The effect of FTY720 at different doses and time-points on LPS-induced acute lung injury in rats.

We sought to assess the protective effect of different doses of Fingolimod (FTY720) in a rat model of acute lung injury (ALI) induced by intratracheal instillation of lipopolysaccharide (LPS) and explored the underlying mechanisms. The ALI model was established in rats and different doses of FTY720 (0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, or 2 mg/kg) were injected intraperitoneally. Lung computed tomography and blood gas analyses were performed at 6 h, 24 h, and 48 h after intraperitoneal injection, and the lung tissues were extracted to prepare paraffin sections for histopathological examination. The levels of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) were detected by ELISA, and the expressions of inflammatory pathway proteins in each group were measured by Western blot analysis. A single intraperitoneal injection of FTY720 inhibited LPS-induced NF-κB activation, reduced the level of inflammatory cytokines, and decreased the infiltration of inflammatory cells. Moreover, it alleviated lung tissue injury, as shown by marked attenuation of pulmonary oedema and improved arterial partial pressure of oxygen (PaO2) and the general condition of ALI rats. In conclusion, our results demonstrate the protective effect of FTY720 against LPS-induced ALI. The underlying mechanism of the protective effect may involve inhibition of LPS-induced activation of NF-κB and regulation of the inflammatory pathway to alleviate barrier dysfunction of alveolar capillaries.

Ruscogenin attenuates sepsis-induced acute lung injury and pulmonary endothelial barrier dysfunction via TLR4/Src/p120-catenin/VE-cadherin signalling pathway.

OBJECTIVES: Sepsis-associated acute lung injury (ALI) occurs with the highest morbidity and carries the highest mortality rates among the pathogenies of ALI. Ruscogenin (RUS) has been found to exhibit anti-inflammation property and rescue lipopolysaccharide-induced ALI, but little is known about its role in sepsis-triggered ALI. The aim of this study was to investigate the potential role of RUS in sepsis-induced ALI and the probable mechanism. METHODS: Mice model of cecal ligation and puncture (CLP) was replicated, and three doses of RUS (0.01, 0.03 and 0.1 mg/kg) were administrated 1 h before CLP surgeries. KEY FINDINGS: RUS significantly extended the survival time and attenuated the lung pathological injury, oedema and vascular leakage in sepsis-induced ALI mice. RUS efficiently decreased the level of MPO in lung tissue and the WBC, NEU counts in BALF. In addition, RUS rescued the expression of VE-cadherin and p120-catenin and suppressed the TLR4/Src signalling in lung tissue. CONCLUSIONS: RUS attenuated sepsis-induced ALI via protecting pulmonary endothelial barrier and regulating TLR4/Src/p120-catenin/VE-cadherin signalling pathway.

Acetylharpagide Protects Mice from Staphylococcus Aureus-Induced Acute Lung Injury by Inhibiting NF-κB Signaling Pathway.

Staphylococcus aureus (S. aureus)-induced acute lung injury (ALI) is a serious disease that has a high risk of death among infants and teenagers. Acetylharpagide, a natural compound of Ajuga decumbens Thunb. (family Labiatae), has been found to have anti-tumor, anti-inflammatory and anti-viral effects. This study investigates the therapeutic effects of acetylharpagide on S. aureus-induced ALI in mice. Here, we found that acetylharpagide alleviated S. aureus-induced lung pathological morphology damage, protected the pulmonary blood-gas barrier and improved the survival of S. aureus-infected mice. Furthermore, S. aureus-induced myeloperoxidase (MPO) activity of lung homogenate and pro-inflammatory factors in bronchoalveolar lavage (BAL) fluid were suppressed by acetylharpagide. Mechanically, acetylharpagide inhibited the interaction between polyubiquitinated receptor interacting protein 1 (RIP1) and NF-κB essential modulator (NEMO), thereby suppressing NF-κB activity. In summary, these results show that acetylharpagide protects mice from S. aureus-induced ALI by suppressing the NF-κB signaling pathway. Acetylharpagide is expected to become a potential treatment for S. aureus-induced ALI.

Ultrastructural Changes in the Air-Blood Barrier of Rats in Acute Intoxication with Furoplast Pyrolysis Products.

