Statement on smoking cessation in COPD and other pulmonary diseases and in smokers with comorbidities who find it difficult to quit.

Chronic obstructive pulmonary disease (COPD), lung cancer, asthma and pulmonary tuberculosis are common pulmonary diseases that are caused or worsened by tobacco smoking. Growing observational evidence suggests that symptoms and prognosis of these conditions improve upon smoking cessation. Despite increasing numbers of (small) randomised controlled trials suggesting intensive smoking cessation treatments work in people with pulmonary diseases many patients are not given specific advice on the benefits or referred for intensive cessation treatments and, therefore, continue smoking. This is a qualitative review regarding smoking cessation in patients with COPD and other pulmonary disorders, written by a group of European Respiratory Society experts. We describe the epidemiological links between smoking and pulmonary disorders, the evidence for benefits of stopping smoking, how best to assess tobacco dependence and what interventions currently work best to help pulmonary patients quit. Finally, we describe characteristics and management of any "hardcore" smoker who finds it difficult to quit with standard approaches.

Novel targeted therapies for eosinophil-associated diseases and allergy.

Eosinophil-associated diseases often present with life-threatening manifestations and/or chronic organ damage. Currently available therapeutic options are limited to a few drugs that often have to be prescribed on a lifelong basis to keep eosinophil counts under control. In the past 10 years, treatment options and outcomes in patients with clonal eosinophilic and other eosinophilic disorders have improved substantially. Several new targeted therapies have emerged, addressing different aspects of eosinophil expansion and inflammation. In this review, we discuss available and currently tested agents as well as new strategies and drug targets relevant to both primary and secondary eosinophilic diseases, including allergic disorders.

TREAT-AND-EXTEND REGIMENS WITH ANTI-VEGF AGENTS IN RETINAL DISEASES: A Literature Review and Consensus Recommendations

PURPOSE A review of treat-and-extend regimens (TERs) with intravitreal anti-vascular endothelial growth factor agents in retinal diseases. METHODS There is a lack of consensus on the definition and optimal application of TER in clinical practice. This article describes the supporting evidence and subsequent development of a generic algorithm for TER dosing with anti-vascular endothelial growth factor agents, considering factors such as criteria for extension. RESULTS A TER algorithm was developed; TER is defined as an individualized proactive dosing regimen usually initiated by monthly injections until a maximal clinical response is observed (frequently determined by optical coherence tomography), followed by increasing intervals between injections (and evaluations) depending on disease activity. The TER regimen has emerged as an effective approach to tailoring the dosing regimen and for reducing treatment burden (visits and injections) compared with fixed monthly dosing or monthly visits with optical coherence tomography-guided regimens (as-needed or pro re nata). It is also considered a suitable approach in many retinal diseases managed with intravitreal anti-vascular endothelial growth factor therapy, given that all eyes differ in the need for repeat injections. CONCLUSION It is hoped that this practical review and TER algorithm will be of benefit to health care professionals interested in the management of retinal diseases.

Hereditary diseases in the horse: I. Monogenetic diseases

Overall, monogenetic hereditary diseases are less important for the breeding industry than polygenetic diseases because they are relatively rare. For the individual animal, however, these diseases have often a dramatic outcome and many of these diseases presently known are lethal. For several of them the exact pathogenesis is known and DNA-tests are available to confirm the exact diagnosis.

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

Postnatal persistent infection with classical Swine Fever virus and its immunological implications.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.

Immune Responses Against Classical Swine Fever Virus: Between Ignorance and Lunacy.

