Airway ciliated cells in adult lung homeostasis and COPD.

Cilia are organelles emanating from the cell surface, consisting of an axoneme of microtubules that extends from a basal body derived from the centrioles. They are either isolated and nonmotile (primary cilia), or grouped and motile (motile cilia). Cilia are at the centre of fundamental sensory processes and are involved in a wide range of human disorders. Pulmonary cilia include motile cilia lining the epithelial cells of the conductive airways to orchestrate mucociliary clearance, and primary cilia found on nondifferentiated epithelial and mesenchymal cells acting as sensors and cell cycle keepers. Whereas cilia are essential along the airways, their regulatory molecular mechanisms remain poorly understood, resulting in a lack of therapeutic strategies targeting their structure or functions. This review summarises the current knowledge on cilia in the context of lung homeostasis and COPD to provide a comprehensive overview of the (patho)biology of cilia in respiratory medicine with a particular emphasis on COPD.

The Nicotinic Receptor Polymorphism rs16969968 Is Associated with Airway Remodeling and Inflammatory Dysregulation in COPD Patients.

Genome-wide association studies unveiled the associations between the single nucleotide polymorphism rs16969968 of CHRNA5, encoding the nicotinic acetylcholine receptor alpha5 subunit (α5SNP), and nicotine addiction, cancer, and COPD independently. Here, we investigated α5SNP-induced epithelial remodeling and inflammatory response in human COPD airways. We included 26 α5SNP COPD patients and 18 wild-type α5 COPD patients in a multi-modal study. A comparative histologic analysis was performed on formalin-fixed paraffin-embedded lung tissues. Isolated airway epithelial cells from bronchial brushings were cultivated in the air-liquid interface. Broncho-alveolar fluids were collected to detect inflammatory mediators. Ciliogenesis was altered in α5SNP COPD bronchial and bronchiolar epithelia. Goblet cell hyperplasia was exacerbated in α5SNP small airways. The broncho-alveolar fluids of α5SNP COPD patients exhibited an increase in inflammatory mediators. The involvement of the rs16969968 polymorphism in airway epithelial remodeling and related inflammatory response in COPD prompts the development of innovative personalized diagnostic and therapeutic strategies.

Clinical and virological study of dengue cases and the members of their households: the multinational DENFRAME Project.

BACKGROUND: Dengue has emerged as the most important vector-borne viral disease in tropical areas. Evaluations of the burden and severity of dengue disease have been hindered by the frequent lack of laboratory confirmation and strong selection bias toward more severe cases. METHODOLOGY: A multinational, prospective clinical study was carried out in South-East Asia (SEA) and Latin America (LA), to ascertain the proportion of inapparent dengue infections in households of febrile dengue cases, and to compare clinical data and biological markers from subjects with various dengue disease patterns. Dengue infection was laboratory-confirmed during the acute phase, by virus isolation and detection of the genome. The four participating reference laboratories used standardized methods. PRINCIPAL FINDINGS: Among 215 febrile dengue subjects-114 in SEA and 101 in LA-28 (13.0%) were diagnosed with severe dengue (from SEA only) using the WHO definition. Household investigations were carried out for 177 febrile subjects. Among household members at the time of the first home visit, 39 acute dengue infections were detected of which 29 were inapparent. A further 62 dengue cases were classified at early convalescent phase. Therefore, 101 dengue infections were found among the 408 household members. Adding these together with the 177 Dengue Index Cases, the overall proportion of dengue infections among the study participants was estimated at 47.5% (278/585; 95% CI 43.5-51.6). Lymphocyte counts and detection of the NS1 antigen differed significantly between inapparent and symptomatic dengue subjects; among inapparent cases lymphocyte counts were normal and only 20% were positive for NS1 antigen. Primary dengue infection and a specific dengue virus serotype were not associated with symptomatic dengue infection. CONCLUSION: Household investigation demonstrated a high proportion of household members positive for dengue infection, including a number of inapparent cases, the frequency of which was higher in SEA than in LA.

Probing the fluctuation-dissipation theorem in a Perrin-like experiment.

In this Letter, we present a new experimental approach to investigate the effective temperature concept as a generalization of the fluctuation-dissipation theorem (FDT) for nonequilibrium systems. Simultaneous measurements of diffusion coefficient and sedimentation velocity of heavy colloids, embedded in a Laponite clay suspension, are performed with a fluorescence-recovery-based setup. This nonperturbative dual measurement, performed at a single time in a single sample, allows for a direct application of the FDT to the tracer velocity observable. It thus provides a well-defined derivation of the effective temperature in this ageing colloidal gel. For a wide range of concentrations and ageing times, we report no violation of the FDT, with effective temperature agreeing with bath temperature. This result is consistent with recent theoretical predictions on the coupling between the velocity observable and nonequilibrium gels dynamics.

Size dependence of tracer diffusion in a laponite colloidal gel.

Using a fluorescence recovery after photobleaching (FRAP) technique, we present measurements of probe diffusion in a colloidal glass-a Laponite suspension. By varying the probe size over 2 orders of magnitude, as well as the concentration of the colloidal glass, we evidence and quantify the deviations of the probe diffusivity from the bulk Stokes-Einstein expectations. These experiments suggest that the probe diffusion in the dynamically arrested Laponite structure is mainly controlled by the ratio between the probe size and the typical clay platelets interdistance. Comparing with a simple hindered diffusion mechanism, the reduction of tracer diffusion is discussed in terms of the hydrodynamic interaction of the probe with the Laponite structure. Finally, these results can be interpreted in terms of a scale dependent viscosity of the colloidal glass.