Rats were exposed to fluoroplast-4 pyrolysis products (sample weight 2.6 g, pyrolysis temperature 440-750°C, pyrolysis duration 4 min) containing perfluoroisobutylene over 15 min. Lung tissue samples for histological and electron microscopic examination were isolated in 3 and 30 min after intoxication and processed routinely. Histological examination revealed no structural changes in the lungs. In ultrathin sections of rat lungs, some changes in the structure of type I pneumocytes were detected in 3 min after the exposure: detachment of cytoplasmic processes and the appearance of transcytosis pores. These changes attested to impaired cell-cell interactions and their adhesion to the basement membrane, where structural disorganization and edema of the collagen matrix were observed. In 30 min following exposure, the signs of damage to type I pneumocytes became more pronounced. The increase in the equivalents of transcellular and paracellular permeability in the alveolar lining profile was observed. No changes in the pulmonary capillary endotheliocytes were detected, which suggest that type I pneumocytes are the primary target of the toxic effect of perfluoroisobutylene. The vulnerability of a particular cell population, in view of specific metabolism of these cells, can be the key to deciphering of the mechanisms of the toxic effect of pyrolysis products of fluorinated polymer materials.

Mechanisms of Pulmonary Toxicity of Perfluoro-n-Alkane Pyrolysis Products with Consideration of the Structural Features of the Blood-Air Barriers.

Perfluoroisobutylene a is pulmonotoxic chemical generated during pyrolysis of perfluoro-nalkanes (polytetrafluoroethylene). The mechanisms of acute pulmonary toxicity induced by perfluoroisobutylene have not been studied yet. The analysis of tissues of brown frogs showed that the products of polytetrafluoroethylene pyrolysis induce typical inflammatory response in the lungs (fluid accumulation, erythrocyte stasis, desquamation of the epithelium, and capillary plethora in lung septa) and oropharyngeal cavity (degeneration of ciliated epithelium, hyperemia of underlying vessels with plasmatic imbibition of the connective tissue, and margination of segmented leukocytes and monocytes). The absence of surfactant is a specific feature of the blood-air barrier of the oropharyngeal cavity in frogs compared to the lungs. It can be hypothesized that toxic effects of perfluoroisobutylene are determined by its influence on epithelial (pneumocytes and cells of nonkeratinized stratified ciliated epithelium) and endothelial cells. Even though the effects of the agent on surfactant cannot be excluded, they do not determine the probability of development of inflammatory response.

In vitro exposure of a 3D-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires.

BACKGROUND: The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 µm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 µg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated. RESULTS: Significant cytotoxic effect was observed for all three types of AgNMs at the highest tested doses. The increased mRNA levels of the pro-apoptotic gene CASP7 suggests that apoptosis may occur after exposure to AgNWs. All three types of AgNMs increased the mRNA level of the anti-oxidant enzyme HMOX-1 and of the metal-binding anti-oxidant metallothioneins (MTs), with AgNWs being the most potent inducer. Even though all types of AgNMs induced the nuclear translocation of NF-kB, only AgNWs increased the mRNA level of pro-inflammatory mediators. The pro-inflammatory response elicited by AgNWs was further confirmed by the increased secretion of the 10 evaluated interleukins. CONCLUSION: In the current study, we demonstrated that the direct exposure of a complex tetra-culture alveolar model to different types of AgNMs at ALI induces shape- and size-specific biological responses. From the three AgNMs tested, AgNWs were the most potent in inducing biological alterations. Starting from 50 ng/cm2, a dose representative for an acute exposure in a high exposure occupational setting, AgNWs induced prominent changes indicative for a pro-inflammatory response. Even though the acute responses towards a dose representative for a full-lifetime exposure were also evaluated, chronic exposure scenarios at low dose are still unquestionably needed to reveal the human health impact of AgNMs during realistic conditions.

Cobra Venom Factor-induced complement depletion protects against lung ischemia reperfusion injury through alleviating blood-air barrier damage.

