patients (P = 0.005) and 21.0% of the control patients (P = 0.008). Urinary RBC were predominantly dysmorphic in all GCA patients in whom RBC morphology was assessed (n = 7). Presenting symptoms, renal function, arterial blood pressure and degree of leukocyturia did not differ significantly between GCA patients with or without haematuria. After the initiation of corticosteroid therapy, microscopic haematuria was no longer detectable in 25 of 35 GCA patients (71.4%). Microscopic haematuria of renal origin is frequent but generally benign in patients with GCA. Its presence, if unassociated with blood pressure elevation or renal function deterioration, helps to rule in rather than to rule out the diagnosis of GCA. In the typical setting invasive urologic and nephrologic work-up may not be warranted.

Sleeve-related thrombosis: a new form of catheter-related thrombosis.

In a detailed study of central venous catheter-related sleeve and thrombosis in experimental animals, a new form of thrombosis was detected and termed sleeve-related thrombosis. A silastic catheter was placed in the jugular vein and the anterior vena cava of 22 rabbits and 54 rats. After intervals of 1, 3, 7 days, 2, 3, 4 weeks and 1, 2, 4, 6 months the veins were examined by light microscopy and by transmission electron microscopy. In about 50% of the rats a thrombus was observed at the end of the catheter sleeve. Consecutive cutting allowed the visualization of a transition from a sleeve via part of sleeve and part of thrombus to a pure thrombus. This thrombus was separated from the vein wall and could not be considered a mural thrombus. As the thrombus was only attached to the terminal part of the organized catheter sleeve we propose the name sleeve-related thrombosis.

Classifying interstitial lung diseases in a fractal lung: a morphologist's view "anno Domini 2000".

Interstitial lung diseases (ILDs) remain a challenging problem for the pathologist. New insights in aetiology and pathogenesis, new diagnostic tools and successful research have led to a renewed interest in ILDs during the last few years, and highlighted the need for a novel classification, particularly of the chronic and/or idiopathic categories of interstitial pneumonias. The present paper compares the terminology of the latter categories in current and previous classifications and briefly discusses the pathological basis for the classifications of ILDs in general, and for the idiopathic interstitial pneumonias (IIPs) in particular. The difference between high versus low morphological specificity determines the pathological classifications. The classification of lIPs relies upon a pattern recognition taking temporal and spatial distribution into consideration. The last section of this paper discusses recent research opposing the conventional pathological approach, analogous to the mechanical two-compartment model of the lung, in which a discontinuity is considered between these two compartments, and thus, a distinction is made between interstitial lung diseases with and without bronchiolitis. In the recent "fractal" concept, the continuity of the lung architecture is emphasized: the lung is a so-called fractal tree with noninteger dimensions. In this fractal model, an interstitial lung disease effects a peripheral part of the pulmonary fractal tree and this may or may not include bronchioles.

Interstitial lung diseases: an epidemiological overview.

Epidemiological studies on interstitial lung diseases (ILDs) may be schematically subdivided into the following major types: 1) quantifications of disease, broken down into incidence, prevalence and mortality data; 2) identification of aetiological factors; and 3) clinical epidemiological studies. Epidemiological data may be obtained from different sources or population groups, using different study designs such as systematic national statistics, population-based data and registries, and large case series of specific diseases. Differences in results between epidemiological studies may be due to real differences in incidence, but may also be due to changes in disease definitions and classifications, differences in the epidemiological design of the studies, or even registration bias. Comparative epidemiological data of different ILDs are almost limited to the general population study in Bernalillo County and to national mortality statistics, which should be interpreted with great caution. Also, some, mostly national registries of the different ILDs have been carried out by specific medical profession groups (especially pulmonologists), which clearly underestimate the real incidence of ILDs, but in which the comparison of the relative frequencies is probably accurate. Based on all these comparative studies, sarcoidosis and idiopathic pulmonary fibrosis appear to be the most frequent ILDs, followed by hypersensitivity pneumonitis and ILD in collagen vascular disease, when classical pneumoconioses are not included. There is also a relatively large group of nonspecific fibrosis. Much more data have been published on the epidemiology of specific forms of interstitial lung disease. Most information is available on the epidemiology of sarcoidosis, and those data are probably the most accurate. Data on idiopathic pulmonary fibrosis have the disadvantage of the recent changes in definition and classification of this disease. Hypersensitivity pneumonitis has been studied epidemiologically, especially in some exposure groups such as farmers and pigeon breeders, and in some regions in North America, UK, France and Scandinavia. Estimates of frequencies of interstitial lung disease in collagen vascular disease or of drug-induced interstitial lung disease are less accurate and more variable, depending on diagnostic criteria. Notwithstanding the aforementioned problems, this report tries to provide a balanced overview of the epidemiology of different interstitial lung diseases.

Giant cell interstitial pneumonia (hard metal lung disease, cobalt lung).

