EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a designed ankyrin repeat protein (DARPin) specific for the epithelial cell adhesion molecule (EpCAM) as a carrier for small interfering RNA (siRNA) complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in Escherichia coli in soluble form, yet, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing four to five siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as shown on MCF-7 breast carcinoma cells. In contrast to unspecific LipofectAMINE transfection, down-regulation of antiapoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as shown by increased sensitivity to the anticancer agent doxorubicin.

Anti-infective therapy of peri-implantitis with adjunctive local drug delivery or photodynamic therapy: 12-month outcomes of a randomized controlled clinical trial

OBJECTIVE: The objective of the study is to compare the clinical, microbiological and host-derived effects in the non-surgical treatment of initial peri-implantitis with either adjunctive local drug delivery (LDD) or adjunctive photodynamic therapy (PDT) after 12 months. MATERIALS AND METHODS: Forty subjects with initial peri-implantitis, that is, pocket probing depths (PPD) 4-6 mm with bleeding on probing (BoP) and radiographic bone loss ≤2 mm, were randomly assigned to two treatment groups. All implants were mechanically debrided with titanium curettes and with a glycine-based powder airpolishing system. Implants in the test group (N = 20) received adjunctive PDT, whereas minocycline microspheres were locally delivered into the peri-implant pockets of control implants (N = 20). At sites with residual BoP, treatment was repeated after 3, 6, 9 and 12 months. The primary outcome variable was the change in the number of peri-implant sites with BoP. Secondary outcome variables included changes in PPD, clinical attachment level (CAL), mucosal recession (REC) and in bacterial counts and crevicular fluid (CF) levels of host-derived biomarkers. RESULTS: After 12 months, the number of BoP-positive sites decreased statistically significantly (P < 0.05) from baseline in both groups (PDT: 4.03 ± 1.66-1.74 ± 1.37, LDD: 4.41 ± 1.47-1.55 ± 1.26). A statistically significant (P < 0.05) decrease in PPD from baseline was observed at PDT-treated sites up to 9 months (4.19 ± 0.55 mm to 3.89 ± 0.68 mm) and up to 12 months at LDD-treated sites (4.39 ± 0.77 mm to 3.83 ± 0.85 mm). Counts of Porphyromonas gingivalis and Tannerella forsythia decreased statistically significantly (P < 0.05) from baseline to 6 months in the PDT and to 12 months in the LDD group, respectively. CF levels of IL-1β decreased statistically significantly (P < 0.05) from baseline to 12 months in both groups. No statistically significant differences (P > 0.05) were observed between groups after 12 months with respect to clinical, microbiological and host-derived parameters. CONCLUSIONS: Non-surgical mechanical debridement with adjunctive PDT was equally effective in the reduction of mucosal inflammation as with adjunctive delivery of minocycline microspheres up to 12 months. Adjunctive PDT may represent an alternative approach to LDD in the non-surgical treatment of initial peri-implantitis.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

What are the implications of changing treatment delivery models for patients with inflammatory bowel disease: a discussion paper.

An integrated model of care has been used effectively to manage chronic diseases; however, there is limited, yet encouraging evidence on its introduction in the management of inflammatory bowel disease (IBD), a chronic gastrointestinal condition. Here, the rationale for and implications of introducing an integrated model of care for patients with IBD are discussed, with a particular focus on psychology input, patient-centred care, efficiency as perceived by patients and doctors, financial implications and the possible means of model introduction. This is a discussion paper on the integrated model of care for IBD against a background of what has been learned from an integrated model of care established in other chronic conditions. Although limited, the emerging data on an integrated model of care in IBD are encouraging with respect to patient outcomes and savings in healthcare costs. In other conditions, the model has been well received by both patients and practitioners, although the loss of autonomy by doctors is listed among its drawbacks. The cost-effectiveness data are now sufficiently convincing to recommend the model's acceptance in principle. The model should be promoted at the policy level rather than by individual practitioners to facilitate equal access for patients with IBD on a larger scale than currently.