The purpose of this study was to study whether complement depletion induced by pretreatment with Cobra Venom Factor (CVF) could protect against lung ischemia reperfusion injury (LIRI) in a rat model and explore its molecular mechanisms. Adult Sprague-Dawley rats were randomly assigned to five groups (n = 6): Control group, Sham-operated group, I/R group, CVF group, I/R + CVF group. CVF (50 µg/kg) was injected through the tail vein 24 h before anesthesia. Lung ischemia reperfusion (I/R) was induced by clamping the left hilus pulmonis for 60 minutes followed by 4 hours of reperfusion. Measurement of complement activity, pathohistological lung injury score, inflammatory mediators, pulmonary permeability, pulmonary edema, integrity of tight junction and blood-air barrier were performed. The results showed that pretreatment with CVF significantly reduced complement activity in plasma and BALF. Evaluation in histomorphology showed that complement depletion induced by CVF significantly alleviated the damage of lung tissues and inhibited inflammatory response in lung tissues and BALF. Furthermore, CVF pretreatment had the function of ameliorating pulmonary permeability and preserving integrity of tight junctions in IR condition. In conclusion, our results indicated that complement depletion induced by CVF could inhibit I/R-induced inflammatory response and alleviate lung I/R injury. The mechanisms of its protective effects might be ameliorated blood-air barrier damage.

Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice.

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.

The 3-methyl-4-nitrophenol (PNMC) compromises airway epithelial barrier function.

BACKGROUND AND AIMS: It is recognized that the air pollution is associated with the pathogenesis of airway diseases. This study aims to elucidate the role of the 3-methyl-4-nitrophenol (PNMC), one of the components of diesel-exhaust particles, in compromising the airway epithelial barrier integrity. METHODS: A549 cells, an airway epithelial cell line, were cultured to monolayers to be used as an in vitro epithelial barrier model. BALB/c mice were treated with nasal drops containing PNMC to test the effects of PNMC on alternating the airway epithelial barrier functions. RESULTS: Exposure of mice to PNMC induced nasal epithelial cell apoptosis and increased the permeability of the nasal epithelial barrier. PNMC increased casp8 and casp3 activities in nasal epithelial cells. Exposure to PNMC up regulated Fas and FasL expression in airway epithelial cells. Inhibition of caspase abolished the PNMC-induced airway epithelial barrier dysfunction. CONCLUSION: Exposure of airway mucosa to PNMC induces epithelial cell apoptosis and compromises the epithelial barrier function, which can be prevented by the inhibition of caspases.

Instillation of hyaluronan reverses acid instillation injury to the mammalian blood gas barrier.

Acid (HCl) aspiration during anesthesia may lead to acute lung injury. There is no effective therapy. We hypothesized that HCl instilled intratracheally in C57BL/6 mice results in the formation of low-molecular weight hyaluronan (L-HA), which activates RhoA and Rho kinase (ROCK), causing airway hyperresponsiveness (AHR) and increased permeability. Furthermore, instillation of high-molecular weight hyaluronan (H-HA; Yabro) will reverse lung injury. We instilled HCl in C57BL/6 wild-type (WT), myeloperoxidase gene-deficient (MPO-/-) mice, and CD44 gene-deficient (CD44-/-) mice. WT mice were also instilled intranasally with H-HA (Yabro) at 1 and 23 h post-HCl. All measurements were performed at 1, 5, or 24 h post-HCl. Instillation of HCl in WT but not in CD44-/- resulted in increased inflammation, AHR, lung injury, and L-HA in the bronchoalveolar lavage fluid (BALF) 24 h post-HCl; L-HA levels and lung injury were significantly lower in HCl-instilled MPO-/- mice. Isolated perfused lungs of HCl instilled WT but not of CD44-/- mice had elevated values of the filtration coefficient ( Kf). Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer decreased barrier resistance ( RT). H-HA significantly mitigated inflammation, AHR, and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT, as well as ROCK2 phosphorylation. Increased H- and L-HA levels were found in the BALF of mechanically ventilated patients but not in healthy volunteers. HCl instillation-induced lung injury is mediated by the L-HA-CD44-RhoA-ROCK2 signaling pathway, and H-HA is a potential novel therapeutic agent for acid aspiration-induced lung injury.

Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

RAGE inhibition reduces acute lung injury in mice.

