RESUMO
INTRODUCTION: Management of cystic fibrosis has recently stepped forward with the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, although data on potential adverse effects are lacking for many categories of patients, such as pregnant women. METHODS: We report one of the first reports on the outcome of pregnancy in a woman treated with Elexacaftor/Tezacaftor/Ivacaftor during the second and third trimester of pregnancy, showing a significant improvement of respiratory status, compared with the first trimester when the medication was discontinued due to unknown and, therefore, potential teratogenic effects. Also, we performed the review of the existing literature on the topic. RESULTS: The course of pregnancy was uneventful, with reference to major obstetric complications, and the patient delivered a healthy neonate. These results were similar to those coming from other short series of pregnant women affected by cystic fibrosis and treated with CFTR modulators during pregnancy. CONCLUSIONS: Thus, despite the lack of evidence on the topic, the use of Elexacaftor/Tezacaftor/Ivacaftor in pregnancy seems to be apparently not associated with major adverse events, thus opening optimistic scenarios in terms of management of these patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Gravidez , Recém-Nascido , Humanos , Feminino , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos adversos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/induzido quimicamente , Mutação , Método Duplo-CegoRESUMO
BACKGROUND: Cystic fibrosis (CF), which is caused by mutations in the CF transmembrane conductance regulator (CFTR), is characterised by chronic bacterial lung infection and inflammation. In CF, monocytes and monocyte-derived macrophages have been shown to display defective phagocytosis and antimicrobial activity against relevant lung pathogens, including Pseudomonas aeruginosa. Thus, we addressed the effect of CFTR triple modulator therapy (elexacaftor/tezacaftor/ivacaftor (ETI)) on the activity of CF monocytes against P. aeruginosa. METHODS: Monocytes from people with CF (PWCF) before and after 1 and 6â months of ETI therapy were isolated from blood and infected with P. aeruginosa to assess phagocytic activity and intracellular bacterial killing. The oxidative burst and interleukin-6 secretion were also determined. Monocytes from healthy controls were also included. RESULTS: Longitudinal analysis of the clinical parameters confirmed an improvement of lung function and lung microbiology by ETI. Both the phagocytic and microbicidal deficiencies of CF monocytes also improved significantly, although not completely. Furthermore, we measured an exuberant oxidative burst in CF monocytes before therapy, which was reduced considerably by ETI. This led to an improvement of reactive oxygen species-dependent bactericidal activity. Inflammatory response to bacterial stimuli was also lowered compared with pre-therapy. CONCLUSIONS: PWCF on ETI therapy, in a real-life setting, in addition to clinical recovery, showed significant improvement in monocyte activity against P. aeruginosa, which may have contributed to the overall effect of ETI on pulmonary disease. This also suggests that CF monocyte dysfunctions may be specifically targeted to ameliorate lung function in CF.
Assuntos
Anti-Infecciosos , Fibrose Cística , Humanos , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Monócitos , Anti-Infecciosos/uso terapêutico , MutaçãoRESUMO
In Gram-negative pathogens, the stringent response regulator DksA controls the expression of hundreds of genes, including virulence-related genes. Interestingly, Pseudomonas aeruginosa has two functional DksA paralogs: DksA1 is constitutively expressed and has a zinc-finger motif, while DksA2 is expressed only under zinc starvation conditions and does not contain zinc. DksA1 stimulates the production of virulence factors in vitro and is required for full pathogenicity in vivo. DksA2 can replace these DksA1 functions. Here, the role of dksA paralogs in P. aeruginosa tolerance to H2O2-induced oxidative stress has been investigated. The P. aeruginosa dksA1 dksA2 mutant showed impaired H2O2 tolerance in planktonic and biofilm-growing cultures and increased susceptibility to macrophages-mediated killing compared to the wild type. Complementation with either dksA1 or dksA2 genes restored the wild type phenotypes. The DksA-dependent tolerance to oxidative stress involves, at least in part, the positive transcriptional control of both katA and katE catalase-encoding genes. These data support the hypothesis that DksA1 and DksA2 are eco-paralogs with indistinguishable function but optimal activity under different environmental conditions, and highlight their mutual contribution to P. aeruginosa virulence.
