Estelle Escudier

Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria.

Bacterial infection of the lung is associated with mucin overproduction. In partial explanation of this phenomenon, we recently reported that supernatant from the Gram-negative organism Pseudomonas (P.) aeruginosa contained an activity that upregulated transcription of the MUC 2 mucin gene [J.-D. Li, A. Dohrman, M. Gallup, S. Miyata, J. Gum, Y. Kim, J. Nadel, A. Prince, C. Basbaum, Transcriptional activation of mucin by P. aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease, Proc. Natl. Acad. Sci. U.S.A., 94 (1997) 967-972]. The purpose of the present study was to determine whether mucin genes other than MUC 2 are so regulated and whether Gram-positive organisms also contain mucin stimulatory activity. Results from in situ hybridization and RNase protection assays showed that P. aeruginosa upregulates MUC 5AC as well as MUC 2 in both bronchial explants and cultured airway epithelial cells. The upregulation of both genes by P. aeruginosa can be mimicked by lipopolysaccharide (LPS) and can be blocked by the tyrosine kinase inhibitor genistein. In addition, both genes are upregulated by a variety of Gram-positive as well as Gram-negative organisms showing the same rank order of potency. These data indicate the existence of a general mechanism by which epithelial cells respond to the presence of bacteria by increasing mucin synthesis.

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

Radiofrequency Is a Safe and Effective Treatment of Turbinate Hypertrophy

Objective To evaluate the safety and efficacy of radiofrequency for reduction of inferior turbinate volume.

Loss-of-Function Mutations in the Human Ortholog of Chlamydomonas reinhardtii ODA7 Disrupt Dynein Arm Assembly and Cause Primary Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved structures that play various physiological roles in diverse cell types. Defects in motile cilia result in primary ciliary dyskinesia (PCD), the most prominent ciliopathy, characterized by the association of respiratory symptoms, male infertility, and, in nearly 50% of cases, situs inversus. So far, most identified disease-causing mutations involve genes encoding various ciliary components, such those belonging to the dynein arms that are essential for ciliary motion. Following a candidate-gene approach based on data from a mutant strain of the biflagellated alga Chlamydomonas reinhardtii carrying an ODA7 defect, we identified four families with a PCD phenotype characterized by the absence of both dynein arms and loss-of-function mutations in the human orthologous gene called LRRC50. Functional analyses performed in Chlamydomonas reinhardtii and in another flagellated protist, Trypanosoma brucei, support a key role for LRRC50, a member of the leucine-rich-repeat superfamily, in cytoplasmic preassembly of dynein arms.

A 20-year experience of electron microscopy in the diagnosis of primary ciliary dyskinesia

Transmission electron microscopy (TEM) analysis of ciliary ultrastructure is classically used for the diagnosis of primary ciliary dyskinesia (PCD). We report our extensive experience of TEM analysis in a large series of patients in order to evaluate its feasibility and results. TEM analysis performed in 1,149 patients with suspected PCD was retrospectively reviewed. Biopsies (1,450) were obtained from nasal (44%) or bronchial (56%) mucosa in children (66.5%) and adults (33.5%). TEM analysis was feasible in 71.4% of patients and showed a main defect suggestive of PCD in 29.9%. TEM was more feasible in adults than in children, regardless of the biopsy site. Main defects suggestive of PCD were found in 76.9% of patients with sinopulmonary symptoms and in only 0.4% of patients with isolated upper and 0.4% with isolated lower respiratory tract infections. The defect pattern was similar in children and adults, involving dynein arms (81.2%) or central complex (CC) (18.8%). Situs inversus was never observed in PCD patients with CC defect. Kartagener syndrome with normal ciliary ultrastructure was not an exceptional condition (10.2% of PCD). In conclusion, TEM analysis is feasible in most patients and is particularly useful for PCD diagnosis in cases of sinopulmonary syndrome of unknown origin.

Loss-of-Function Mutations in LRRC6, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought.

