Airways Of Cystic Fibrosis Patients Research Articles

Nα-methyl coprogen B, a potential marker of the airway colonization byScedosporium apiospermumin patients with cystic fibrosis

Scedosporium apiospermum is an emerging pathogen colonizing the airways of patients with cystic fibrosis (CF). While usually responsible for chronic colonization without clinical signs, this fungus may cause severe and often lethal infections in lung transplant recipients. Early diagnosis of its airway colonization and appropriate treatment are required to eradicate the fungus when a lung transplantation is planned. Here we propose an alternative to mycological examination of sputum samples based on extraction of siderophores by chromatography on Amberlite XAD-4, followed by high performance liquid chromatography analysis of the siderophore extract. Improvement of the extraction procedure was performed in a fractional factorial design which revealed the importance of prior ammonium sulfate precipitation of the proteins, alkalinization of the obtained solution and stirring during extraction. In order to verify the specificity of N(α)-methyl coprogen B for S. apiospermum, the method was applied on culture supernatants of different filamentous fungi colonizing the airways of CF patients, including some aspergilli and Exophiala dermatitidis. N(α)-methyl coprogen B was detected exclusively for species of the S. apiospermum complex. Likewise, sputum samples from colonized and non-colonized CF patients were analyzed, and the siderophore was detected exclusively in three out of the five specimens which were found by culture to contain S. apiospermum. Together these results confirmed N(α)-methyl coprogen B as a marker of the airway colonization by species of the S. apiospermum complex.

Mutators in cystic fibrosis chronic lung infection: Prevalence, mechanisms, and consequences for antimicrobial therapy

Hypermutable (or mutator) microorganisms are those that have an increased spontaneous mutation rate due to defects in DNA repair or error avoidance systems. Over last decade, several studies have provided strong evidence of a relevant role of mutators in the evolution of natural bacterial populations, with a special emphasis in the field of infectious diseases. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis (CF) patients was the first natural environment revealing a high prevalence and important role of mutators. High frequencies of mutators have also been noted for other pathogens in the CF setting, particularly Staphylococcus aureus and Haemophilus influenzae. In addition to a strong statistical link between hypermutation and antibiotic resistance (i.e., mutators are more frequently resistant against antibiotics) obtained from the analysis of collections of clinical isolates from CF patients, several in vitro and in vivo experiments have further highlighted the dramatic consequences of hypermutation, especially when acting on microorganisms that are genetically equipped to acquire efficient resistance to most antibiotics by mutations in chromosomal genes such as P. aeruginosa. Recent studies additionally showed that mutators accelerate genetic adaptation of P. aeruginosa to the airways of CF patients and that patients infected by mutator strains have a poorer lung function. Further prospective clinical studies are nevertheless needed for a deep evaluation of the impact of mutators in disease progression and outcome. Future research should particularly focus on the investigation of the optimal therapeutical strategies to prevent the emergence of mutator strains in the lungs of CF patients and to avoid multiple-antimicrobial resistance development if they have already been selected.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs, Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRDeltaF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRDeltaF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRDeltaF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRDeltaF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.

Effect of Th2 type cytokines on hCLCA1 and mucus expression in cystic fibrosis airways

Correlations between expression of interleukin (IL)-9, the calcium-activated chloride channel hCLCA1 and mucus expression in cystic fibrosis (CF) airways have suggested a causal relationship. To verify this hypothesis mucosal tissue from upper airways of CF patients ( N = 5) was stimulated with the Th2 type cytokines IL-4, IL-9, or IL-13. Expression of hCLCA1 mRNA and protein as well as mucus and mucin (MUC5AC) gene expression was quantified using real time PCR, immunohistochemistry (hCLCA1) and PAS staining (mucus). Th2 type cytokines significantly increased hCLCA1 protein expression ( P < 0.05) whereas increase in hCLCA1 mRNA expression failed to reach statistical significance ( P > 0.05). Mucin protein and MUC5AC mRNA expression were not significantly changed ( P > 0.05). These data suggest that Th2 type cytokines may increase hCLCA1 expression in CF but may not have a significant effect on mucus expression. Therefore the role of hCLCA1 as a mediator of mucus overexpression in CF has to be questioned.