Assuntos
Peróxido de Hidrogênio , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Macrófagos/metabolismo , Pseudomonas aeruginosa/fisiologia , Zinco/metabolismoRESUMO
The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients' treatment by epigenetic targeting.
Assuntos
Fibrose Cística , Curcumina/farmacologia , Curcumina/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo/genética , Epigênese Genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Humanos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiopatologiaRESUMO
The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype-phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, ß and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5'-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.
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Metilação de DNA , Canais Epiteliais de Sódio/biossíntese , Regulação da Expressão Gênica , Linhagem Celular Tumoral , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , HumanosRESUMO
Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a prominent cause of nosocomial infections associated with high rates of morbidity and mortality, particularly in oncological patients. The hypermucoviscous (HMV) phenotype and biofilm production are key factors for CRKP colonization and persistence in the host. This study aims at exploring the impact of CRKP virulence factors on morbidity and mortality in oncological patients. A total of 86 CRKP were collected between January 2015 and December 2019. Carbapenem resistance-associated genes, antibiotic susceptibility, the HMV phenotype, and biofilm production were evaluated. The median age of the patients was 71 years (range 40-96 years). Clinically infected patients were 53 (61.6%), while CRKP colonized individuals were 33 (38.4%). The most common infectious manifestations were sepsis (43.4%) and pneumonia (18.9%), while rectal surveillance swabs were the most common site of CRKP isolation (81.8%) in colonized patients. The leading mechanism of carbapenem resistance was sustained by the KPC gene (96.5%), followed by OXA-48 (2.3%) and VIM (1.2%). Phenotypic CRKP characterization indicated that 55.8% of the isolates were strong biofilm-producers equally distributed between infected (54.2%) and colonized (45.8%) patients. The HMV phenotype was found in 22.1% of the isolates, which showed a significant (P<0.0001) decrease in biofilm production as compared to non-HMV strains. The overall mortality rate calculated on the group of infected patients was 35.8%. In univariate analysis, pneumoniae significantly correlated with death (OR 5.09; CI 95% 1.08-24.02; P=0.04). The non-HMV phenotype (OR 4.67; CI 95% 1.13-19.24; P=0.03) and strong biofilm-producing strains (OR 5.04; CI95% 1.39-18.25; P=0.01) were also associated with increased CRKP infection-related mortality. Notably, the multivariate analysis showed that infection with strong biofilm-producing CRKP was an independent predictor of mortality (OR 6.30; CI 95% 1.392-18.248; P=0.004). CRKP infection presents a high risk of death among oncological patients, particularly when pneumoniae and sepsis are present. In infected patients, the presence of strong biofilm-producing CRKP significantly increases the risk of death. Thus, the assessment of biofilm production may provide a key element in supporting the clinical management of high-risk oncological patients with CRKP infection.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Pessoa de Meia-IdadeRESUMO
Cystic fibrosis (CF) is an inherited disease that is characterised by susceptibility to bacterial infections and chronic lung inflammation. Recently, it was suggested that macrophages contribute to impaired host defence and excessive inflammatory responses in CF. Indeed, dysfunction attributed to CF macrophages includes decreased bacterial killing and exaggerated inflammatory responses. However, the mechanisms behind such defects have only been partially defined. MicroRNAs (miRNAs) have emerged as key regulators of several macrophage functions, including their activation, differentiation and polarisation. The goal of this study was to investigate whether miRNA dysregulation underlies the functional abnormalities of CF macrophages. MiRNA profiling of macrophages was performed, with 22 miRNAs identified as differentially expressed between CF and non-CF individuals. Among these, miR-146a was associated with significant enrichment of validated target genes involved in responses to microorganisms and inflammation. As miR-146a dysregulation has been reported in several human inflammatory diseases, we analysed the impact of increased miR-146a expression on inflammatory responses of CF macrophages. These data show that inhibition of miR-146a in lipopolysaccharide-stimulated CF macrophages results in increased interleukin-6 production, which suggests that miR-146a overexpression in CF is functional, to restrict inflammatory responses.