Fluorescent or not? Size-dependent fluorescence switching for polymer-stabilized gold clusters in the 1.1-1.7 nm size range.

The synthesis of fluorescent water-soluble gold nanoparticles by the reduction of a gold salt in the presence of a designed polymer ligand is described, the size and fluorescence of the particles being controlled by the polymer to gold ratio; the most fluorescent nanomaterial has a 3% quantum yield, a 1.1 nm gold core and a 6.9 nm hydrodynamic radius.

Evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using NS1 antigen detection in human serum.

BACKGROUND: We compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection-an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)-with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad). METHODS: We tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included. RESULTS: The sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%). CONCLUSION: Our findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP-the first rapid diagnostic test for DENV infection-was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

Regulation of human CETP gene expression: role of SP1 and SP3 transcription factors at promoter sites -690, -629, and -37.

Cholesteryl ester transfer protein (CETP) is a key factor in plasma reverse cholesterol transport and is implicated in the pathophysiology of atherogenic dyslipidemia. Variations observed in plasma CETP mass and activity in both normolipidemic and dyslipidemic individuals may reflect differences in CETP gene expression. We evaluated the respective roles of the Sp1 and Sp3 transcription factors on the promoter activity of the human CETP gene at a new Sp1/Sp3 site identified at position -690, and at two previously described Sp1/Sp3 sites at positions -37 and -629. In transient transfection in HepG2 cells, site-directed mutagenesis using luciferase reporter constructs containing a promoter fragment from +32 to -745 indicated that the new -690 site acts as a repressive element in reducing CETP promoter activity (-22%; P < 0.05); equally, this site exerts an additive effect with the -629 site, inducing marked repression (-42%; P < 0.005). In contrast, in NCTC cells that display a 16-fold lower level of Sp3, the repressive effect at the -690 site was enhanced 2-fold (-45%; P < 0.05), whereas the -629 site exerted no effect. Cotransfection of Sp1 and/or Sp3 in SL2 insect cells lacking endogenous Sp factors demonstrated that Sp1 and Sp3 act as activators at the -690 and -37 sites, whereas Sp3 acts as a repressor at the -629 site. Taken together, our data demonstrate that Sp1 and Sp3 regulate human CETP promoter activity through three Sp1/Sp3 binding sites in a distinct manner, and that the Sp1/Sp3 ratio is a key factor in determining the relative contribution of these sites to total promoter activity.

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo.

BACKGROUND: Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature. METHODS AND RESULTS: We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino-sugar-based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo. CONCLUSIONS: These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo.

Association of postmenopausal hormone replacement therapy with carotid atherosclerosis and soluble thrombomodulin: the vascular aging (EVA) study. Etude du Vieillissement Artériel.

Hormone replacement therapy (HRT) may reduce atherosclerosis among postmenopausal women, partly by reducing vascular endothelium damage. We have tested this hypothesis by evaluating the association of HRT with firstly, carotid intima media thickness (IMT) and plaques, and secondly, with endothelial cell damage, indicated by soluble thrombomodulin (sTM). Then, we tested the association between the two markers of atherosclerosis and the levels of sTM. Among 747 postmenopausal women included into the EVA study, we compared 154 HRT users (including 80% transdermal treatment) with 593 never users. Carotid IMT and plaques were measured with B-mode ultrasonography and sTM with ELISA. At least one plaque was detected among 13.6% of HRT users and 27.3% of never users. After adjustment for confounding factors, the odds ratio for the presence of plaque was 0.45 (95% confidence interval, 0.25-0.78, P=0.005) in HRT users in comparison with nonusers. HRT users had a slightly lower crude mean IMT than nonusers, but the difference was not significant. sTM was positively associated with mean IMT (P for trend=0.001) but not with plaques. Finally, estrogen users had a lower sTM level than nonusers (difference 0.14 ng/ml, P=0.03). As HRT was associated with sTM and plaques, but not with IMT, while sTM was only associated with IMT, our hypothesis was not confirmed. This suggests that the possible beneficial effects of HRT on atherosclerosis may not go through the endothelial cell damage assessed by plasma thrombomodulin.

Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example.

Artificial self-assembling systems are currently widely investigated as an alternative approach to recombinant viruses for gene transfection in vitro and in vivo. Cationic lipids are particularly attractive, as a great variety of well-characterized reagents can be synthesized from there. Over the last few years, numerous cationic lipid systems have been developed and shown to be efficient for in vitro transfection. However, although some promising results have been reported in the in vivo setting (even in clinical gene therapy trials in man), the in vivo use of cationic lipid-based systems is still problematic, especially when considering the systemic route of administration. Herein, we summarize our own research on a particular class of cationic lipids, cholesterol derivatives characterized by polar headgroups with guanidinium functions, in order to illustrate the basic principles of and the positive results already obtained by cationic lipid-mediated gene delivery as well as the remaining problems that need to be urgently resolved, particularly as regards the systemic administration. In this forward-looking review, we also discuss the present efforts to develop modular systems for improved in vivo transfection. Indeed, lipid-based vectors offer the possibility to create sophisticated modular gene delivery systems capable of self-assembly via hydrophobic interaction between their components, the role of the different functional elements being to help in overcoming the distinct extracellular and cellular barriers to in vivo gene transfection into the various somatic target tissues.