Early adaptive developments of Pseudomonas aeruginosa after the transition from life in the environment to persistent colonization in the airways of human cystic fibrosis hosts

Pseudomonas aeruginosa is an opportunistic pathogen ubiquitous to the natural environment but with the capability of moving to the host environment. Long-term infection of the airways of cystic fibrosis patients is associated with extensive genetic adaptation of P. aeruginosa, and we have studied cases of the initial stages of infection in order to characterize the early adaptive processes in the colonizing bacteria. A combination of global gene expression analysis and phenotypic characterization of longitudinal isolates from cystic fibrosis patients revealed well-known characteristics such as conversion to a mucoid phenotype by mucA mutation and increased antibiotic resistance by nfxB mutation. Additionally, upregulation of the atu operon leading to enhanced growth on leucine provides a possible example of metabolic optimization. A detailed investigation of the mucoid phenotype uncovered profound pleiotropic effects on gene expression including reduction of virulence factors and the Rhl quorum sensing system. Accordingly, mucoid isolates displayed a general reduction of virulence in the Caenorhabditis elegans infection model, altogether suggesting that the adaptive success of the mucoid variant extends beyond the benefits of alginate overproduction. In the overall perspective the global phenotype of the adapted variants appears to place them on paths in direction of fully adapted strains residing in long-term chronically infected patients.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

The Streptococcus milleri Population of a Cystic Fibrosis Clinic Reveals Patient Specificity and Intraspecies Diversity

The genetic relatedness of Streptococcus milleri group isolates from the airways of cystic fibrosis patients was determined by using pulsed-field gel electrophoresis. This study reveals no evidence for patient-to-patient transmission in our patient population; however, within individual patients, complex inter- and intraspecies diversity and dynamics can be observed.

Macrolide and Clindamycin Resistance inStreptococcus milleriGroup Isolates from the Airways of Cystic Fibrosis Patients

Organisms belonging to the Streptococcus milleri group (SMG) are known for their role in pyogenic infections but have recently been implicated as etiological agents of pulmonary exacerbation in adult patients with cystic fibrosis (CF). The prolonged exposure of CF patients to antibiotics prompted us to investigate the susceptibility profiles of 118 SMG isolates from the airways of CF patients to 12 antibiotics compared to 43 SMG isolates from patients with invasive infections. We found that approximately 60% of all isolates failed to grow using the standard medium for disc diffusion, Mueller-Hinton blood agar (MHBA), so we explored the usefulness of brain heart infusion (BHI) agar for susceptibility testing. Zone-of-inhibition comparisons between BHI and MHBA showed strong correlations for six antibiotics, and interpretations were similar for both medium types. For ceftriaxone and cefepime, both groups of isolates were highly susceptible. Tetracycline resistance levels were comparable between the two groups (22% in CF isolates and 17.4% in invasive isolates). However, more than half of the CF isolates were not susceptible to azithromycin, erythromycin, and clindamycin, compared to 11%, 13%, and 6.5% of invasive isolates, respectively. There were 5-fold and 8-fold increased risks of azithromycin and clindamycin resistance, respectively, for the isolates from the airways of CF patients relative to the invasive isolates. Macrolide resistance was strongly linked to chronic azithromycin therapy in CF patients. This study shows that BHI agar is a suitable alternative for antimicrobial susceptibility testing for the SMG and that SMG isolates from the airways of CF patients are more resistant to macrolides and clindamycin than strains isolated from patients with invasive infections.

In vitro antifungal activity of aminosterols against moulds isolated from cystic fibrosis patients

