Intronic Variants in the NFKB1 Gene May Influence Hearing Forecast in Patients with Unilateral Sensorineural Hearing Loss in Meniere's Disease

Meniere''s disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10⁻⁸), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.     

Cell surface properties of two differently virulent strains of Acinetobacter baumannii isolated from a patient

The aim of this study was to unravel, by focusing on cell surface properties, the underlying virulence factors contributing to the difference in the pathogenicity observed in two Acinetobacter baumannii strains isolated from the same patient. The two strains were phenotypically different: (i) a mucoid strain (AB-M), highly virulent in a mouse model of pneumonia, and (ii) a nonmucoid strain (AB-NM), moderately virulent in the same model. The study of the cell surface properties included the microbial adhesion to solvents method, the measurement of the electrophoretic mobility of bacteria, the analysis of biofilm formation by calcofluor white staining, the adherence to silicone catheters, and scanning electron microscopy. The AB-NM strain was more hydrophobic, more adherent to silicone catheters, and produced more biofilm than the AB-M strain. Scanning electron microscopy showed bacterial cells with a rough surface and the formation of large cell clusters for AB-NM whereas the AB-M strain had a smooth surface and formed only a few cell clusters. Contrary to the results of most previous studies, cell surface properties were not correlated to the virulence described in our experimental model, indicating that mechanisms other than adherence may be involved in the expression of A.?baumannii virulence.     

A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

Background

Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2''-deoxy-2''-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo.

Methods

Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.

Results

In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.

Conclusion

These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.     

Ferritin,iron homeostasis,and oxidative damage

Ferritin is one of the major proteins of iron metabolism. It is almost ubiquitous and tightly regulated by the metal. Biochemical and structural properties of the ferritins are largely conserved from bacteria to man, although the role in the regulation of iron trafficking varies in the different organisms. Recent studies have clarified some of the major aspects of the reaction between iron and ferritin, which results in the formation of the iron core and production of hydrogen peroxide. The characterization of cellular models in which ferritin expression is modulated has shown that the ferroxidase catalytic site on the H-chain has a central role in regulating iron availability. In turn, this has secondary effects on a number of cellular activities, which include proliferation and resistance to oxidative damage. Moreover, the response to apoptotic stimuli is affected by H-ferritin expression. Altered ferritin L-chain expression has been found in at least two types of genetic disorders, although its role in the determination of the pathology has not been fully clarified. The recent discovery of a new ferritin specific for the mitochondria, which is functionally similar to the H-ferritin, opens new perspectives in the study of the relationships between iron, oxidative damage and free radicals.     

Data quality aware analysis of differential expression in RNA-seq with NOISeq R/Bioc package

As the use of RNA-seq has popularized, there is an increasing consciousness of the importance of experimental design, bias removal, accurate quantification and control of false positives for proper data analysis. We introduce the NOISeq R-package for quality control and analysis of count data. We show how the available diagnostic tools can be used to monitor quality issues, make pre-processing decisions and improve analysis. We demonstrate that the non-parametric NOISeqBIO efficiently controls false discoveries in experiments with biological replication and outperforms state-of-the-art methods. NOISeq is a comprehensive resource that meets current needs for robust data-aware analysis of RNA-seq differential expression.     

Bronchodilator Responsiveness and Reported Respiratory Symptoms in an Adult Population

Background

The relationship between patient-reported symptoms and objective measures of lung function is poorly understood.

Aim

To determine the association between responsiveness to bronchodilator and respiratory symptoms in random population samples.

Methods

4669 people aged 40 years and older from 8 sites in Canada completed interviewer-administered respiratory questionnaires and performed spirometry before and after administration of 200 ug of inhaled salbutamol. The effect of anthropometric variables, smoking exposure and doctor-diagnosed asthma (DDA) on bronchodilator responsiveness in forced expiratory volume in 1 second (FEV₁) and in forced vital capacity (FVC) were evaluated. Multiple logistic regression was used to test for association between quintiles of increasing changes in FEV₁ and in FVC after bronchodilator and several respiratory symptoms.

Results

Determinants of bronchodilator change in FEV₁ and FVC included age, DDA, smoking, respiratory drug use and female gender [p<0.005 to p<0.0001 ]. In subjects without doctor-diagnosed asthma or COPD, bronchodilator response in FEV₁ was associated with wheezing [p for trend<0.0001], while bronchodilator response for FVC was associated with breathlessness. [p for trend <0.0001].

Conclusions

Bronchodilator responsiveness in FEV₁ or FVC are associated with different respiratory symptoms in the community. Both flow and volume bronchodilator responses are useful parameters which together can be predictive of both wheezing and breathlessness in the general population.     

Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway

Introduction

The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.

Methods and Results

Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALP^low OPN^low RUNX2^high OSX ^high CD166^high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.

Conclusions

Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.