Differential invasion of respiratory epithelial cells by members of the Burkholderia cepacia complex

To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I–V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B . cepacia epidemic strain marker was identified.     

Meropenem in cystic fibrosis patients infected with resistant Pseudomonas aeruginosa or Burkholderia cepacia and with hypersensitivity to β-lactam antibiotics

Objective: To assess the efficacy and safety of meropenem, administered on a compassionate basis to 62 cystic fibrosis (CF) patients (age: 24±6 years) with hypersensitivity reactions to β-lactam antibiotics and/or infection by bacteria resistant to other antibiotics.
Methods: Fifty-seven patients were chronically infected with Pseudomonas aeruginosa and 5 with Burkholderia cepacia . In total, 124 courses (1 to 6/patient) of meropenem, 2 g three times a day by intravenous infusion (10 to 15 min) for 14 days, were administered.
Results: During treatment for P. aeruginosa the mean increase in pulmonary function (as a percentage of the predictive values) was 5.6% for FEV₁ (forced expiratory volume in the first second) and 8.6% for FVC (forced vital capacity). C-reactive protein and erythrocyte sedimentation rate (ESR) and leukocyte count decreased significantly. In courses administered for chronic infection with B. cepacia the post treatment FEV₁ and FVC values were higher than the pre-treatment values, and all the inflammatory parameters decreased.
The geometric means of minimal inhibitory concentration (MICs) (μg/mL) for P. aeruginosa ( B. cepacia ) were: tobramycin 6 (59), ciprofloxacin 1.2 (9.7), piperacillin 49 (16.3), ceftazidime 26 (23), aztreonam 26 (35), imipenem 6.4 (not determined) and meropenem 5.1 (4.8). No statistically significant increase in the MICs of meropenem for either pathogen occurred during therapy. Of the 124 courses, 115 were tolerated without any clinical complaint. The following side effects were observed: nausea (0.8%), itching (4%), rash (3.2%), drug fever (1.6%).
Conclusions: Meropenem proved to be a valuable drug in the treatment of CF patients with chronic pulmonary infection with multiresistant P. aeruginosa and B. cepacia and with hypersensitivity reactions to other β-lactam drugs.     

洋葱伯克霍尔德菌耐药性和金属β-内酰胺酶的检测

目的探讨洋葱伯克霍尔德菌耐药情况及其多重耐药特点。方法用微量琼脂稀释法检测2004年1月至2006年12月临床分离不重复75株洋葱伯克霍尔德菌对15种抗菌药物的抗菌活性及其产金属β-内酰胺酶情况，比较产酶株与非产酶株抗药活性。结果2004—2006年洋葱伯克霍尔德菌检出率呈上升趋势，痰标本分离率较高。感染者分布于临床各个科室，并患有各种严重基础疾病，并使用过抗菌药物。葱伯克霍尔德菌对多数抗菌药物耐药，仅对复方新喏明、美罗培南、头孢哌酮／舒巴坦、哌拉西林／他巴唑坦敏感，敏感率分别为95％、82．8％、74．7％、70．7％；而对头孢噻肟、头孢哌酮、头孢曲松、阿米卡星、氨曲南、哌拉西林耐药率超过50％。产金属β-内酰胺酶株与非产金属β-内酰胺酶株比较，产酶株对含酶抑制剂β-内酰胺类抗生素敏感性较高。结论洋葱伯克霍尔德菌对多数抗菌药物耐药，仅对复方新喏明、头孢哌酮／舒巴坦、美罗培南、哌拉西林／他巴唑坦敏感．建议临床医生根据药敏试验合理用药，以减少耐药菌株的产生。     

Elafin重组气道上皮细胞对铜绿假单胞菌生物被膜的抗菌作用

目的探讨Elafin重组气道上皮细胞对铜绿假单胞菌(Pseudomonas aeruginosa,Pa)生物被膜(biofilm,BF)的抗菌作用。方法体外平板法制备Pa生物被膜模型,用银染法及扫描电镜鉴定。体外培养A549细胞,将pEGEP-N1-Elafin重组到A549细胞,倒置荧光显微镜观察转染情况。采用猪胰弹性蛋白酶(NE组)、铜绿假单胞菌(Pa组)上清、大肠埃希杆菌(E．coli组)上清分别诱导孵育重组细胞24 h,另设转染未含Elafin基因空载体的A549细胞作为对照组。将成熟BF载体放入4组中继续孵育8 h,扫描电镜观察4组BF结构的变化,并于第8小时用四甲基偶氮唑盐(MTT)法测定细菌存活数。结果扫描电镜下观察到3个诱导组的BF结构较对照组的BF明显松散,尤以Pa组和NE组为甚;NE组[(3．238±0．316)×106 CFU／cm2]、Pa组细菌数量[(3．317±0．247)×106 CFU／cm2]均较对照组[(5．946±0,453)×106 CFU／cm2]显著减少(P<0．01),E．coli组细菌数量[(5．138±0．391)×106 CFU／cm2]较Pa组和NE组减少,差异均有统计学意义(P<0．01)。3个诱导组BF细菌清除率与对照组相比分别为45．6％、44．2％和13．6％。结论Elafin重组气道上皮细胞能有效清除Pa生物被膜中的细菌,而Pa产物也是Elafin表达的有效诱导物。     