The receptor for advanced glycation end-products (RAGE) is involved in inflammatory response during acute respiratory distress syndrome (ARDS). Growing body of evidence support strategies of RAGE inhibition in experimental lung injury, but its modalities and effects remain underinvestigated. Anesthetised C57BL/6JRj mice were divided in four groups; three of them underwent orotracheal instillation of acid and were treated with anti-RAGE monoclonal antibody (mAb) or recombinant soluble RAGE (sRAGE), acting as a decoy receptor. The fourth group served as a control. Lung injury was assessed by the analysis of blood gases, alveolar permeability, histology, AFC, and cytokines. Lung expression and distribution epithelial channels ENaC, Na,K-ATPase, and aquaporin (AQP)-5 were assessed. Treatment with either anti-RAGE mAb or sRAGE improved lung injury, arterial oxygenation and decreased alveolar inflammation in acid-injured animals. Anti-RAGE therapies were associated with restored AFC and increased lung expression of AQP-5 in alveolar cell. Blocking RAGE had potential therapeutic effects in a translational mouse model of ARDS, possibly through a decrease in alveolar type 1 epithelial cell injury as shown by restored AFC and lung AQP-5 expression. Further mechanistic studies are warranted to describe intracellular pathways that may control such effects of RAGE on lung epithelial injury and repair.

Impact of zinc oxide nanoparticles on an in vitro model of the human air-blood barrier.

The inhalation of zinc oxide nanoparticles (nZnO) may induce systemic diseases, damages to the alveolar epithelium and inflammatory response to endothelial cells. In this work the use of an in vitro air-blood barrier (ABB) model provided a tool to elucidate the biological mechanisms underlying the potential effects of inhaled nanoparticles (NPs). The ABB model used is composed of a Transwell co-culture of a lung epithelial cell line (NCI-H441) and an immortalized pulmonary microvascular endothelial cell line (HPMEC-ST1.6R). In addition, a tri-culture model was developed by adding monocytes (THP-1) on the basal compartment of the inserts. These models have been set up to analyse the importance of the interplay among the different cell types on various responses after nZnO exposure: inflammation, endothelial damage and modulation of the immune system. The barrier integrity was assessed by measuring the transepithelial electrical resistance (TEER); the pro-inflammatory and immune cells responses were analysed by ELISA. The results have evidenced that nZnO do not affect the barrier integrity, since no TEER reduction was measured after 24h of exposure, but an activation of endothelial cells, which released pro-inflammatory mediators (IL-6, IL-8), and endothelial dysfunction markers (sICAM-1 and sVCAM-1) were induced. These results confirm that apical exposure to NPs promote endothelium activation. The in vitro-ABB model here used is thus a useful tool able to evidence the interaction between lung epithelium and endothelium in inducing biological response, and the role of endothelium dysfunction following NPs inhalation.

Nanoencapsulation of a glucocorticoid improves barrier function and anti-inflammatory effect on monolayers of pulmonary epithelial cell lines.

The anti-inflammatory effect of polymeric deflazacort nanocapsules (NC-DFZ) was investigated, and possible improvement of epithelial barrier function using filter grown monolayers of Calu-3 cells was assessed. NC prepared from poly(ε-caprolactone) (PCL) had a mean size around 200nm, slightly negative zeta potential (∼-8mV), and low polydispersity index (<0.10). Encapsulation of DFZ had an efficiency of 85%. No cytotoxic effects were observed at particle concentration of 9.85×1011NC/ml, which was therefore chosen to evaluate the effect of NC-DFZ at 1% (w/v) of PCL and 0.5% (w/v) of DFZ on the epithelial barrier function of Calu-3 monolayers. Nanoencapsulated drug at 0.5% (w/v) increased transepithelial electrical resistance and decreased permeability of the paracellular marker sodium fluorescein, while non-encapsulated DFZ failed to improve these parameters. Moreover, NC-DFZ reduced the lipopolysaccharide (LPS) mediated secretion of the inflammatory marker IL-8. In vitro dissolution testing revealed controlled release of DFZ from nanocapsules, which may explain the improved effect of DFZ on the cells. These data suggest that nanoencapsulation of pulmonary delivered corticosteroids could be advantageous for the treatment of inflammatory conditions, such as asthma and chronic obstructive pulmonary diseases.

Platelet-released growth factors induce psoriasin in keratinocytes: Implications for the cutaneous barrier.