European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia

The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no “gold standard” reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia. International ERS guidelines recommend a combination of tests to diagnose primary ciliary dyskinesia http://ow.ly/sJhH304InBN

Increased expression of matrix metalloproteinase‐9 in nasal polyps

To investigate the role of gelatinases in nasal polyposis, a common and disabling airway disease characterized by chronic inflammation and tissue remodelling, matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 expression was investigated in the nasal polyps (NP) of 24 patients undergoing ethmoidectomy and compared with 15 control nasal mucosal (CM) samples obtained from snorers during turbinectomy. Tissue samples were either frozen for enzymatic analysis or paraffin wax‐embedded for immunohistochemistry. Zymography and quantitative image analysis showed that MMP‐9 active forms were significantly increased (p<0.05) in NPs compared to CM (44±40 versus 13±19×103 AU/10 µg protein), while MMP‐2 expression was similar in both tissues. Concomitant studies of gelatinase immunoexpression showed that MMP‐9 expression was enhanced (4‐ to 16‐fold) in surface epithelium, glands (p<0.05), and submucosal inflammatory cells (p<0.05). In addition, MMP‐9 positivity was markedly increased in endothelial cells (p<0.01). In situ zymography demonstrated marked gelatinolytic activity, consistent with the immunolocalization of MMP‐2 and MMP‐9. These results suggest up‐regulation of active MMP‐9 in the glands and vessels characteristic of NPs. It is concluded that MMP‐9 may play a role in the upper airway remodelling observed during nasal polyposis. Copyright

Myofibroblast accumulation induced by transforming growth factor-β is involved in the pathogenesis of nasal polyps

Myofibroblasts that express α‐smooth muscle actin(α‐SMA) are detected in many chronic inflammatory diseases. Transforming growth factor‐β (TGF‐β) is a potent inducer of myofibroblast accumulation in tissues. In this study, scattered myofibroblasts and TGF‐β were quantified and localized in nasal polyps (NPs) and normal nasal mucosa(NM). NPs were sampled in 16 patients during ethmoidectomy and NM was obtained from 10 control subjects during rhinoplasty. α‐SMA and TGF‐β were detected using immunohistochemistry and the numbers of labeled cells were quantified (α‐SMA and TGF‐β indices) and compared between NPs and NM. In eight NPs, in which the pedicle was preserved, α‐SMA and TGF‐β were evaluated and compared in the pedicle, central, and tip areas. Finally, TGF‐β expression was compared between low (zone 1), moderate(zone 2), and high (zone 3) zones of α‐SMA positivity. α‐SMA and TGF‐β indices were significantly higher in NPs than in NM. In the eight selected NPs, α‐SMA‐positive cells were significantly more abundant in the pedicle than in the central and tip areas, whereas TGF‐β‐positive cells were significantly more numerous in the pedicle than in the tip area. The number of TGF‐β‐positive cells was significantly higher in zone 3 than in zone 1 of α‐SMA positivity. Myofibroblasts, which are abundant in NPs but rare in NM, could be involved in the growth of NPs by inducing extracellular matrix accumulation. The local development of myofibroblasts in NPs could be controlled by TGF‐β, locally produced by inflammatory cells.

Bronchoalveolar lavage during neutropenic episodes: diagnostic yield and cellular pattern

Few data are available concerning the relationship between alveolar and blood cell populations during neutropenia. We wanted to compare the value of pulmonary endoscopic procedures with lavage in neutropenic (polymorphonuclear (PMN) count < or = 1,000.mm-3) and non-neutropenic settings. We therefore, retrospectively, reviewed the results of 118 investigations for pneumonia in patients with malignant haematological diseases. All had bronchoalveolar lavage (BAL), and some had additional studies with protected bacteriological samples. Each BAL specimen was studied after cytocentrifugation by cytological examination for opportunistic infections, haemorrhage, virus, legionellae, and bacteriological cultures. The diagnostic yield of all endoscopic procedures (BAL, telescoping plugged catheter and protected specimen brush) was 53% in neutropenic (Group 1) and 61% in non-neutropenic (Group 2) patients. The aetiological pattern of pneumonia was nearly the same in the two groups, except for more alveolar proteinosis in Group 1 and more cytomegalovirus (CMV) in Group 2. The absolute number of alveolar cells recovered through BAL (total number, macrophages, lymphocytes and PMNs) was significantly lower in neutropenic patients. We conclude that: 1) neutropenic patients with pneumonia require the same investigative approach as non-neutropenic patients; 2) profound neutropenia may be concomitant with a decreased cellularity of alveoli, which may reflect the consequences of marrow aplasia on the pulmonary cell population and/or direct effect of chemotherapy on the lung.