人气管上皮细胞对绿脓杆菌抗菌作用观察

目的：探讨呼吸道上皮细胞与呼吸道致病菌之间的相互关系,观察呼吸道上皮细胞对绿脓杆菌的抗菌作用。方法：（1）贴壁生长的人呼吸道上皮细胞株HBE-16与绿脓杆菌标准株ATCC27853共孵育,庆大霉素杀死胞外菌,动态观察上皮细胞内活细菌数;（2）HBE-16细胞与绿脓杆菌共同悬浮于细胞培养基,在不同孵育时间点用平板菌落计数法计数活菌数。结果：绿脓杆菌不能在HBE-16细胞内生长,并被细胞逐渐清楚。悬浮状态下的HBE-16细胞对绿脓杆菌有一定杀菌作用。结论：呼吸道上皮细胞对胞内外绿脓杆菌的抗菌作用,可能是呼吸道抵抗细菌感染的一种天然免疫防御机制。     

Invasion of murine respiratory epithelial cells in vivo by Burkholderia cepacia.

Pulmonary infections caused by Burkholderia cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy and a propensity to cause bacteraemic infections in patients with CF. A mouse respiratory challenge model was used to investigate the invasion phenotype of B. cepacia in vivo. After intratracheal inoculation, epidemic B. cepacia strains translocated from lung to liver and spleen; however, all bacteria were cleared from all organs within 7 days. B. cepacia strains, irrespective of cable piliation, were capable of attaching to and then invading murine respiratory tract epithelial cells. Histopathological examination of lungs showed interstitial infiltrates comprised mainly of polymorphonuclear leucocytes and were associated with widened alveolar septa. Electron microscopy demonstrated B. cepacia within epithelial cells and pulmonary macrophages. This study provides support for in-vitro observations that B. cepacia strains from patients with CF adhere to and then invade respiratory epithelial cells. The invasion phenotype in B. cepacia may be an important virulence factor in CF infections.     

Invasion of respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia.

Pulmonary infections caused by Burkholderia (Pseudomonas) cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of cellular invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy to which the organism demonstrates in vitro susceptibility and a propensity to cause bacteremic infections in patients with CF. Epithelial cell invasion was demonstrated in vitro in A549 cells by a modified gentamicin protection assay. The kinetics of invasion appear to be saturable. Electron microscopy of invaded monolayers showed intracytoplasmic bacteria enclosed by membrane-bound vacuoles. No lysosomal fusion with these vacuoles was observed. Intraepithelial cell replication was suggested by electron microscopy and confirmed by both a quantitative assay and a visual assay. Cytochalasin D, but not colchicine, inhibited invasion, suggesting a role for microfilaments but not microtubules. The invasion phenotype in B. cepacia may be an important virulence factor for CF infections.     

Conditional regulation of cyclooxygenase-2 in tracheobronchial epithelial cells modulates pulmonary immunity

Cyclooxygenase-2 (COX-2) gene expression in the lung is induced in pathological conditions such as asthma and pneumonia; however, the exact impact of COX-2 gene expression in the airway in regulating inflammatory and immunological response in the lung is not understood. To define a physiological role of inducible COX-2 in airway epithelial cells, we developed a novel line of transgenic mice, referred to as CycloOxygenase-2 TransActivated (COTA) mice, that overexpress a COX-2 transgene in the distribution of the CC-10 promoter in response to doxycycline. In response to doxycycline treatment, COX-2 expression was increased in airway epithelium of COTA mice and whole lung tissue contained a three- to sevenfold increase in prostaglandin E(2) (PGE(2)), prostaglandin D(2) (PGD(2)) thromboxane B(2) (TXB(2)) and 6-Keto prostaglandin F(2alpha) (PGF(2alpha)) compared to wild-type and untreated COTA mice. Interestingly, primary mouse tracheal epithelial cells from COTA mice produced only PGE(2) by doxycycline-induced COX-2 activation, providing an indication of cellular specificity in terms of mediator production. In the ovalbumin model, in which doxycycline was given at the sensitization stage, there was an increase in interleukin (IL)-4 level in lung tissue from COTA mice compared to untreated COTA and wild-type mice. In addition, COTA mice that were treated with doxycycline had impaired clearance of Pseudomonas aeruginosa pneumonia compared to wild-type mice. COX-2 gene expression in airway epithelial cells has an important role in determining immunological response to infectious and allergic agents.     

