Neonatal dexamethasone induces premature microvascular maturation of the alveolar capillary network.

Postnatal glucocorticoid treatment of preterm infants was mimicked by treating newborn rats with dexamethasone (0.1-0.01 microg/g, days 1-4). This regimen has been shown to cause delayed alveolarization. Knowing that microvascular maturation (transformation of double- to single-layered capillary networks in alveolar septa) and septal thinning prevent further alveolarization, we measured septal maturation on electron photomicrographs in treated and control animals. In treated rats and before day 10, we observed a premature nonreversing microvascular maturation and a transient septal thinning, which both appeared focally. In vascular casts of both groups, we observed contacts between the two capillary layers of immature alveolar septa, which were predictive for capillary fusions. Studying serial electron microscopic sections of human lungs, we were able to confirm the postulated fusion process for the first time. We conclude that alveolar microvascular maturation indeed occurs by capillary fusion and that the dexamethasone-induced impairment of alveolarization is associated with focal premature capillary fusion.

Impairment of rat postnatal lung alveolar development by glucocorticoids: involvement of the p21CIP1 and p27KIP1 cyclin-dependent kinase inhibitors

It has been shown that glucocorticoids accelerate lung development by limiting alveolar formation resulting from a premature maturation of the alveolar septa. Based on these data, the aim of the present work was to analyze the influence of dexamethasone on cell cycle control mechanisms during postnatal lung development. Cell proliferation is regulated by a network of signaling pathways that converge to the key regulator of cell cycle machinery: the cyclin-dependent kinase (CDK) system. The activity of the various cyclin/CDK complexes can be modulated by the levels of the cyclins and their CDKs, and by expression of specific CDK inhibitors (CKIs). In the present study, newborn rats were given a 4-d treatment with dexamethasone (0.1-0.01 microg/g body weight dexamethasone sodium phosphate daily on d 1-4), or saline. Morphologically, the treatment caused a significant thinning of the septa and an acceleration of lung maturation on d 4. Study of cyclin/CDK system at d 1-36 documented a transient down-regulation of cyclin/CDK complex activities at d 4 in the dexamethasone-treated animals. Analysis of the mechanisms involved suggested a role for the CKIs p21CIP1 and p27KIP1. Indeed, we observed an increase in p21CIP1 and p27KIP1 protein levels on d 4 in the dexamethasone-treated animals. By contrast, no variations in either cyclin and CDK expression, or cyclin/CDK complex formation could be documented. We conclude that glucocorticoids may accelerate lung maturation by influencing cell cycle control mechanisms, mainly through impairment of G1 cyclin/CDK complex activation.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

The potential of microfluidic lung epithelial wounding: towards in vivo-like alveolar microinjuries

Idiopathic pulmonary fibrosis (IPF) remains a major clinical challenge to date. Repeated alveolar epithelial microinjuries are considered as the starting point and the key event in both the development and the progression of IPF. Various pro-fibrotic agents have been identified and shown to cause alveolar damage. In IPF, however, no leading cause of alveolar epithelial microinjuries can be identified and the exact etiology remains elusive. New results from epidemiologic studies suggest a causal relation between IPF and frequent episodes of gastric refluxes resulting in gastric microaspirations into the lung. The effect of gastric contents on the alveolar epithelium has not been investigated in detail. Here, we present a microfluidic lung epithelial wounding system that allows for the selective exposure of alveolar epithelial cells to gastric contents. The system is revealed to be robust and highly reproducible. The thereby created epithelial microwounds are of tiny dimensions and best possibly reproduce alveolar damage in the lung. We further demonstrate that exposure to gastric contents, namely hydrochloric acid (HCl) and pepsin, directly damages the alveolar epithelium. Together, this novel in vitro wounding system allows for the creation of in vivo-like alveolar microinjuries with the potential to study lung injury and alveolar wound repair in vitro.