Assuntos
Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Interleucina-6/metabolismo , Macrófagos/metabolismo , MicroRNAs/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , TranscriptomaRESUMO
Sustained pulmonary delivery of tobramycin from microparticles composed of drug/polymer nanocomplexes offers several advantages against traditional delivery methods. Namely, in patients with cystic fibrosis, microparticle delivery can protect the tobramycin being delivered from strong mucoadhesive interactions, thus avoiding effects on its diffusion toward the infection site. Polymeric ion-pair complexes were obtained starting from two synthetic polyanions, through impregnation of their solid dissociated forms with tobramycin in aqueous solution. The structure of these polymeric systems was characterized, and their activities were examined against various biofilm-forming Pseudomonas aeruginosa. Once dried, the nanocomplexes can change their aggregation state, to form microparticle-based aggregates with a spherical shape and a micrometer size. In aqueous dispersions, the ion-pair complexes produced had nanometric size, negative ζ potential, and high biocompatibility toward human bronchial epithelium cells. The antibiofilm activity of these formulations was more efficient than for free tobramycin, with the antibiofilm activity against P. aeruginosa mucoid and nonmucoid end-stage strains isolated from cystic fibrosis lungs being of particular relevance.
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Antibacterianos/administração & dosagem , Fibrose Cística/tratamento farmacológico , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Tobramicina/administração & dosagem , Biofilmes , Linhagem Celular , Humanos , Muco/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologiaRESUMO
Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.
Assuntos
Brônquios/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Vetores Genéticos/genética , Plasmídeos/genética , Mucosa Respiratória/citologia , Brônquios/metabolismo , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/terapia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Terapia Genética/métodos , Humanos , Mucosa Respiratória/metabolismo , Transfecção/métodosRESUMO
PURPOSE: Development of a reliable, simple method to monitor lung condition in cystic fibrosis (CF) patients. Lung functionality assessment in CF patients is relevant, as most of them still die of respiratory failure. In lung mucus (sputum) of CF patients, components such as proteins, biopolymers, DNA, bacteria, and mucin are pathologically increased. As lung functionality is related to the amount of the pathological components in the sputum, their determination can help clinicians in monitoring lung condition and planning therapy. METHODS: Low-field NMR was used to evaluate the variation of the relaxation time (T2m ) of the water hydrogens present in CF sputum in relation to the amounts of the pathological components. Low-field NMR was tested in artificial samples (mucin or alginates), then in conditional sputum (saliva from healthy volunteers, added by different amounts of the pathological components), and finally in 12 patients' sputums, in which T2m was correlated to a commonly used lung monitoring test (i.e., forced expiratory volume in the first second). RESULTS: T2m significantly (P < 0.05) differed between samples with and without pathological components and between healthy and CF patients (P < 0.05), in which T2m correlated (r = 0.87) with FEV1 . CONCLUSIONS: The presented method can potentially become a valuable lung-monitoring tool in CF patients. Magn Reson Med 79:2323-2331, 2018. © 2017 International Society for Magnetic Resonance in Medicine.
Assuntos
Fibrose Cística/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Espectroscopia de Ressonância Magnética , Escarro/química , Adulto , Biopolímeros/química , DNA/análise , Feminino , Humanos , Pulmão/microbiologia , Masculino , Infecções por Pseudomonas/diagnóstico por imagem , Pseudomonas aeruginosa , Escarro/microbiologia , Água , Adulto JovemRESUMO
Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656T. Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts.