Sir, We have recently demonstrated that aminosterol derivatives (ASDs) possess an interesting in vitro antibacterial activity against multidrug-resistant bacteria recovered from cystic fibrosis (CF) patients. However, mould infections are also a serious threat for CF patients. Indeed, the fragile pulmonary environment of these patients facilitates the development of bacterial and fungal infections. Repeated bacterial infections, intensive antibiotic therapy and lung transplantation are considered as risk factors for airway infection and/or colonization by moulds in CF patients. Major problems are related to Aspergillus spp. and Scedosporium spp. that may cause chronic disorders such as asthma, bronchitis and aspergilloma. Amphotericin B, caspofungin and azoles are currently used in the management of fungal infections. However, in addition to acquired resistance resulting from antifungal treatment pressure as noted for Aspergillus spp., intrinsic resistance to available antifungals reported in some fungi such as Fusarium, Rhizopus, Rhizomucor and Scedosporium spp. represents a major issue, highlighting the need to develop new antifungal compounds. ASDs have been evaluated only against reference fungal strains. We evaluated herein for the first time the in vitro antifungal activity of squalamine and one of our compounds (ASD 1; the chemical structure is available in Alhanout et al.) possessing an in vitro antibacterial effect, against a panel of clinical mould isolates recovered from sputa of CF patients. All tested isolates were identified at the species level using partial internal transcribed spacer PCR amplification, and the sequencing procedure and sequences have been deposited in GenBank under accession numbers GU594733– GU594779. The in vitro antifungal activities of currently available systemic antifungal drugs were evaluated for comparison purposes. MICs of all tested compounds were determined by the reference broth dilution method as previously reported, and found to be reproducible and in agreement with previous reports for standard antifungal agents as noted using reference fungal strains including four yeasts and one mould (Aspergillus brasiliensis ATCC 16404) (Table 1). Squalamine and ASD 1 displayed MICs for all tested isolates ranging from 8 to 16 mg/L and from 2 to 4 mg/L, respectively. Since no internationally agreed antifungal resistance breakpoints are currently available, we adopted breakpoints proposed in a recent work suggesting MICs .2 mg/L for azoles to designate resistance in Aspergillus spp. For simplicity, we used this MIC to assign resistance to other antifungal drugs for all fungal isolates. In keeping with previous studies, Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus isolates were susceptible to all tested antifungals. Conversely, Aspergillus terreus isolates were resistant to amphotericin B as previously reported and, surprisingly, to itraconazole for our CF clinical isolates, which is unusual. As has been documented for other Aspergillus spp., the resistance of A. terreus to itraconazole probably may be acquired since this fungus chronically colonizes the airways of CF patients that are exposed to various antimicrobial treatments. According to previous reports, Aspergillus ustus was resistant to itraconazole, voriconazole and caspofungin. Resistance to caspofungin and voriconazole was noted for Emericella quadrilineata. Antifungal resistance in Penicillium and Cladosporium spp. was previously reported. Accordingly, two out of four Penicillium griseofulvum isolates were resistant to voriconazole and caspofungin while the other two isolates were resistant to all tested available antifungals. Moreover, Cladosporium spp. were resistant to all antifungals except amphotericin B, while Alternaria spp. were susceptible to all tested antifungals. In accordance with previous findings, resistance to all antifungals was found for all tested isolates of Fusarium proliferatum, Scedosporium prolificans, Pseudallescheria boydii, Scedosporium apiospermum, Rhizopus oryzae and Rhizomucor tauricus. Hence, airway colonization with these moulds is quite worrying, calling for an effective therapy to be available. Conversely, tested aminosterols and particularly ASD 1 demonstrated a homogeneous in vitro activity against all susceptible and resistant isolates. This finding suggests that novel mechanistic aspects, distinct from those of available drugs, underlie the antifungal activity of ASDs. While it is known that squalamine acts by disrupting bacterial membranes, the antifungal mechanism of action of ASDs remains unknown. Squalamine and ASD 1 previously demonstrated comparable activities against various bacterial isolates, whilst the present data interestingly show that ASD 1 was superior to squalamine, indicating a probable different effect due to a different chemical structure of the two tested compounds. While the safety of squalamine has been demonstrated in clinical studies, no in vitro or in vivo toxicity studies have been reported for synthesized aminosterols. Nevertheless, since ASDs were reported to act as membrane disruptors, such a wide antimicrobial spectrum may encompass a potential toxicity that should be further investigated. Overall, we have demonstrated herein that ASDs possess an interesting in vitro antifungal activity, advocating their development for local administration, for example as aerosols especially in the context of CF. Further studies are warranted in order to evaluate their in vivo antimicrobial activities in animal models.

Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3

Airway defenses are regulated by a complex purinergic signaling network located on the epithelial surfaces, where ATP stimulates the clearance of mucin and pathogens. The present study shows that the obstructive disease cystic fibrosis (CF) affects the activity, expression, and tissue distribution of two ectonucleotidases found critical for the regulation of ATP on airway surfaces: NTPDase1 and NTPDase3. Functional polarities and mRNA expression levels were determined on primary cultures of human bronchial epithelial (HBE) cells from healthy donors and CF patients. The in vitro model of the disease was completed by exposing CF HBE cultures for 4 days to supernatant of the mucopurulent material (SMM) collected from the airways of CF patients. We report that NTPDase1 and NTPDase3 are coexpressed on HBE cultures, where they regulate physiological and excess nucleotide concentrations, respectively. In aseptic conditions, CF epithelia exhibit >50% lower NTPDase1 activity, protein, and mRNA levels than normal epithelia, whereas these parameters are threefold higher for NTPDase3. Exposure to SMM induced opposite polarity shifts of the two NTPDases on both normal and CF epithelia, apical NTPDase1 being mobilized to basolateral surfaces and bilateral NTPDase3 to the apical surface. Their immunolocalization in human tissue revealed that NTPDase1 is expressed in epithelial, inflammatory, and endothelial cells, whereas NTPDase3 is restricted to epithelial cells. Furthermore, the SMM-exposed CF HBE cultures reproduced the impact of the disease on their in vivo distribution. This study provides evidence that an extensive remodeling of the enzymatic network regulating clearance occurs in the airways of CF patients.

IL-17 primes airway epithelial cells lacking functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) to increase NOD1 responses

In Cystic Fibrosis (CF), the absence of functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is key to better therapies for CF. In this manuscript, we have found that the presence of IL-17 in the airways of CF patients not only exacerbates inflammation through the recruitment of neutrophils via secretion of CXCL8, but also by priming airway epithelial cells lacking functional CFTR to increase response to the bacterial sensor NOD1. IL-17 stimulation of airway epithelial cells (AECs) lacking functional CFTR increased the expression of NOD1, NOD2, TLR4 and its own receptors IL-17RA and IL-17RC. Moreover, prior stimulation of AECs expressing the CFTRΔF508 mutant with IL-17 showed much greater CXCL8 secretion in response to a NOD1 agonist and Pseudomonas aeruginosa diffusible material. Taken together our results show that IL-17 primes AECs expressing CFTRΔF508 to increase host defence response to bacteria through the up-regulation of PRRs, and in particular of NOD1, and identifies another mechanism of action through which the CFTRΔF508 mutation leads to increase inflammation in response to bacterial ligands. Therefore preventing IL-17 function in CF may prove an important strategy in decreasing lung inflammation due to both direct and indirect effects.

Biofilm-forming Pseudomonas aeruginosa bacteria undergo lipopolysaccharide structural modifications and induce enhanced inflammatory cytokine response in human monocytes

To determine whether growth of bacteria in biofilms triggers a specific immune response, we compared cytokine induction in human monocytes and mouse macrophages by planktonic and biofilm bacteria. We compared Pseudomonas aeruginosa and Staphylococcus aureus, two bacteria often colonizing the airways of cystic fibrosis patients. Planktonic and biofilm S. aureus induced equivalent amounts of cytokine in human monocytes. In contrast, biofilm-forming P. aeruginosa induced a higher production of tumor necrosis factor and interleukin-6 than their planktonic counterpart, both for clinical isolates and laboratory strains. This increased cytokine production was partly dependent on phagocytosis. In contrast, no difference in cytokine induction was observed with mouse macrophages. We investigated the structures of the lipopolysaccharides (LPSs) of these Gram-negative bacteria in biofilm and planktonic cultures of P. aeruginosa. Switch between the two life-styles was shown to cause several reversible LPS structure modifications affecting the lipid A and polysaccharide moieties of both clinical isolates and laboratory strains. In addition, LPS isolated from biofilm-grown bacteria induced slightly more inflammatory cytokines than that extracted from its planktonic counterpart. Our results, therefore, show that P. aeruginosa biofilm LPS undergoes structural modifications that only partially contribute to an increased inflammatory response from human monocytes.

