Short-term benefit of smoking cessation in patients with coronary heart disease: estimates based on self-reported smoking data and serum cotinine measurements.

AIMS: To assess the short-term impact of smoking and smoking cessation measured by self-report and by serum cotinine on the risk of secondary cardiovascular disease events (CVD events). METHODS AND RESULTS: Cohort study among participants of an in-patient 3-week rehabilitation programme following an acute coronary syndrome or coronary artery revascularization. Smoking status at baseline was assessed by self-report (beginning of the rehabilitation programme, rehab) and serum cotinine (end of rehab). Active follow-up was conducted one year later. Subsequent CVD events were observed in 139 of the 967 patients. Both self-reported smoking status (odds ratio (OR) compared to continued smokers: recent quitters 0.96, former smokers 0.83, never smokers 0.54, p for trend 0.04) and serum cotinine (OR 0.59 (95% confidence interval (CI) 0.36-0.97) for cotinine-negative compared to cotinine-positive subjects) were associated with the occurrence of a secondary CVD event. After reclassification of all cotinine-positive subjects to continued smokers and cotinine-negative self-reported smokers to recent quitters, this association became even stronger. The OR now reached 0.71 (95% CI interval 0.38-1.33) for recent quitters, 0.64 (0.36-1.11) for former smokers and 0.44 (0.24-0.81) for never smokers (p-value for trend=0.009). CONCLUSION: The benefits of non-smoking and smoking cessation in cardiac patients are beyond controversy and might even be larger than suggested by previous studies which exclusively relied on self-reported smoking status.     

Outcome and prognostic factors of multimodal therapy for pulmonary large-cell neuroendocrine carcinomas

Background

There is controversy whether patients diagnosed with large-cell neuroendocrine carcinoma (LCNEC) should be treated according to protocols for non-small cell lung cancers (NSCLC) or small cell lung cancers (SCLC), especially with regard to the administration of prophylactic cranial irradiation (PCI). This study was set up to determine the incidence of brain metastases and to investigate the outcome following multimodal treatment in 70 patients with LCNEC.

Methods

Seventy patients with histologically confirmed LCNEC were treated at the University Hospital of Heidelberg between 2001 and 2014. Data were collected retrospectively. Al most all patients received thoracic surgery as initial treatment (94 %). Chemotherapy was administered in 32 patients as part of the initial treatment. Fourteen patients were treated with adjuvant or definitive thoracic radiotherapy according to NSCLC protocols. Cranial radiotherapy due to brain metastases, mostly given as whole brain radiotherapy (WBRT), was received by fourteen patients. Statistical analysis was performed using the long-rank test and the Kaplan–Meier method.

Results

Without PCI, the detected rate for brain metastases was 25 % after a median follow-up time of 23.4 months, which is comparable to NSCLC patients in general. Overall (OS), local (LPFS), brain metastases-free survival (BMFS) and extracranial distant progression-free survival (eDPFS) was 43, 50, 63 and 50 % at 5 years, respectively. Patients with incomplete resection showed a survival benefit from adjuvant radiotherapy. The administration of adjuvant chemotherapy improved the general worse prognosis in higher pathologic stages.

Conclusion

In LCNEC patients, the administration of radiotherapy according to NSCLC guidelines appears reasonable and contributes to acceptable results of multimodal treatment regimes. The low incidence of spontaneous brain metastases questions a possible role of PCI.     

Effects of Piclamilast,a Selective Phosphodiesterase-4 Inhibitor,on Oxidative Burst of Sputum Cells From Mild Asthmatics and Stable COPD Patients

Oxidative stress associated with increased presence of neutrophils is an important feature of inflammatory airways diseases like asthma or chronic obstructive pulmonary disease. We studied the in vitro effect of piclamilast (RP73401), a selective phosphodiesterase (PDE)-4 inhibitor, compared to theophylline and prednisolone, on respiratory burst of sputum cells from mild asthmatics and COPD patients. Sputum cells were harvested from mild asthmatics and stable COPD patients and treated with piclamilast, theophylline or prednisolone. Respiratory burst was assessed by luminol-dependent chemoluminescence after stimulation with 10 M n-formyl-met-leu-phe (FMLP). Piclamilast inhibited FMLP-induced respiratory burst of sputum cells in a concentration-dependent manner (asthma: EC₅₀ approximately 100 nM, max. inhibition: 97.5±5% at 100 M; COPD: EC₅₀ approximately 1 M, max. inhibition: 70.6±4.5% at 100 M), whereas maximal inhibition observed with theophylline (asthma: max. inhib. 27±15%; COPD: 6±2%, both p < 0.05 vs. piclamilast) and prednisolone (asthma: 16±6%; COPD: 7.8±6.2%, both p < 0.05 vs. piclamilast) was weaker. Inhibition by piclamilast was largely reversed through pretreatment of cells with the adenylcyclase inhibitor SQ22536. We concluded that piclamilast, a selective PDE-4 inhibitor, attenuates the respiratory burst of sputum cells from mild asthmatics and COPD patients in vitro. These data underline the potential of PDE-4 inhibition as a novel therapeutic approach to inflammatory airway diseases like asthma or COPD.     