Assuntos
Burkholderia cenocepacia/fisiologia , Microbiologia Ambiental , Aptidão Genética , Infecções Respiratórias/microbiologia , Adaptação Fisiológica , Animais , Carga Bacteriana , Biofilmes , Burkholderia cenocepacia/patogenicidade , Doença Crônica , Células Clonais , Contagem de Colônia Microbiana , Estimativa de Kaplan-Meier , Larva/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Técnica de Amplificação ao Acaso de DNA Polimórfico , Inoculações Seriadas , VirulênciaRESUMO
Campylobacter jejuni (C. jejuni) bacteremia is difficult to diagnose in individuals with hematological disorders undergoing chemotherapy. The cause can be attributed to the rarity of this infection, to the variable clinical presentation, and to the partial overlapping symptoms underlying the disease. Here, we report a case of a fatal sepsis caused by C. jejuni in a 76-year-old Caucasian man with non-Hodgkin's lymphoma. After chemotherapeutic treatment, the patient experienced fever associated with severe neutropenia and thrombocytopenia without hemodynamic instability, abdominal pain, and diarrhea. The slow growth of C. jejuni in the blood culture systems and the difficulty in identifying it with conventional biochemical phenotyping methods contributed to the delay of administering a targeted antimicrobial treatment, leading to a fatal outcome. Early recognition and timely intervention are critical for the successful management of C. jejuni infection. Symptoms may be difficult to recognize in immunocompromised patients undergoing chemotherapy. Thus, it is important to increase physician awareness regarding the clinical manifestations of C. jejuni to improve therapeutic efficacy. Moreover, the use of more aggressive empirical antimicrobial treatments with aminoglycosides and/or carbapenems should be considered in immunosuppressed patients, in comparison to those currently indicated in the guidelines for cancer-related infections supporting the use of cephalosporins as monotherapy.
Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/isolamento & purificação , Linfoma não Hodgkin/microbiologia , Sepse/etiologia , Idoso , Anti-Infecciosos/uso terapêutico , Diagnóstico Precoce , Evolução Fatal , Humanos , Hospedeiro Imunocomprometido , Linfoma não Hodgkin/imunologia , Masculino , Tempo para o TratamentoRESUMO
Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 µM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 µM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients.
Assuntos
Antibacterianos/administração & dosagem , Reposicionamento de Medicamentos , Pneumopatias/tratamento farmacológico , Niclosamida/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Administração por Inalação , Animais , Antibacterianos/química , Química Farmacêutica , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos/tendências , Pneumopatias/microbiologia , Pneumopatias/patologia , Masculino , Nanopartículas/administração & dosagem , Nanopartículas/química , Niclosamida/química , Pós , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Ratos , Ratos Wistar , Virulência/efeitos dos fármacosRESUMO
Among polymeric polycations, chitosan has emerged as a powerful carrier for gene delivery. Only a few studies have focused on the stability of the chitosan/DNA complex under storage, although this is imperative for nanomedicinal applications. Here, we synthesized polyelectrolyte complexes at a charge ratio of 10 using 50 kDa chitosan and plasmid (p)DNA that encodes a GFP reporter. These preparations were stable up to 3 months at 4 °C and showed reproducible transfection efficiencies in vitro in HEK293 cells. In addition, we developed a methodology that increases the in vitro transfection efficiency of chitosan/pDNA complexes by 150% with respect to standard procedures. Notably, intracellular pDNA release and transfected cells peaked 5 days following transection of mitotically active cells. These new developments in formulation technology enhance the potential for polymeric nanoparticle-mediated gene therapy.