Characterization and differentiation of body fluids, putrefaction fluid, and blood using Hounsfield unit in postmortem CT

The purpose of the present study was to evaluate the ranges of Hounsfield unit (HU) found in body fluids, putrefaction fluids, and blood on postmortem CT and how these ranges are affected by postmortem interval, temperatures, and CT beam energy. Body fluids, putrefaction fluids, and blood from a total of 53 corpses were analyzed to determine the ranges of HU values from postmortem CT images that were taken prior to autopsy. The fluids measured in CT images were obtained at autopsy and examined in terms of macroscopic and microscopic appearances. Body fluids and blood were also collected in plastic bottles, which were subjected to CT scans at different beam energies (80-130 kV) and at various fluid temperatures (4 to 40 °C). At a postmortem interval of 1 to 4 days, the ranges of HU values of the serous fluids (13-38 HU) and the nonsedimented blood (40-88 HU) did not overlap. In the sedimented blood, the upper serum layer exhibited HU value ranges that overlapped with those of the serous fluids. The putrefaction fluids exhibited a range of HU values between 80 and -130 HU. Elevated HU values were observed in fluids with accretive cell impurities. HU values decreased slightly with increasing temperature and CT beam energy. We concluded that serous fluids and blood in fresh corpses can be characterized and differentiated from each other based on HU value ranges. In contrast, body fluids in decomposed corpses cannot be differentiated by their HU value ranges. Different beam energies and corpse temperatures had only minor influences on HU value ranges and therefore should not be obstacles to the differentiation and characterization of body fluids and blood.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Immunoblotting for the serodiagnosis of alveolar echinococcosis in alive and dead Eurasian beavers (Castor fiber)

A novel species-specific anti-beaver-IgG-alkaline-phosphatase conjugate was synthesized for the development of a new serological test for echinococcosis in beavers. Two different ELISAs conventionally used for human Echinococcus multilocularis serology (Em18-ELISA and Em2-ELISA) yielded diagnostic sensitivities of 0% and 46%, respectively. In contrast, the subsequently developed immunoblotting assay gave an 85% diagnostic sensitivity (11 out of 13 beavers with alveolar echinococcosis were immunoblotting-positive, i.e. showed reactivity with a specific 21 Mr band), and maximal specificity. In conclusion, this immunoblotting assay should be the method of choice for use in serological studies on E. multilocularis in Eurasian beavers, and the test proved suitable to investigate both animals alive and post-mortem.

Hepatic alveolar hydatid disease (Echinococcus multilocularis) in a boxer dog from southern Ontario.

A 2-year-old boxer dog from southern Ontario was evaluated because of acute onset lethargy. Exploratory laparotomy revealed a hemorrhagic, destructive, liver mass. Histology, immunohistochemistry, and polymerase chain reaction confirmed Echinococcus multilocularis as the cause of the hepatic mass. This constitutes the first description of endemic E. multilocularis in Ontario.

Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region.

An adult dog that lived in central British Columbia was examined because of a history of lethargy and vomiting. Histology, immunohistochemistry, and polymerase chain reaction (PCR) examination of a hepatic mass confirmed the presence of an alveolar hydatid cyst, the first description of Echinococcus multilocularis in British Columbia. We provide recommendations for case management and remind practitioners in endemic areas of western Canada that dogs can serve as definitive and, rarely, intermediate hosts for E. multilocularis.

Influence of inspiration to expiration ratio on cyclic recruitment and derecruitment of atelectasis in a saline lavage model of acute respiratory distress syndrome*