Potent Ex Vivo Activity of Naphthoquine and Methylene Blue against Drug-Resistant Clinical Isolates of Plasmodium falciparum and Plasmodium vivax

The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC₅₀]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC₅₀s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic.     

DNA copy number alterations mark disease progression in paediatric chronic myeloid leukaemia

Early recognition of children with chronic phase chronic myeloid leukaemia (CML‐CP) at risk for developing a lymphoid blast crisis (LyBC) is desirable, because therapy options in CML‐LyBC are limited. We used Multiplex Ligation‐dependent Probe Amplification to determine whether B‐cell lymphoid leukaemia‐specific copy number alterations (CNAs) (e.g. IKZF1, PAX5, CDKN2A deletions) could be detected in CML‐CP and may be used to predict disease progression to LyBC. CNAs were detected in all patients with CML‐LyBC, but in none of the 77 patients with CML‐CP. Based on this study we conclude that CNAs remain a hallmark of disease progression.     

Quantifying the atomic-level mechanics of single long physisorbed molecular chains

Individual in situ polymerized fluorene chains 10–100 nm long linked by C–C bonds are pulled vertically from an Au(111) substrate by the tip of a low-temperature atomic force microscope. The conformation of the selected chains is imaged before and after manipulation using scanning tunneling microscopy. The measured force gradient shows strong and periodic variations that correspond to the step-by-step detachment of individual fluorene repeat units. These variations persist at constant intensity until the entire polymer is completely removed from the surface. Calculations based on an extended Frenkel–Kontorova model reproduce the periodicity and magnitude of these features and allow us to relate them to the detachment force and desorption energy of the repeat units. The adsorbed part of the polymer slides easily along the surface during the pulling process, leading to only small oscillations as a result of the high stiffness of the fluorenes and of their length mismatch with respect to the substrate surface structure. A significant lateral force also is caused by the sequential detachment of individual units. The gained insight into the molecule–surface interactions during sliding and pulling should aid the design of mechanoresponsive nanosystems and devices.Ever since the invention of the atomic force microscope (AFM) (1) and the first imaging applications, force spectroscopy has been applied to study the mechanical behavior of polymers (2); more complex chain-like biomolecules, e.g., DNA complementary strands (3); and proteins, subject to controlled extension (2) or applied force (4), mostly in solution and at room temperature. Reactive groups are chemically inserted at the ends and/or along each molecule to firmly bind some of them to suitably functionalized tips and sample surfaces. Irreversible jumps in curves of force vs. vertical separation may be associated in this way with the rupture of bonds or the unfolding of coiled subunits. If reproducible, the lowest peak in the histogram of the forces attained just before each jump is attributed to such an event in a single molecular chain or complementary pair. In the case of homogeneous polymers or protein segments, simulations based on two-state rate theory combined with a standard model of polymer nonlinear elasticity can reproduce such events, whereas reversible plateaus or continuous rises in the force may be associated with fast binding–rebinding processes or with large thermal fluctuations (2). Attention thus has focused on conformational changes strongly influenced by pulling speed or imposed force jumps (4) and also by external stimuli, e.g., optical excitation of inserted chromophores (5) or specific reactants or enzymes (6). Furthermore, mechanical forces recently were discovered by chemists as a unique stimulus to induce specific chemical reactions. In this so-called mechanochemistry, sonication typically is applied to polymer systems and is believed to result in a strong force acting on the weakest link in the chain, where the reaction takes place (7, 8). Regardless of the direct or indirect exposure to force, it is clear that the mechanics of polymer chains constrained in their surrounding environment is of utmost importance for a variety of biophysical and chemical processes as well as self-healing materials applications (9, 10).A few pulling studies have been conducted on polyelectrolytes unspecifically adsorbed on self-assembled monolayers via tunable electrostatic interactions (11), including DNA (12). They merely revealed noisy force plateaus, interpreted as continuous partial desorption of single chains, terminated by a drop to zero upon complete detachment from the surface. Despite the undisputed merit of these studies, little is known about the mechanical behavior of single molecular chains pulled off a surface, both defined and characterized on the atomic scale, in the absence of significant thermal fluctuations and drifts. Measurements at low temperature reduce the diffusion of adsorbates and provide an opportunity to determine the energetic landscape of specific molecules interacting with a surface under controlled conditions. As demonstrated here, the sliding and detachment mechanisms of individual polymer repeat units can then be inferred from the analysis of pulling experiments. A detailed interpretation of our results, based on a modified Frenkel–Kontorova (FK) model (13), also is presented.     

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.     

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.