Assuntos
Quitosana/metabolismo , DNA/metabolismo , Técnicas de Transferência de Genes , Plasmídeos , Transfecção/métodos , Linhagem Celular , Estabilidade de Medicamentos , Células Epiteliais/metabolismo , Humanos , Reprodutibilidade dos Testes , Temperatura , Fatores de Tempo , Transformação GenéticaRESUMO
OBJECTIVES: Acute lung rejection (ALR) is a relatively frequent complication during the first year after lung transplantation (LT). It is characterized by perivascular/bronchial mononuclear inflammation mediated by several cytokines. The aim of our study was to monitor a panel of cytokines extracted from the bronchoalveolar lavage (BAL) during the first year after LT and correlate them with clinical ALR. METHODS: Twenty double lung transplant recipients were prospectively assessed. Fifteen (75%) were affected by cystic fibrosis (CF). BAL was collected at seven different steps (pretransplant, immediately post-transplant, after 1 week, 1, 3, 6 months and 1 year). A panel of six cytokines was analysed: tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α and IL-10. We correlated the cytokine levels with clinical ALR episodes, bacterial and cytomegalovirus (CMV) infections. RESULTS: One hundred and thirty-eight BAL samples were collected and analysed. In CF patients, the levels of proinflammatory cytokines significantly dropped immediately after the transplant while they increased in all the other patients. Four patients (20%) died between 6 months and 1 year. Nine patients (45%) showed one clinical ALR episode within 6 months; in 6 (30%) patients, a bacterial pneumonia was diagnosed and 5 (25%) developed CMV infection. No differences with the complication rate between CF and non-CF patients were observed. During the infection episodes, all proinflammatory cytokines increased with low levels of IL-10; in case of ALR, levels of IL-1ß and MIP-1α increased significantly (P = 0.01 and P < 0.0001), IL-10 levels were higher compared with the infection episodes (P = 0.03). No significant changes were observed for TNF-α, IL-6 and IL-8. CONCLUSIONS: The BAL cytokine profile (IL-1ß, MIP-1α and IL-10) seems useful to differentiate ALR and infections.
Assuntos
Citocinas/análise , Rejeição de Enxerto/epidemiologia , Transplante de Pulmão/efeitos adversos , Transplante de Pulmão/estatística & dados numéricos , Infecções Respiratórias/epidemiologia , Doença Aguda , Adulto , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Rejeição de Enxerto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Infecções Respiratórias/imunologiaRESUMO
The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.
Assuntos
Recombinação Genética , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Células HEK293 , Resolvases de Junção Holliday/metabolismo , Humanos , Plasmídeos , Recombinases/metabolismo , Homeostase do Telômero , Proteína 2 de Ligação a Repetições Teloméricas/genética , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , DNA/genética , Instabilidade Genômica/genética , Homeostase do Telômero/genética , Telômero/genética , Animais , HumanosRESUMO
Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.
Assuntos
Fibrose Cística/microbiologia , Fibrose Cística/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Viabilidade Microbiana , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Adolescente , Adulto , Separação Celular , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Espaço Intracelular/microbiologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Masculino , Viabilidade Microbiana/efeitos dos fármacos , Pessoa de Meia-Idade , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. METHODS: Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. RESULTS: Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. CONCLUSIONS: We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis.
Assuntos
Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/genética , Vetores Genéticos , Lentivirus/genética , RNA Interferente Pequeno/genética , Mucosa Respiratória/metabolismo , Linhagem Celular , Células Epiteliais/virologia , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/virologia , Transdução GenéticaRESUMO
Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infection, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines, 16HBE14o(-) and CFBE41o(-), which have a non-CF and a CF phenotype, respectively. The environmental strains showed a significantly lower level of invasion into both CF and non-CF cells in comparison with the clinical B. cenocepacia LMG16656(T) strain. Exposure of polarized CFBE41o(-) or 16HBE14o(-) cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable with that observed following exposure to the clinical strain. A different mechanism of tight junction disruption in CF versus non-CF epithelia was found. In the 16HBE41o(-) cells, the environmental strains resulted in a drop in TER without any apparent effect on tight junction proteins such as zonula occludens-1 (ZO-1). In contrast, in CF cells, the amount of ZO-1 and its localization were clearly altered by the presence of both the environmental strains, comparable with the effect of LMG16656. This study demonstrates that even if the environmental strains are significantly less invasive than the clinical strain, they have an effect on epithelial integrity comparable with that of the clinical strain. Finally, the tight junction regulatory protein ZO-1 appears to be more susceptible to the presence of environmental strains in CF cells than in cells which express a functional cystic fibrosis transmembrane regulator (CFTR).