Hard metal lung disease is a rare form of occupational lung disease that can occur in workers engaged in the manufacture, utilization, or maintenance of tools composed of hard metal [a material composed mainly of tungsten carbide (WC) and cobalt] or diamond-cobalt. Clinically, the condition resembles hypersensitivity pneumonitis, with subacute presentations and possible evolution to pulmonary fibrosis. However, this interstitial lung disease is uniquely characterized by the presence of bizarre ;;cannibalistic'' multinucleated giant cells in the alveoli and the bronchoalveolar lavage. A pathological diagnosis of giant cell interstitial pneumonitis (GIP) is, therefore, specific for hard metal lung disease, even though not all affected subjects exhibit this pathognomonic feature. Cobalt is the critical toxic component causing hard metal lung disease, hence also the term cobalt-lung. Hard metal lung disease is more likely to occur in poorly regulated workplaces, but its occurrence depends mainly on individual susceptibility, rather than on cumulative exposure, so that even young subjects may be affected.

Intimal hyperplasia after long-term venous catheterization.

OBJECTIVE: Intimal hyperplasia is a well-known consequence of arterial injury and arterialization in vein grafts. However, the subacute and chronic vein wall changes which occur after catheterization have not been well studied. In this animal study, intimal hyperplasia in the vein wall after catheterization was examined. METHODS: A silicon catheter was placed in the anterior caval vein of 54 rats. After in situ fixation at scheduled intervals (1 day to 6 months), the pathologic changes in the vein wall were studied on semi-serial histology sections by means of light microscopy. RESULTS: Three forms of intimal hyperplasia could be observed: plaque-like, papillary-like and incorporation of the mural part of the sleeve into the underlying vein wall. Although the appearance of each was different, their composition was identical. All were mainly composed of alpha-actin-positive cells and collagen localized above the internal elastin layer, and covered by endothelium if facing the lumen. The plaque-like and papillary-like forms were mainly localized in the anterior vena cava, while sleeve incorporation mainly occurred in the jugular vein. Plaque-like and papillary-like intimal hyperplasia could be seen together on the same slide, but these two forms were never seen together with sleeve incorporation. CONCLUSION: Intimal hyperplasia occurs after venous catheterization and is probably caused by chronic injury to the vein wall due to knocking and rubbing movements of the catheter against the wall.

Tracheal side effects of endoscopic balloon tracheal occlusion in the fetal lamb model.

OBJECTIVE: To evaluate effects of in-utero endoluminal balloon tracheal occlusion (TO) as suggested for the treatment of Congenital Diaphragmatic Hernia (CDH) on the higher airways of a fetal lamb model. STUDY DESIGN: Fetuses from time-dated pregnant ewes underwent at 94 days (term=145 days) in-utero tracheal occlusion. In study animals an endoluminal, detachable balloon was placed by tracheoscopy. For that purpose a 1.2mm fibre-optic, semi-rigid endoscope and a medically graded latex balloon were used. In group I (n=9) lambs were delivered after 2 weeks. In group II (n=8) the tracheal occlusion was released after 2 weeks, to allow in-utero recovery until term. In positive control animals (group III; n=5) the trachea was clipped at 98 days and fetuses were harvested near term by cesarean section. A total of 17 contralateral littermates in multiple pregnancies served as negative controls. After macroscopic inspection of the trachea, sections were evaluated by light microscopy. Alterations were scored with an empirical interval score for each of the different anatomical elements in the fetal trachea (epithelium, submucosa, cartilage, pars membranacea). RESULTS: For the animal experiments in group I, all balloons were found in place and according to the pulmonary response they were obstructive. Tracheas were macroscopically dilated by the plug mainly due to elongation of the pars membranacea. The total histologic score was correlated to the increase in circumference (mean increase: 3.0mm). In nearly all cases, the tracheal epithelium at the level of the plug had lost its typical folding pattern. In 44% of cases, local epithelial defects were observed and in 33% of cases there was squamous metaplasia. A chronic inflammatory response was present in over half of the cases, sometimes with giant cell reaction. In group II (the in-utero recovery group) the total score was significantly lower than in group I, with much less prominent unfolding and absence of epithelial defects. Squamous metaplastia was still present in half of the cases; whereas inflammatory responses were less frequent. In group III the trachea expanded normally after removal of the clip. The epithelium had compacted folds, and cilia were well preserved. In two animals however, intraluminal synechia were observed. Below the level of occlusion animals of groups I and II all showed areas of unfolding, but without metaplasia or epithelial defects. CONCLUSION: Tracheal obstruction by means of endoluminal plugging has been suggested as an alternative in-utero treatment for congenital diagphragmatic hernia. The balloon causes mild epithelial changes, such as unfolding, limited epithelial defects (<25% of the exposure surface) and local inflammatory changes. These changes disappear nearly completely following in-utero unplugging during the rest of gestation. Unfolding of the epithelium is also seen in the trachea under the plug.