Millions of patients around the world suffer minor or major extremity amputation due to progressive wound healing complications of chronic or infected wounds, the therapy of which remains a challenge. One emerging therapeutic option for the treatment of these complicated wounds is the local application of an autologous thrombocytes concentrate lysate (e.g. platelet-released growth factors ((PRGF)) or Vivostat PRF®) that contains a multitude of chemokines, cytokines and growth factors and is therefore supposed to stimulate the complex wound healing process. Although PRGF and Vivostat PRF® are already used successfully to support healing of chronic, hard-to-heal and infected wounds the underlying molecular mechanisms are not well understood. Psoriasin, also termed S100A7, is a multifunctional antimicrobial protein expressed in keratinocytes and is involved in various processes such as wound-healing, angiogenesis, innate immunity and immune-modulation. In this study, we investigated the influence of PRGF on psoriasin expression in human primary keratinocytes in vitro and the influence of Vivostat PRF® on psoriasin expression in experimentally generated skin wounds in vivo. PRGF treatment of primary keratinocytes caused a significant concentration- and time-dependent increase of psoriasin gene and protein expression in vitro that were partially mediated by the epidermal growth factor receptor (EGFR) and the interleukin-6 receptor (IL-6R). In accordance with these cell culture data, Vivostat PRF® induced a significant psoriasin gene and protein expression when applied to artificially generated skin wounds in vivo. The observed psoriasin induction in keratinocytes may contribute to the wound healing-promoting effects of therapeutically used thrombocyte concentrate lysates.

Sevoflurane Abolishes Oxygenation Impairment in a Long-Term Rat Model of Acute Lung Injury.

BACKGROUND: Patients experiencing acute lung injury (ALI) often need mechanical ventilation for which sedation may be required. In such patients, usually the first choice an intravenously administered drug. However, growing evidence suggests that volatile anesthetics such as sevoflurane are a valuable alternative. In this study, we evaluate pulmonary and systemic effects of long-term (24-hour) sedation with sevoflurane compared with propofol in an in vivo animal model of ALI. METHODS: Adult male Wistar rats were subjected to ALI by intratracheal lipopolysaccharide (LPS) application, mechanically ventilated and sedated for varying intervals up to 24 hours with either sevoflurane or propofol. Vital parameters were monitored, and arterial blood gases were analyzed. Inflammation was assessed by the analysis of bronchoalveolar lavage fluid (BALF), cytokines (monocyte chemoattractant protein-1 [MCP-1], cytokine-induced neutrophil chemoattractant protein-1 [CINC-1], interleukin [IL-6], IL-12/12a, transforming growth factor-ß, and IL-10) in blood and lung tissue and inflammatory cells. The alveolocapillary barrier was indirectly assessed by wet-to-dry ratio, albumin, and total protein content in BALF. Results are presented as mean ± standard deviation. RESULTS: After 9 hours of ventilation and sedation, oxygenation index was higher in the LPS/sevoflurane (LPS-S) than in the LPS/propofol group (LPS-P) and reached 400 ± 67 versus 262 ± 57 mm Hg after 24 hours (P < .001). Cell count in BALF in sevoflurane-treated animals was lower after 18 hours (P = .001) and 24 hours (P < .001) than in propofol controls. Peak values of CINC-1 and IL-6 in BALF were lower in LPS-S versus LPS-P animals (CINC-1: 2.7 ± 0.7 vs 4.0 ± 0.9 ng/mL; IL-6: 9.2 ± 2.3 vs 18.9 ± 7.1 pg/mL, both P < .001), whereas IL-10 and MCP-1 did not differ. Also messenger RNAs of CINC-1, IL-6, IL-12a, and IL-10 were significantly higher in LPS-P compared with LPS-S. MCP-1 and transforming growth factor-ß showed no differences. Wet-to-dry ratio was lower in LPS-S (5.4 ± 0.2 vs 5.7 ± 0.2, P = .016). Total protein in BALF did not differ between P-LPS and S-LPS groups. CONCLUSIONS: Long-term sedation with sevoflurane compared with propofol improves oxygenation and attenuates the inflammatory response in LPS-induced ALI. Our findings suggest that sevoflurane may improve lung function when used for sedation in patients with ALI.