Significance of human β-defensins in the epithelial lining fluid of patients with chronic lower respiratory tract infections

Human beta-defensins (hBDs) are the most abundant antimicrobial peptides in epithelial cells, and function in the host immune system. Respiratory epithelial cells express hBDs to inhibit bacterial proliferation during respiratory tract infections. The aim of this study was to investigate the release of hBDs into the respiratory tract and their benefit as a host defence system in chronic Pseudomonas aeruginosa infections. The levels of four hBD peptides (hBD-1-hBD-4) were measured in the bronchial epithelial lining fluid (ELF) of nine patients with chronic lower respiratory tract infection caused by P. aeruginosa. Eight patients with idiopathic pulmonary fibrosis and eight volunteers free of pulmonary disease were recruited as controls. ELF was obtained by bronchoscopic microsampling and hBD levels were measured by radioimmunoassays. The antimicrobial effects of hBDs were studied individually and in combination using an in-vitro colony count assay for P. aeruginosa. Concentrations of hBD-1 and hBD-3 tended to be higher in patients with chronic lower respiratory tract infection than in the controls. hBD-2 and hBD-4 were detected in ELF from five and four of nine patients, respectively, but the hBD levels in controls were all below the limits of detection. All patients with infection caused by mucoid P. aeruginosa had detectable hBD-2 and hBD-4 levels in ELF. In-vitro colony count assays showed a potential synergism between hBD-2 and hBD-4 in inhibiting bacterial proliferation. The findings indicate that hBDs, especially hBD-2 and hBD-4, are pathophysiologically important in infections caused by mucoid strains of P. aeruginosa.     

铜绿假单胞菌活菌对呼吸道上皮细胞表达IL-8的影响

目的:探讨铜绿假单胞菌活菌与人呼吸道上皮细胞的相互关系,细菌对呼吸道上皮炎症反应的影响。方法:采用PAO1及ATCC 27853两株铜绿假单胞菌,在体外与培养的呼吸道上皮细胞株A549及无血清培养的人支气管上皮原代细胞相互作用,收集细胞培养上清,ELISA检测上清IL-8浓度。结果:两株绿脓杆菌均能诱导呼吸道上皮细胞IL-8分泌增加,在细菌刺激下,A549细胞IL-8分泌比对照高出5倍(P<0.05),原代上皮细胞IL-8分泌比对照高出8倍(P<0.05)。结论:铜绿假单胞菌呼吸道感染的过程中,细菌与上皮细胞的直接作用可能是呼吸道炎症反应的重要原因。铜绿假单胞菌刺激上皮细胞炎症的分子机制和信号传导值得进一步探讨。     

Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity

To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.     

Evaluation of susceptibility testing methods for Burkholderia cepacia complex: a comparison of broth microdilution,agar dilution,gradient strip and EUCAST disc diffusion

ObjectivesTo evaluate the accuracy and reproducibility of antimicrobial susceptibility testing methods in Burkholderia cepacia complex (BCC).MethodsMinocycline, ciprofloxacin, trimethoprim/sulphamethoxazole, meropenem, ceftazidime and chloramphenicol were tested against 155 BCC strains using broth microdilution at 35 ± 1°C (BMD₃₅) in triplicate, then BMD at 30 ± 1°C (BMD₃₀), agar dilution at 30°C and 35°C (AD₃₀ and AD₃₅), gradient strip (GS) and EUCAST standardized disc diffusion (DD) testing methods once.ResultsBMD₃₅ reproducibility ranged from 70% to 84.5% for all agents. Correlations of MICs from BMD₃₅ with BMD₃₀ ranged from 63% to 85%, with AD₃₅ from 32.9% to 87% and with GS methods from 36% to 83.9%. Essential agreement (EA) of MICs by GS with BMD₃₅ ranged from 62.6% (trimethoprim-sulphamethoxazole) to 83.9% (minocycline). EA of EUCAST DD zone diameters using CLSI breakpoint criteria was between 85.8% and 97.4%, however Very Major Errors (VME) for trimethoprim/sulphamethoxazole were 31%.ConclusionsBMD at 35 ± 1°C was poorly reproducible for most agents and no method showed acceptable performance. Of particular concern were the GS results. Although this is the most commonly used method for determining MICs in laboratories, there was poor correlation with BMD₃₅ for meropenem and trimethoprim/sulphamethoxazole. EUCAST DD correlated poorly with BMD₃₅ MICs. This study confirms that no susceptibility method is capable of providing reproducible and accurate MICs when testing BCC.     