Cystic fibrosis infections: treatment strategies and prospects

Pseudomonas aeruginosa and Burkholderia cepacia are the two major Gram-negative rods that colonize/infect the lungs of patients with cystic fibrosis (CF). These organisms may cause progressive respiratory failure, although occasionally more rapid infections result in the 'Cepacia' syndrome. Many antibiotics have been used against Pseudomonas and Burkholderia, but once chronic colonization has been established, eradication of these organisms is rare. Drug therapy for CF patients is compromised by a number of bacterial factors that render the infectious agents resistant to antibiotics, including efflux pumps that remove antibiotics, lack of penetration of antibiotics into bacterial biofilms, and changes in the cell envelope that reduce the permeability of antibiotics. Any combination of these mechanisms increases the likelihood of bacterial survival. Therefore, combinations of antibiotics or of antibiotic and nonantibiotic compounds are currently being tested against Pseudomonas and Burkholderia. However, progress has been slow, with only occasional combinations showing promise for the eradication of persistent Gram-negative rods in the airways of CF patients. This review will summarize the current knowledge of CF infections and speculate on potential future pathways to treat these chronic infections.

The GO system prevents ROS-induced mutagenesis and killing inPseudomonas aeruginosa

Inactivation of the Pseudomonas aeruginosa mutM, mutY, or mutT gene conferred a 2.4-, 17.2-, or 38.1-fold increase in spontaneous mutation frequency, respectively. Importantly, the mutY and mutT strains each displayed a robust H(2)O(2)-induced mutation frequency. In addition, the mutM, mutY, and mutT mutations severely sensitized P. aeruginosa to killing by H(2)O(2), suggesting that these gene products act to repair one or more cytotoxic lesions in P. aeruginosa. Nucleotide sequence analysis of a fragment of the rpoB gene from rifampicin resistant mutM-, mutY-, and, mutT-deficient strains was consistent with this conclusion. These findings are discussed in terms of possible roles for mutM, mutY, and mutT in contributing to survival and mutagenesis of P. aeruginosa colonizing the airways of cystic fibrosis patients.

Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C(4)-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C(12)-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

Role of the amiloride‐sensitive epithelial Na+ channel in the pathogenesis and as a therapeutic target for cystic fibrosis lung disease

Increased airway Na(+) absorption mediated by the amiloride-sensitive epithelial Na(+) channel (ENaC) is a basic defect in cystic fibrosis (CF) lung disease. Cystic fibrosis is one of the most common lethal hereditary diseases and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR acts as a cAMP-dependent Cl(-) channel and regulator of ENaC, and CFTR dysfunction causes impaired Cl(-) secretion and increased Na(+) absorption in the airways of CF patients. Evidence from in vitro studies suggested that increased Na(+) absorption produces airway surface liquid (ASL) volume depletion and led to the generation of transgenic mice with airway-specific overexpression of ENaC to elucidate the role of this mechanism in the in vivo pathogenesis of lung disease. Studies of the pulmonary phenotype of betaENaC-overexpressing mice demonstrated that increased airway Na(+) absorption caused ASL depletion and reduced mucus transport, producing a CF-like lung disease with airway mucus plugging, chronic airway inflammation and pulmonary mortality. Further, recent pharmacological studies demonstrated that preventive, but not late, inhibition of increased airway Na(+) absorption with the ENaC blocker amiloride reduced morbidity and mortality in this murine model of CF lung disease. These results support a critical role of ENaC in the in vivo pathogenesis of CF lung disease and suggest that amiloride may be an effective preventive therapy for CF patients.