Aerosol generation and characterization of multi-walled carbon nanotubes exposed to cells cultured at the air-liquid interface.

Aerosol generation and characterization are critical components in the assessment of the inhalation hazards of engineered nanomaterials (NMs). An extensive review was conducted on aerosol generation and exposure apparatus as part of an international expert workshop convened to discuss the design of an in vitro testing strategy to assess pulmonary toxicity following exposure to aerosolized particles. More specifically, this workshop focused on the design of an in vitro method to predict the development of pulmonary fibrosis in humans following exposure to multi-walled carbon nanotubes (MWCNTs). Aerosol generators, for dry or liquid particle suspension aerosolization, and exposure chambers, including both commercially available systems and those developed by independent researchers, were evaluated. Additionally, characterization methods that can be used and the time points at which characterization can be conducted in order to interpret in vitro exposure results were assessed. Summarized below is the information presented and discussed regarding the relevance of various aerosol generation and characterization techniques specific to aerosolized MWCNTs exposed to cells cultured at the air-liquid interface (ALI). The generation of MWCNT aerosols relevant to human exposures and their characterization throughout exposure in an ALI system is critical for extrapolation of in vitro results to toxicological outcomes in humans.

[EFFECT OF FLIXOTIDE ON ELECTRON-MICROSCOPIC CHANGES IN LUNG TISSUE OF GUINEA PIGS WITH BRONCHIAL ASTHMA MODEL.]

Chronic experiments on nonlinear short-hair guinea pigs with bronchial asthma model caused by administration of ovalbumin without treatment showed the ap- pearance of electron-microscopic changes of the lungs tissue in the form of chronic allergic inflammation. Significant changes in air - blood barrier with a loo- sening of intercellular contacts, degenerative changes in alveolocytes, and circulatory disorders with symptoms of vascular dilatation and stasis of blood cor- puscles were revealed. Treatment with inhaled fluticasone propionate in the form of flixotide preparation (GlaxoSmithKline, UK) for 3 months (2 times a day for 30 - 45 sec) partially reduced disorders of circulation and transcapillary exchange, decreased edema and degenerative changes in the cells, and restored in- tercellular contacts and pinocytic activity of the air - blood barrier. The obtained results show the expediency of further studies for determining the optimal du- ration of basic treatment during remission of bronchial asthma.

Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction.

The confluent pulmonary endothelium plays an important role as a semi-permeable barrier between the vascular space of blood vessels and the underlying tissues, and it contributes to the maintenance of circulatory fluid homeostasis. Pulmonary endothelial barrier dysfunction is a pivotal early step in the development of a variety of high mortality diseases, such as acute lung injury (ALI). Endothelium barrier dysfunction in response to inflammatory or infectious mediators, including lipopolysaccharide (LPS), is accompanied by invertible cell deformation and interendothelial gap formation. However, specific pharmacological therapies aiming at ameliorating pulmonary endothelial barrier function in patients are still lacking. A full understanding of the fundamental mechanisms that are involved in the regulation of pulmonary endothelial permeability is essential for the development of barrier protective therapeutic strategies. Therefore, this review summarizes several important molecular mechanisms involved in LPS-induced changes in pulmonary endothelial barrier function. As for barrier-disruption, the activation of myosin light chain kinase (MLCK), RhoA and tyrosine kinases; increase of calcium influx; and apoptosis of the endothelium lead to an elevation of lung endothelial permeability. Additionally, the activation of Rac1, Cdc42, protease activated receptor 1 (PAR1) and adenosine receptors (ARs), as well as the increase of cyclic AMP and sphingosine-1-phosphate (S1P) content, protect against LPS-induced lung endothelial barrier dysfunction. Furthermore, current regulatory factors and strategies against the development of LPS-induced lung endothelial hyper-permeability are discussed.

Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant protein D in human models of airway epithelium.

BACKGROUND: Surfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in host defense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light of infection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-D make it relevant in the development and pathogenesis of asthma. METHODS: Expression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC) grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic and non-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, and cytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epithelium and its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmatic and asthmatic donors. RESULTS: Airway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D as demonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures (fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 days to 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6. CONCLUSIONS: SP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients. Future functional studies on the role of SP-D in asthma can provide better insight into defects in the structure and regulation of SP-D.