12－烷基化壳聚糖纳米粒-反义内皮素转换酶核酸表达质粒的制备及其性质的研究

摘要：目的：气道给予12－烷基化壳聚糖纳米粒（12－ACSs）包裹的反义内皮素转换酶（ECE）核酸表达质粒,观察对OVA致敏的小鼠变应性气道炎症的影响。方法：通过透射电镜观察12－ACSs/ 反义 ECE质粒复合体纳米粒的形成、形态及大小;应用凝胶阻滞、结合平衡、DNA沉淀和DNA酶消化实验等检测12－ACSs对反义ECE核酸表达质粒的结合保护作用;通过MTT实验检测12－ACSs对细胞的毒性;通过离体培养细胞及活体动物转染实验观察12－ACSs能否携带反义ECE核酸表达质粒成功转染。结果：电镜观察示纳米粒粒径在100－150 nm之间。12－ACSs与反义ECE核酸表达质粒在质量比为1：1时,全部反义ECE质粒被结合。应用DNase I消化后可见,12－ACSs可保护核酸免受破坏。MTT检测结果显示12－ACSs 对16HBE细胞在低浓度下几乎没有毒性。12－ACSs包裹的反义ECE核酸表达质粒的纳米粒能成功转染离体培养的气道上皮细胞及活体动物。结论：12－ACSs能够成功包裹反义ECE质粒并且成功转染16HBE及小鼠,其有可能作为一种基因治疗的载体选择之一。 关键词：哮喘 壳聚糖 纳米粒 内皮素转换酶 基因治疗     

Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model

Summary Nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different ³H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23° C and 37° C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 μl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.     

Dynamic interaction between airway epithelial cells and Staphylococcus aureus

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent (10(4), 10(6), and 10(8) CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the alpha-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus (<8%) adhered to airway cells, and no airway epithelial cell damage was observed. In contrast, after 24 h of incubation, more bacteria adhered to airway epithelial cells, internalized bacteria were observed, and a bacterial concentration-dependent effect on airway cell damage was observed. At 24 h, most airway cells incubated with bacteria at 10(8) CFU/ml exhibited a necrotic phenotype. The necrosis was preceded by a transient apoptotic process. In parallel, we observed a time- and bacterial concentration-dependent decrease in SLPI and increase in alpha-toxin expression. These results suggest that airway cells can defend against S. aureus in the early stages of infection. However, in later phases, there is a marked imbalance between the bactericidal capacity of host cells and bacterial virulence. These findings reinforce the potential importance of S. aureus in the pathogenicity of airway infections, including those observed early in CF patients.     

An efficient microtest to study adherence of bacteria to mammalian cells

A simple, fast and highly reproducible microtest was developed for in vitro adherence studies. A rat epithelial cell line was investigated for the adherence of clinical and subclinical ovine and bovine Staphylococcus aureus strains isolated from mastitis. Staphylococcus aureus strains differed in their ability to adhere to epithelial cells, the degree of adherence being dependent on the concentration of bacteria used in the test.     

Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.     

Adherence to HEp-2 cells and replication in macrophages of Salmonella derby of human origin.

Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.     

Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.     

Distribution of basement membrane pores in bronchus revealed by microscopy following epithelial removal

The basement membrane of the bronchial epithelium separates the epithelial and mesenchymal compartments. Basement membrane pores allow cells to cross this boundary. We present a method for preparation of samples of human basement membrane allowing us easy visualisation and characterisation of the distribution and persistence of these pores. Columnar epithelial cells were removed from airway samples with gentle scraping with a circular glass coverslip. In contrast, the underlying basal cells required incubation once in dithiothreitol and twice in ethylenediaminetetraacetic acid. Scanning electron microscopy (SEM) at each stage of the epithelial stripping process showed the selective removal of epithelial cells with eventual visualisation of the pores. Using confocal microscopy on blocks of viable tissue, pores were shown to persist in culture for at least 5 days, despite the presence of viable cells in the submucosa. The distribution of pores in tissues determined by SEM was compared to simulations of three distribution patterns (random, clumped, and distributed). The pattern of pores in the samples was consistent with a random distribution. We suggest that basement membrane pores can be generated by the passage of infiltrating cells into the epithelium providing a network suitable for intraepithelial surveillance.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ²) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Repair and regeneration of the airway epithelium

Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions, through several mechanisms including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply extracellular matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors as well as cytokines and growth factors secreted by airway epithelial and mesenchymal cells. The development of in vitro and in vivo models of airway epithelium wound repair allowed the study of the spatio-temporal modulation of these factors during the different steps of epithelial repair and regeneration. In this context, several studies have demonstrated that the matrix and secretory environment are markedly involved in these mechanisms and that their dysregulation may induce remodelling of the airway mucosa. A better knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to regenerative therapeutics allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.     

Staphylococcal adherence to rabbit epithelial cells

The adherence of Staphylococcus aureus strains to rabbit epithelial cells has been studied. The strains have been shown to possess similar adhesiveness with respect to the epithelium of the mouth cavity of rabbits. The investigation, carried out with the use of one staphylococcal strain taken as a model, has revealed that the cells of this strain adhere to different areas of the epithelium in the mouth cavity in varying amounts, the amount of adhering bacteria depending on the age of rabbits. The data presented in this work suggest that in staphylococci adhering to rabbit epithelial cells the adhesive function is performed by thermostable and trypsin-resistant staphylococcal cell-wall surface structures of nonprotein nature.     

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

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

Adherence of airway neutrophils and inflammatory response are increased in CF airway epithelial cell-neutrophil interactions

Persistent presence of PMN in airways is the hallmark of CF. Our aim was to assess PMN adherence, percentage of apoptotic airway PMN (aPMN), and IL-6 and IL-8 production when aPMN are in contact with airway epithelial cells. Before coculture, freshly isolated CF aPMN have greater spontaneous and TNF-alpha-induced apoptosis compared with blood PMN from the same CF patients and from aPMN of non-CF patients. We then examined cocultures of PMN isolated from CF and non-CF airways with bronchial epithelial cells bearing mutated cftr compared with cftr-corrected bronchial epithelial cells. After 18-h coculture, the number of CF aPMN adhered on cftr-deficient bronchial epithelial cells was 2.3-fold higher compared with the coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. The percentage of CF apoptotic aPMN (9.5 +/- 0.2%) adhered on cftr-deficient bronchial epithelial cells was similar to the percentage of non-CF apoptotic aPMN adhered on cftr-corrected bronchial epithelial cells (10.3 +/- 0.7%). IL-6 and IL-8 levels were enhanced 6.5- and 2.9-fold, respectively, in coculture of CF aPMN adhered on cftr-deficient bronchial epithelial cells compared with coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. Moreover, blocking surface adhesion molecules ICAM-1, VCAM-1, and E-selectin on cftr-deficient bronchial epithelial cells with specific MAbs inhibited the adherence of CF aPMN by 64, 51, and 50%, respectively. Our data suggest that in CF patients a high number of nonapoptotic PMN adhered on airway epithelium associated with elevated IL-6 and IL-8 levels may contribute to sustained and exaggerated inflammatory response in CF airways.     

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.     

Silica directly increases permeability of alveolar epithelial cells.

Alveolar epithelial cell injury and increased alveolar-capillary membrane permeability are important features of acute silicosis. To determine whether silica particles contribute directly to this increased permeability, we measured paracellular permeability of rat alveolar epithelium after exposure to silica, in vitro, using markers of the extracellular space. Silica (Minusil) markedly increased permeability in a dose- and time-dependent manner. This was not the result of cytolytic injury, because lactate dehydrogenase release from monolayers exposed to silica was not increased. Pretreatment of the silica with serum, charged dextrans, or aluminum sulfate blocked the increase in permeability. Scanning electron microscopy demonstrated adherence of the silica to the surface of the alveolar epithelial cells. Thus silica can directly increase permeability of alveolar epithelium.