Classical swine fever virus infection of pigs causes disease courses from life-threatening to asymptomatic, depending on the virulence of the virus strain and the immunocompetence of the host. The virus targets immune cells, which are central in orchestrating innate and adaptive immune responses such as macrophages and conventional and plasmacytoid dendritic cells. Here, we review current knowledge and concepts aiming to explain the immunopathogenesis of the disease at both the host and the cellular level. We propose that the interferon type I system and in particular the interaction of the virus with plasmacytoid dendritic cells and macrophages is crucial to understand elements governing the induction of protective rather than pathogenic immune responses. The review also concludes that despite the knowledge available many aspects of classical swine fever immunopathogenesis are still puzzling.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Medical image computing in diagnosis and intervention of spinal diseases

Spinal image analysis and computer assisted intervention have emerged as new and independent research areas, due to the importance of treatment of spinal diseases, increasing availability of spinal imaging, and advances in analytics and navigation tools. Among others, multiple modality spinal image analysis and spinal navigation tools have emerged as two keys in this new area. We believe that further focused research in these two areas will lead to a much more efficient and accelerated research path, avoiding detours that exist in other applications, such as in brain and heart.

LC-MS/MS based assay and reference intervals in children and adolescents for oxysterols elevated in Niemann-Pick diseases

BACKGROUND Niemann-Pick type C (NP-C) is a rare progressive neurodegenerative lipid storage disorder with heterogeneous clinical presentation and challenging diagnostic procedures. Recently oxysterols have been reported to be specific biomarkers for NP-C but knowledge on the intra-individual variation and on reference intervals in children and adolescents are lacking. METHODS We established a LC-MS/MS assay to measure Cholestane-3β, 5α, 6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following Steglich esterification. To assess reference intervals and intra-individual variation we determined oxysterols in 148 children and adolescents from 0 to 18 years and repeat measurements in 19 of them. RESULTS The reported method is linear (r>0.99), sensitive (detection limit of 0.03 ng/mL [0.07 nM] for C-triol, and 0.54 ng/mL [1.35 nM] for 7-KC) and precise, with an intra-day imprecision of 4.8% and 4.1%, and an inter-day imprecision of 7.0% and 11.0% for C-triol (28 ng/ml, 67 nM) and 7-KC (32 ng/ml, 80 nM), respectively. Recoveries for 7-KC and C-triol range between 93% and 107%. The upper reference limit obtained for C-triol is 40.4 ng/mL (95% CI: 26.4-61.7 ng/mL, 96.0 nM, 95% CI: 62.8-146.7 nM) and 75.0 ng/mL for 7-KC (95% CI: 55.5-102.5 ng/mL, 187.2 nM, 95% CI: 138.53-255.8 nM), with no age or gender dependency. Both oxysterols have a broad intra-individual variation of 46%±23% for C-triol and 52%±29% for 7-KC. Nevertheless, all Niemann-Pick patients showed increased C-triol levels including Niemann-Pick type A and B patients. CONCLUSIONS The LC-MS/MS assay is a robust assay to quantify C-triol and 7-KC in plasma with well documented reference intervals in children and adolescents to screen for NP-C in the pediatric population. In addition our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases.

A critical evaluation of the clinical evidence for pomegranate preparations in the prevention and treatment of cardiovascular diseases

This study attempts a critical evaluation of the clinical evidence behind the use of dietary pomegranate preparations in the prevention and treatment of cardiovascular diseases. A search of PubMed on August 10, 2014 identified 228 references, which yielded extractable data from 24 clinical studies of pomegranate preparations. Hand searching identified two further studies. The quality of the studies and evidence of effectiveness of pomegranate were assessed by an established set of conventional criteria. Overall, the study quality was poor. Even in the best studies, indications of benefit did not reach the conventional levels of statistical significance. The only study with a definitive design had a biochemical rather than a clinical endpoint: it showed the expected difference in blood concentrations of myeloperoxidase after a single dose of either pomegranate or placebo. Only 10 of the 26 studies provided HPLC data on the amounts of co-active ingredients in the preparations that were consumed by the subjects. If pomegranate has a role in the prevention and treatment of cardiovascular diseases, there is a pressing need for dose-finding and long-term confirmatory studies. The ultimate endpoint for definitive studies would be mortality, but reductions in blood pressure or demonstrable decreases in atherosclerotic plaques would be useful surrogates. Sample sizes for various assumptions are provided. Future studies need to prove the clinical benefit.