Innate and Adaptive Immunity in Orolabial Herpes Simplex

Oral mucosa is a frequent site of primary herpes simplex virus type 1 (HSV-1) infection, whereas intraoral recurrent disease is very rare. Instead, reactivation from latency predominantly results in asymptomatic HSV shedding to saliva or recurrent labial herpes (RLH) with highly individual frequency. The current study aimed to elucidate the role of human oral innate and acquired immune mechanisms in modulation of HSV infection in orolabial region. Saliva was found to neutralize HSV-1, and to protect cells from infection independently of salivary antibodies. Neutralization capacity was higher in saliva from asymptomatic HSV-seropositive individuals compared to subjects with history of RLH or seronegative controls. Neutralization was at least partially associated with salivary lactoferrin content. Further, lactoferrin and peroxidase-generated hypothiocyanite were found to either neutralize HSV-1 or interfere with HSV-1 replication, whereas lysozyme displayed no anti-HSV-1 activity. Lactoferrin was also shown to modulate HSV-1 infection by inhibiting keratinocyte proliferation. RLH susceptibility was further found to be associated with Th2 biased cytokine responses against HSV, and a higher level of anti- HSV-IgG with Th2 polarization, indicating lack of efficiency of humoral response in the control of HSV disease. In a three-dimensional cell culture, keratinocytes were found to support both lytic and nonproductive infection, suggesting HSV persistence in epithelial cells, and further emphasizing the importance of peripheral immune control of HSV. These results suggest that certain innate salivary antimicrobial compounds and Th1 type cellular responses are critically important in protecting the host against HSV disease, implying possible applications in drug, vaccine and gene therapy design.

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.