Rapid effects of extrafine beclomethasone dipropionate/formoterol fixed combination inhaler on airway inflammation and bronchoconstriction in asthma: a randomised controlled trial

Background

Airway Bypass is a catheter-based, bronchoscopic procedure in which new passageways are created that bypass the collapsed airways, enabling trapped air to exit the lungs. The Exhale Airway Stents for Emphysema (EASE) Trial was designed to investigate whether Exhale^® Drug-Eluting Stents, placed in new passageways in the lungs, can improve pulmonary function and reduce breathlessness in severely hyperinflated, homogeneous emphysema patients (NCT00391612).

Methods/Design

The multi-center, randomized, double-blind, sham-controlled trial design was posted on http://www.clinicaltrials.gov in October 2006. Because Bayesian statistics are used for the analysis, the proposed enrollment ranged from 225 up to 450 subjects at up to 45 institutions. Inclusion criteria are: high resolution CT scan with evidence of homogeneous emphysema, post-bronchodilator pulmonary function tests showing: a ratio of FEV₁/FVC < 70%, FEV₁≤50% of predicted or FEV₁ < 1 liter, RV/TLC≥0.65 at screening, marked dyspnea score ≥2 on the modified Medical Research Council scale of 0-4, a smoking history of at least 20 pack years and stopped smoking for at least 8 weeks prior to enrollment. Following 16 to 20 supervised pulmonary rehabilitation sessions, subjects were randomized 2:1 to receive either a treatment (Exhale^® Drug-Eluting Stent) or a sham bronchoscopy. A responder analysis will evaluate the co-primary endpoints of an FVC improvement ≥12% of the patient baseline value and modified Medical Research Council dyspnea scale improvement (reduction) ≥1 point at the 6-month follow-up visit.

Discussion

If through the EASE Trial, Airway Bypass is shown to improve pulmonary function and reduce dyspnea while demonstrating an acceptable safety profile, then homogeneous patients will have a minimally invasive treatment option with meaningful clinical benefit.

Trial Registration

ClinicalTrials.gov: NCT00391612     

Pathway landscapes and epigenetic regulation in breast cancer and melanoma cell lines

Understanding how developmental systems evolve over time is a key question in stem cell and developmental biology research. However, due to hurdles of existing experimental techniques, our understanding of these systems as a whole remains partial and coarse. In recent years, we have been constructing in-silico models that synthesize experimental knowledge using software engineering tools. Our approach integrates known isolated mechanisms with simplified assumptions where the knowledge is limited. This has proven to be a powerful, yet underutilized, tool to analyze the developmental process. The models provide a means to study development in-silico by altering the model’s specifications, and thereby predict unforeseen phenomena to guide future experimental trials. To date, three organs from diverse evolutionary organisms have been modeled: the mouse pancreas, the C. elegans gonad, and partial rodent brain development. Analysis and execution of the models recapitulated the development of the organs, anticipated known experimental results and gave rise to novel testable predictions. Some of these results had already been validated experimentally. In this paper, I review our efforts in realistic in-silico modeling of stem cell research and developmental biology and discuss achievements and challenges. I envision that in the future, in-silico models as presented in this paper would become a common and useful technique for research in developmental biology and related research fields, particularly regenerative medicine, tissue engineering and cancer therapeutics.     

Synthesis of 3,6-diazabicyclo[3.1.1]heptanes as novel ligands for neuronal nicotinic acetylcholine receptors

Alpha series of novel 3,6-diazabicyclo[3.1.1]heptane derivatives 4a-f was synthesized and their affinity and selectivity towards alpha4beta2 and alpha7 nAChR subtypes were evaluated. The results of the current study revealed a number of compounds (4a, 4b and 4c) having a very high affinity for alpha4beta2 (K(i) at alpha4beta2 ranging from 0.023 to 0.056 nM) versus alpha7 nAChR subtypes; among these compounds, the 3-(6-bromopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptane 4c was found to be the most alpha7alpha4beta2 selective term in receptor binding assays (alpha7alpha4beta2=1295). Moreover, compound 4d also had high affinity for the alpha4beta2 nAChR subtype (K(i)=1.2 nM) with considerably high selectivity (alpha7/alpha4beta2=23300).     

Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.     

Conformational changes of G protein-coupled receptors during their activation by agonist binding

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high-resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high-resolution x-ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three-dimensional GPCR structure can be included into pharmaceutical drug design programs.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile

Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect. Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures. Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect. Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1. Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation. Implications for the pathophysiology of C. difficile-induced diarrhea are discussed.     

Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells

The expression of the anti‐apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF‐induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy. J. Cell. Physiol. 226: 2901–2907, 2011. © 2011 Wiley‐Liss, Inc.     

The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.