Endothelial cells and renal epithelial cells do not express the Wegener''s autoantigen, proteinase 3.

In vitro and in vivo T cell responses were determined during the course of bronchopulmonary infection with mucoid Pseudomonas aeruginosa. T cell responses were compared in two inbred mouse strains, namely BALB/c mice, which are resistant to the establishment of chronic bronchopulmonary Ps. aeruginosa infection, and C57Bl/6 mice, which have high numbers of bacteria in the lungs through 14 days post-infection. Unseparated lung cells and lung T cells from BALB/c mice exhibited significantly higher in vitro proliferative responses to both heat-killed Ps. aeruginosa and concanavalin A (Con A) than cells from C57Bl/6 mice through 20 days post-intratracheal infection with 10(4) colony-forming units (CFU) Ps. aeruginosa. Proliferation of unseparated lung cells but not lung T cells from BALB/c mice infected 6 days previously with 10(5) CFU Ps. aeruginosa was suppressed in response to Con A; these cells were unresponsive to specific antigen. Suppression of lymphocyte proliferation in the lungs of C57Bl/6 mice infected with 10(4) CFU Ps. aeruginosa and in BALB/c mice infected with 10(5) CFU was found to be mediated by adherent lung cells via the production of nitric oxide and prostaglandins. Determination of in vivo T cell-mediated responses in infected mice demonstrated that resistant BALB/c mice had high DTH and low Pseudomonas-specific antibody responses, while C57Bl/6 mice had low DTH and high antibody levels, in particular, IgG2b and IgM.     

呼吸道合胞病毒对人支气管上皮细胞通透性的影响

目的探讨呼吸道合胞病毒对支气管上皮细胞通透性的影响。方法通过ELISA、荧光定量PCR检测呼吸道合胞病毒感染人支气管上皮细胞后对其血管内皮生长因子（VEGF）表达的影响,同时,通过检测细胞的跨膜电阻了解呼吸道合胞病毒对人支气管上皮细胞通透性的影响。结果呼吸道合胞病毒增加了人支气管上皮细胞VEGF的表达,增加了支气管上皮细胞的通透性,VEGF的单克隆抗体可以明显阻断这一效应。结论呼吸道合胞病毒通过上调VEGF的表达增加对支气管上皮细胞的通透性。     

T cells,dendritic cells and epithelial cells in intestinal homeostasis

The mucosal immune system of the intestinal tract is continuously exposed to both potential pathogens and beneficial commensal microorganism. A variety of mechanisms contribute to the ability of the gut to either react or remain tolerant to antigen present in the intestinal lumen. Antigens of the gut commensals are not simply ignored, but rather trigger an active immunosuppressive process, which prevents the outcome of immunopathology. The aim of this review is to provide an update on the mechanism of intestinal homeostasis, with particular focus on the complex crosstalk between T cells, dendritic cells and intestinal epithelial cells.     

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.     

Different fates ofLegionella pneumophila pmiandmilmutants within macrophages and alveolar epithelial cells

Alveolar epithelial cells, which constitute the majority of the alveolar surface, may represent a potential niche for intracellular replication ofLegionella pneumophilathat has been largely overlooked. We examined the phenotypes of a bank of 121 macrophage-defective mutants ofL. pneumophila(designated aspmiandmil) for their cytopathogenicity to and intracellular survival and replication within human alveolar epithelial cells. Our data showed that 91 of 121 mutants that were defective (modest–severe) in macrophages exhibited wild type-like phenotypes in human type I alveolar epithelial cells. In contrast, the other 30 mutants were defective in both macrophages and alveolar epithelial cells. Transmission electron microscopy of the intracellular infection by three mutants showed that the defect in intracellular replication in macrophages and epithelial cells was associated with a defect in recruitment of the RER around the phagosome. Differences in attachment to macrophages and epithelial cells were also exhibited by some of the mutants. Pulmonary infection studies of A/J mice showed that a mutant defective in macrophages but not in alveolar epithelial cells replicated like the wild type strain in the lungs of A/J mice. In contrast, a mutant defective in both macrophages and alveolar epithelial cells failed to replicate and was killed. We conclude that certain distinct genetic loci ofL. pneumophilaare uniquely required for intracellular survival and replication within phagocytic but not epithelial cells, which may be important in vivo.