Stable S/MAR-based episomal vectors are regulated at the chromatin level

Episomal vectors assembled from defined genetic components are a promising alternative to traditional gene therapy vectors that integrate in the host genome and may cause insertional mutations. The vector pEPI-eGFP is stably retained in the episomal state in cultured mammalian cells at low copy number for many generations without integration into the host genome. Although pEPI-eGFP is a fully engineered vector, little is known about how it interacts with the host genome and about the molecular mechanisms that are responsible for its transcriptional activity. We have analyzed the expression of the episomal reporter gene eGFP under conditions that affect the chromatin state of the genome. We have also constructed pEPI derivatives carrying a tandem array of lac operator sequences, which allows in vivo visualization and manipulation of the chromatin state of the episome. We show that changes in chromatin state of both the host and pEPI-eGFP induces changes in episomal gene activity and influences the episome’s nuclear distributions. We conclude that episomal genes are subject to control systems of the host, similarly to their counterparts in the host genome

Novel antibacterial strategies to combat biomaterial-associated infection

The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. The risk of infection is a significant problem with any inserted or implanted foreign body material, and is the number one cause of failure of implanted biomaterials. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. This thesis describes the development and characterization of novel antimicrobial agents and delivery systems, and their effectiveness in the prevention of BAI and other difficult-to-treat biofilm infections. The first strategy is based on a direct contact-killing approach. We describe the design and synthesis of a thin layer hydrogel with covalently attached synthetic antimicrobial peptides (AMPs) stabilized against proteolytic degradation. Another strategy to prevent BAI is the controlled release of antimicrobial agents, like chlorhexidine or synthetic AMPs, from a surface coating. All synthetic AMPs developed within this thesis have great potential for clinical application. Release of OP-145 was able to prevent S. aureus osteomyelitis in rabbits. The newly developed synthetic peptides, i.e. SAAP-145, SAAP-148, SAAP-276 and TC19, proved to have broad spectrum activity including activity against highly dangerous multi-drug resistant pathogens, to prevent biofilm formation, and to have in vivo activity, either in preventing biomaterial-associated or wounded skin infections. The results of the studies described in this thesis provide more insight in the pathogenesis of BAI and present novel prevention options in the fight against difficult-to-treat biofilm infection caused by multidrug-resistant pathogens

Current osteomyelitis mouse models, a systematic review

Osteomyelitis is an inflammatory bone disease caused by an infecting microorganism leading to a gradual bone loss. Due to the difficulty in studying osteomyelitis directly in patients, animal models allow researchers to investigate the pathogenesis of the infection and the development of novel prophylactic, anti-inflammatory and antimicrobial treatment strategies. This review is specifically focused on the in vivo mouse osteomyelitis studies available in literature. Thus, a systematic search on Web of Science and PubMed was conducted using the query "(infection) AND (mice OR mouse OR murine) AND (model OR models) AND (arthroplasty OR fracture OR (internal fixator) OR (internal fixation OR prosthesis OR implant OR osteomyelitis)". After critical assessment of the studies according to the inclusion and exclusion criteria, 135 studies were included in the detailed analysis. Based on the model characteristics, the studies were classified into five subject groups: haematogenous osteomyelitis, post-traumatic osteomyelitis, bone-implant-related infection, peri-prosthetic joint infection, fracture-related infection. In addition, the characteristics of the mice used, such as inbred strain, age or gender, the characteristics of the pathogens used, the inoculation methods, the type of anaesthesia and analgesia used during surgery and the procedures for evaluating the pathogenicity of the infecting micro-organism were described. Overall, the mouse is an excellent first step in vivo model to study the pathogenesis, inflammation and healing process of osteomyelitis and to evaluate novel prophylaxis and treatment strategies