Inhibition of NFκB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation

Cystic Fibrosis (CF) is one of the most common autosomal genetic disorders in humans. This disease is caused by mutations within a single gene, coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The phenotypic hallmark of CF is chronic lung infection and associated inflammation from opportunistic microbes such as Pseudomonas aeruginosa (PA), Haemophilus influenzae, and Staphylococcus aureus. This eventually leads to deterioration of lung function and death in most CF patients. Unfortunately, there is no approved therapy for correcting the genetic defect causal to the disease. Hence, controlling inflammation and infection in CF patients are critical to disease management. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most of the anti-inflammatory agents in CF have severe limitations due to adverse side effects, and resistance to antibiotics is becoming an even more prominent problem. Thus, new agents that can be used to control chronic inflammation in CF are needed in the absence of a cure for the disease. Activation of the transcription factor NFκB through Toll-like receptors (TLR) following bacterial infection is principally involved in regulating lung inflammation in CF. NFκB regulates the transcription of several genes that are involved in inflammation, anti-apoptosis and anti-microbial activity, and hyper-activation of this transcription factor leads to a potent inflammatory response. Thus, NFκB is a potential anti-inflammatory drug target in CF. Screening of several compounds from natural sources in an in vitro model of CF-related inflammation wherein NFκB is activated by filtrates of a clinically isolated strain of PA (PAF) led us to Withaferin A (WFA), a steroidal lactone from the plant Withania Somnifera L. Dunal. Our data demonstrate that WFA blocks PAF-induced activation of NFκB as determined using reporter assays, IL-8 measurements and high-content fluorescent imaging of NFκB subunit p65 translocation. Since the airways of CF patients can be specifically targeted for delivery of therapeutics, we propose that WFA should be further studied as an anti-inflammatory agent in models of CF related inflammation mediated by NFκB.

Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation.

In previous work (E. E. Smith, D. G. Buckley, Z. Wu, C. Saenphimmachack, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson, Proc. Natl. Acad. Sci. USA 103:8487-8492, 2006) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.

TLR 5, but neither TLR2 nor TLR4, is involved in lung epithelial cell response toBurkholderia cenocepacia

Burkholderia cenocepacia is known to induce a harmful inflammatory response in the airways of cystic fibrosis patients. Toll-like receptors (TLRs) play key roles in sensing microbial-associated molecular patterns and initiating host innate immunity, but their role in the inflammatory response elicited by B. cenocepacia has not been precisely examined. In this study, we evaluated the contribution of TLR2, TLR4 and TLR5 to the signaling pathways triggered by B. cenocepacia in human bronchial epithelial cells. By quantitative reverse transcriptase (RT)-PCR, we demonstrated that the expression of both TLR2 and TLR4 was significantly upregulated by B. cenocepacia infection, whereas TLR5 expression remained unchanged. Using a dominant-negative approach and airway epithelial cells isolated from MyD88(-/-) mice, we found that B. cenocepacia activated a signaling complex that required the adapter molecule MyD88. Moreover, using epithelial cells from TLR2(-/-), TLR4(-/-) or TLR2/4(-/-) mice or cells overexpressing a functional form of TLR5, we established that TLR5, but neither TLR2 nor TLR4, critically regulated B. cenocepacia-induced lung epithelial inflammatory response.

Sinus surgery in Cystic Fibrosis patients: Comparison of sinus and lower airway cultures

The aim of this study is to determine whether cross-infection occurs between infections in the sinuses and lower airways in Cystic Fibrosis patients, and to determine whether the infections begin in the sinuses before spreading to the lungs. Retrospective study of pediatric Cystic Fibrosis patients who underwent simultaneous sinus surgery and bronchial washings. The results of the cultures were reviewed to determine if the same organisms colonized both the sinuses and lower airways. Staphylococcus aureus (Staph. Aureus) was found in 40.7% of the sinuses but only 33.3% of the lower airways. One patient had Staph. Aureus in the lower airway but not the sinuses. Pseudomonas, Hemophilus Influenza, and Moraxella showed similar patterns: multiple instances of positive sinus cultures and negative bronchial cultures, but only a small number of cases with positive lower airway cultures and negative sinus cultures. The data showed that as patients age, they are more likely to have infections in both upper and lower airways, but infections start in the upper airways at a younger age. In many cases, organisms were cultured from sinuses in patients who had negative lower airway cultures. In only a few instances, organisms grew in the lower airways and not the sinuses. In most cases, the bacteria that caused sinus infections at a young age caused the lung infections in older patients. This is the paper to show that bacteria spread from the sinuses to the lungs to cause infections in Cystic Fibrosis patients, and not vice-versa.