The next generation of fibroblast-based vaccine development

Chicken embryo fibroblasts (CEF) and diploid cells have a long history in vaccine production since their isolation in the 1960s at the Wistar Institute (WI-38 cells) as well as the Medical Research Council (MRC-5 cells). The cells quickly became adopted for a number of vaccines: varicella zoster (VZV), MMR, yellow fever, polio, hepatitis A, rotavirus, rabies, Marek\u27s disease, and dengue virus. Most of these vaccine processes were developed with classical media supplemented with Fetal Bovine Serum (FBS). The Hayflick limit of diploid cells restricted their adaptation to a serum-free process. While some of the vaccines such as polio and rabies have been transitioned to Vero cells, several vaccines continue to be manufactured with CEF and human diploid cells. Currently, FBS from Australia and New Zealand are utilized for the highest level of patient safety for human vaccines. However, this supply of serum is challenged by two factors: growth of existing vaccines to improve global access and the development of new gene therapies that require FBS. In order to reduce dependency on serum, we initiated a medium development program. Using metabolite analysis and DOE, we have developed a serum-reduced growth medium and a serum-free virus production medium for MRC-5 and other fibroblast cells. With a serum reduction of 90-100%, the growth medium can support direct recovery from thaw and adaptation-free expansion, resulting in performance that is comparable to classical medium with 10% serum. We confirmed virus production with VZV and vesicular stomatitis virus in MRC-5 cells as well as Marek’s disease virus in CEFs and demonstrate a higher specific productivity. By switching to a low serum process, vaccine manufacturers can reduce production and purification costs, and increase product consistency and safety

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle

Effectiveness of structured patient-clinician communication with a solution focused approach (DIALOG+) in community treatment of patients with psychosis - a cluster randomised controlled trial

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Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp.

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20–25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reductionThis work was supported by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed III; by Consellería de Educación e Ordenación Universitaria, Xunta de Galicia's Grupos de referencia competitiva (grant number ED431C 2017/09). The authors thank TÜBİTAK (The Scientific and Technical Research Council of Turkey) for supporting this project (Project number : 217S068). S.A acknowledges the financial support for his postdoctoral research by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed IIIS

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made

Overcoming drug resistance with alginate oligosaccharides able to potentiate the action of selected antibiotics

The uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both US and European healthcare systems. We describe the utilization of a low molecular weight oligosaccharide nanomedicine (OligoG) based on the biopolymer alginate, which is able to perturb multi-drug resistant (MDR) bacteria by modulating biofilm formation/persistence and reducing resistance to antibiotic treatment; evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased the efficacy of conventional antibiotics (up to 512-fold) against important MDR pathogens including Pseudomonas, Acinetobacter and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e. macrolides, β-lactams, tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) increasing concentrations of alginate oligomer (2, 6 and 10%) were shown have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This study demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations

Pattern of drug utilization for treatment of uncomplicated malaria in urban Ghana following national treatment policy change to artemisinin-combination therapy