OBJECTIVE Cyclic recruitment and derecruitment of atelectasis can occur during mechanical ventilation, especially in injured lungs. Experimentally, cyclic recruitment and derecruitment can be quantified by respiration-dependent changes in PaO2 (ΔPaO2), reflecting the varying intrapulmonary shunt fraction within the respiratory cycle. This study investigated the effect of inspiration to expiration ratio upon ΔPaO2 and Horowitz index. DESIGN Prospective randomized study. SETTING Laboratory investigation. SUBJECTS Piglets, average weight 30 ± 2 kg. INTERVENTIONS At respiratory rate 6 breaths/min, end-inspiratory pressure (Pendinsp) 40 cm H2O, positive end-expiratory pressure 5 cm H2O, and FIO2 1.0, measurements were performed at randomly set inspiration to expiration ratios during baseline healthy and mild surfactant depletion injury. Lung damage was titrated by repetitive surfactant washout to induce maximal cyclic recruitment and derecruitment as measured by multifrequency phase fluorimetry. Regional ventilation distribution was evaluated by electrical impedance tomography. Step changes in airway pressure from 5 to 40 cm H2O and vice versa were performed after lavage to calculate PO2-based recruitment and derecruitment time constants (TAU). MEASUREMENTS AND MAIN RESULTS In baseline healthy, cyclic recruitment and derecruitment could not be provoked, whereas in model acute respiratory distress syndrome, the highest ΔPaO2 were routinely detected at an inspiration to expiration ratio of 1:4 (range, 52-277 torr [6.9-36.9 kPa]). Shorter expiration time reduced cyclic recruitment and derecruitment significantly (158 ± 85 torr [21.1 ± 11.3 kPa] [inspiration to expiration ratio, 1:4]; 25 ± 12 torr [3.3 ± 1.6 kPa] [inspiration to expiration ratio, 4:1]; p < 0.0001), whereas the PaO2/FIO2 ratio increased (267 ± 50 [inspiration to expiration ratio, 1:4]; 424 ± 53 [inspiration to expiration ratio, 4:1]; p < 0.0001). Correspondingly, regional ventilation redistributed toward dependent lung regions (p < 0.0001). Recruitment was much faster (TAU: fast 1.6 s [78%]; slow 9.2 s) than derecruitment (TAU: fast 3.1 s [87%]; slow 17.7 s) (p = 0.0078). CONCLUSIONS Inverse ratio ventilation minimizes cyclic recruitment and derecruitment of atelectasis in an experimental model of surfactant-depleted pigs. Time constants for recruitment and derecruitment, and regional ventilation distribution, reflect these findings and highlight the time dependency of cyclic recruitment and derecruitment.

Alveolar derecruitment and collapse induration as crucial mechanisms in lung injury and fibrosis.

Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. The purpose of this study was to quantify how surfactant inactivation, alveolar collapse, and collapse induration lead to degradation of lung function. Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days after intratracheal bleomycin-instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Active surfactant subtypes declined by Day 1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared with ventilated alveoli. Collapse induration occurred on Day 7 and Day 14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Before the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points, the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.

Development of a specific tracer for metabolic imaging of alveolar echinococcosis: A preclinical study

Positron emission tomography (PET)-computed tomography (CT) using [18F]-fluorodeoxyglucose (FDG) (FDG-PET/CT) is a valuable method for initial staging and follow up of patients with alveolar echinococcosis (AE). However, the cells responsible for FDG uptake have not been clearly identified. The main goal of our study was to evaluate the uptake of PET tracers by the cells involved in the host-parasite reaction around AE lesions as the first step to develop a specific PET tracer that would allow direct assessment of parasite viability in AE. Candidate molecules ([18F]-fluorotyrosine (FET), [18F]-fluorothymidine (FLT), and [18F]-fluorometylcholine (FMC), were compared to FDG by in vitro studies on human leukocytes and parasite vesicles. Our results confirmed that FDG was mainly consumed by immune cells and showed that FLT was the best candidate tracer for parasite metabolism. Indeed, parasite cells exhibited high uptake of FLT. We also performed PET/CT scans in mice infected intraperitoneally with E. multilocularis metacestodes. PET images showed no FDG or FLT uptake in parasitic lesions. This preliminary study assessed the metabolic activity of human leukocytes and AE cells using radiolabeling. Future studies could develop a specific PET tracer for AE lesions to improve lesion detection and echinococcosis treatment in patients. Our results demonstrated that a new animal model is needed for preclinical PET imaging to better mimic human hepatic and/or periparasitic metabolism.

An international study on the serological differential diagnosis of human cystic and alveolar echinococcosis.

An enzyme-linked immunosorbent assay (ELISA) for the serological differentiation of cystic (Echinococcus granulosus) and alveolar (E. multilocularis) echinococcosis in man has been evaluated. A discrimination rate of 95.1% was found for 82 sera from patients of geographically disparate endemic areas. This rate was essentially the same as that found for 57 Swiss patients, indicating that inter- and intraspecific strain differences do not influence the test results. The assay method is suitable for immunodiagnostic purposes as well as for seroepidemiological studies.