Secondary prevention in the clinical management of patients with cardiovascular diseases. Core components, standards and outcome measures for referral and delivery

Despite major improvements in diagnostics and interventional therapies, cardiovascular diseases remain a major health care and socio-economic burden both in western and developing countries, in which this burden is increasing in close correlation to economic growth. Health authorities and the general population have started to recognize that the fight against these diseases can only be won if their burden is faced by increasing our investment on interventions in lifestyle changes and prevention. There is an overwhelming evidence of the efficacy of secondary prevention initiatives including cardiac rehabilitation in terms of reduction in morbidity and mortality. However, secondary prevention is still too poorly implemented in clinical practice, often only on selected populations and over a limited period of time. The development of systematic and full comprehensive preventive programmes is warranted, integrated in the organization of national health systems. Furthermore, systematic monitoring of the process of delivery and outcomes is a necessity. Cardiology and secondary prevention, including cardiac rehabilitation, have evolved almost independently of each other and although each makes a unique contribution it is now time to join forces under the banner of preventive cardiology and create a comprehensive model that optimizes long term outcomes for patients and reduces the future burden on health care services. These are the aims that the Cardiac Rehabilitation Section of the European Association for Cardiovascular Prevention & Rehabilitation has foreseen to promote secondary preventive cardiology in clinical practice.

Development of light-responsive porous polycarbonate membranes for controlled caffeine delivery

For controlled caffeine release, light-responsive membranes were developed. It was possible to produce membranes that reduced their caffeine permeability resistance by about 97% when irradiated with UV-light compared to measurements at daylight. This was achieved by grafting polymers possessing photochromic units onto track-edged polycarbonate membranes. Covalently linked coatings on porous polycarbonate membranes were obtained by plasma activation of the membrane surface followed by plasma-induced graft polymerization. Copolymerization of spiro-compounds during the coating process as well as postmodification of preformed coatings with spiropyran resulted in photochromic membranes. For the copolymerization process, the synthesis of five photochromic methacrylic and acrylic spiropyrans and spirooxazines was successfully performed. Additionally, a spiropyran with carboxylic acid functionality was synthesized for the postmodification process. This enabled us to postmodify polymeric materials containing alcohol or amine groups to obtain photochromic materials. UV-irradiation of these light-responsive membranes resulted in a strong colouration of the membrane, in a reduction of surface tension, which resulted in a decreased caffeine permeability resistance. The membranes were characterized using XPS for the elemental composition of the coating, contact angle measurements for the surface tension, solid-state UV/VIS measurements for the determination of the kinetic and stability properties, and two-photon microscopy for the localisation of the photochromic substance in the porous membrane.

Delivery of health care at the end of life in cancer patients of four swiss cantons a retrospective database study (SAKK 89/09)

Background The use of cancer related therapy in cancer patients at the end-of-life has increased over time in many countries. Given a lack of published Swiss data, the objective of this study was to describe delivery of health care during the last month before death of cancer patients. Methods Claims data were used to assess health care utilization of cancer patients (identified by cancer registry data of four participating cantons), deceased between 2006-2008. Primary endpoints were hospitalization rate and delivery of cancer related therapies during the last 30 days before death. Multivariate logistic regression assessed the explanatory value of patient and geographic characteristics. Results 3809 identified cancer patients were included. Hospitalization rate (mean 68.5%, 95%CI 67.0-69.9) and percentage of patients receiving anti-cancer drug therapies (ACDT, mean 14.5%, 95%CI 13.4-15.6) and radiotherapy (mean 7.7%, 95%CI 6.7-8.4) decreased with age. Canton of residence and insurance type status most significantly influenced the odds for hospitalization or receiving ACDT. Conclusions The intensity of cancer specific care showed substantial variation by age, cancer type, place of residence and insurance type status. This may be partially driven by cultural differences within Switzerland and the cantonal organization of the Swiss health care system.