<p>Abstract</p> <p>Background</p> <p>Change of first-line treatment of uncomplicated malaria to artemisinin-combination therapy (ACT) is widespread in Africa. To expand knowledge of safety profiles of ACT, pharmacovigilance activities are included in the implementation process of therapy changes. Ghana implemented first-line therapy of artesunate-amodiaquine in 2005. Drug utilization data is an important component of determining drug safety, and this paper describes how anti-malarials were prescribed within a prospective pharmacovigilance study in Ghana following anti-malarial treatment policy change.</p> <p>Methods</p> <p>Patients with diagnosis of uncomplicated malaria were recruited from pharmacies of health facilities throughout Accra in a cohort-event monitoring study. The main drug utilization outcomes were the relation of patient age, gender, type of facility attended, mode of diagnosis and concomitant treatments to the anti-malarial regimen prescribed. Logistic regression was used to predict prescription of nationally recommended first-line therapy and concomitant prescription of antibiotics.</p> <p>Results</p> <p>The cohort comprised 2,831 patients. Curative regimens containing an artemisinin derivative were given to 90.8% (n = 2,574) of patients, although 33% (n = 936) of patients received an artemisinin-based monotherapy. Predictors of first-line therapy were laboratory-confirmed diagnosis, age >5 years, and attending a government facility. Analgesics and antibiotics were the most commonly prescribed concomitant medications, with a median of two co-prescriptions per patient (range 1–9). Patients above 12 years were significantly less likely to have antibiotics co-prescribed than patients under five years; those prescribed non-artemisinin monotherapies were more likely to receive antibiotics. A dihydroartemisinin-amodiaquine combination was the most used therapy for children under five years of age (29.0%, n = 177).</p> <p>Conclusion</p> <p>This study shows that though first-line therapy recommendations may change, clinical practice may still be affected by factors other than the decision or ability to diagnose malaria. Age, diagnostic confirmation and suspected concurrent conditions lead to benefit:risk assessments for individual patients by clinicians as to which anti-malarial treatment to prescribe. This has implications for adherence to policy changes aiming to implement effective use of ACT. These results should inform education of health professionals and rational drug use policies to reduce poly-pharmacy, and also suggest a potential positive impact of increased access to testing for malaria both within health facilities and in homes.</p

Long-Term Results of External Upper Esophageal Sphincter Myotomy for Oropharyngeal Dysphagia

The aim of this work was to assess the efficacy of external myotomy of the upper esophageal sphincter (UES) for oropharyngeal dysphagia. In the period 1991–2006, 28 patients with longstanding dysphagia and/or aspiration problems of different etiologies underwent UES myotomy as a single surgical treatment. The main symptoms were difficulties in swallowing of a solid-food bolus, aspiration, and recurrent incidents of solid-food blockages. Pre- and postoperative manometry and videofluoroscopy were used to assess deglutition and aspiration. Outcome was defined as success in the case of complete relief or marked improvement of dysphagia and aspiration and as failure in the case of partial improvement or no improvement. Initial results showed success in 21 and failure in 7 patients. The best outcomes were observed in patients with dysphagia of unknown origin, noncancer-related iatrogenic etiology, and neuromuscular disease. No correlation was found between preoperative constrictor pharyngeal muscle activity and success rate. After follow-up of more than 1 year, 20 patients were marked as success and 3 as failure. All successful patients had full oral intake with a normal bolus consistency without clinically significant aspiration. We conclude that in select cases of oropharyngeal dysphagia success may be achieved by UES myotomy with restoration of oral intake of normal bolus consistency

Self-Interest versus Group-Interest in Antiviral Control

Antiviral agents have been hailed to hold considerable promise for the treatment and prevention of emerging viral diseases like H5N1 avian influenza and SARS. However, antiviral drugs are not completely harmless, and the conditions under which individuals are willing to participate in a large-scale antiviral drug treatment program are as yet unknown. We provide population dynamical and game theoretical analyses of large-scale prophylactic antiviral treatment programs. Throughout we compare the antiviral control strategy that is optimal from the public health perspective with the control strategy that would evolve if individuals make their own, rational decisions. To this end we investigate the conditions under which a large-scale antiviral control program can prevent an epidemic, and we analyze at what point in an unfolding epidemic the risk of infection starts to outweigh the cost of antiviral treatment. This enables investigation of how the optimal control strategy is moulded by the efficacy of antiviral drugs, the risk of mortality by antiviral prophylaxis, and the transmissibility of the pathogen. Our analyses show that there can be a strong incentive for an individual to take less antiviral drugs than is optimal from the public health perspective. In particular, when public health asks for early and aggressive control to prevent or curb an emerging pathogen, for the individual antiviral drug treatment is attractive only when the risk of infection has become non-negligible. It is even possible that from a public health perspective a situation in which everybody takes antiviral drugs is optimal, while the process of individual choice leads to a situation where nobody is willing to take antiviral drugs