Pieter Schoen

Treatment outcome of bone-anchored craniofacial prostheses after tumor surgery

Percutaneous endosseous implants have acquired an important place in the prosthetic rehabilitation of patients with craniofacial defects. The objective of this study was to evaluate the clinical outcome of the use of endosseous implants in the orbital and auricular region as well as to assess the satisfaction of patients with implant‐retained craniofacial prostheses after tumor surgery.

Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles

Hemagglutinin, the membrane fusion protein of influenza virus, is known to mediate a low-pH-dependent fusion reaction between the viral envelope and the limiting membrane of the endosomal cell compartment following cellular uptake of the virus particles by receptor-mediated endocytosis. Here we exploited this activity of hemagglutinin to achieve efficient gene delivery to cultured cells. Hemagglutinin was reconstituted in the presence of the monocationic lipid dioleoyldimethylammonium chloride (DODAC) to permit plasmid binding to the virosome surface. Virosomes with 30 mol% DODAC exhibited a distinct binding capacity for plasmid without causing aggregation. The virosome fusion activity was not affected by the cationic lipid DODAC as demonstrated by low-pH-dependent lipid mixing with erythrocyte ghosts. Efficient cell transfection of BHK-21 cells was observed with virosomes containing 30 mol% DODAC and plasmid encoding for β-galactosidase (pCMV β-gal) associated to their surface. The transfection activity observed was dependent on the functional activity of hemagglutinin. Contrary to DNA/cationic lipid complexes the transfection was not dependent on the cationic lipid to DNA charge ratio. Importantly, transfection of BHK-21 cells with pCMV β-gal by DODAC-containing virosomes did not show any significant signs of cytotoxicity that is commonly observed with DNA/cationic lipid complexes. Together with the high levels of expression of the transgene this highlights the potential of DODAC-containing virosomes as a novel approach in nonviral gene transfer.

Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.

Role of Hemagglutinin Surface Density in the Initial Stages of Influenza Virus Fusion: Lack of Evidence for Cooperativity

ABSTRACT Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.

Benefits of dental implants installed during ablative tumour surgery in oral cancer patients: a prospective 5-year clinical trial.

OBJECTIVE This prospective study assessed treatment outcome and patient satisfaction of oral cancer patients with a mandibular overdenture on implants up to 5 years after treatment. MATERIALS AND METHODS At baseline, 50 consecutive edentulous oral cancer patients, in whom prosthetic problems were expected after oncological treatment, were evaluated by standardized questionnaires and clinical assessments. All implants were installed during ablative tumour surgery in native bone in the interforaminal area. About two-thirds of the patients (n=31) had radiotherapy post-surgery (dose >40 Gy in the interforaminal area). RESULTS At the 5-year evaluation, 26 patients had passed away and four patients had to be excluded from the analyses, because superstructures were not present, due to persistent local irritation (n=2), loss of three implants (n=1) and the impossibility of making an overdenture related to tumour and oncological surgery-driven anatomical limitations (n=1). In the remaining 20 patients, the prosthesis was still in function (76 implants). During the 5-year follow-up, total 14 implants were lost, 13 in irradiated bone (survival rate 89.4%, dose >40 Gy) and one in non-irradiated bone (survival rate 98.6%). Peri-implant tissues had a healthy appearance and remained healthy over time. Patients were satisfied with their dentures. CONCLUSIONS It was concluded that oral cancer patients can benefit from implants installed during ablative surgery, with a high survival rate of the implants, a high percentage of rehabilitated patients and a high denture satisfaction up to 5 years after treatment.

Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA.

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA-virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA-virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA-virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

Fusion of alphaviruses with liposomes is a non-leaky process

It has been reported that low‐pH‐induced fusion of influenza virus with liposomes results in rapid and extensive release of both low‐ and high‐molecular‐weight substances from the liposomes [Günther‐Ausborn et al., J. Biol. Chem. 270 (1995) 29279–29285; Shangguan et al., Biochemistry 35 (1996) 4956–4965]. Here, we demonstrate retention of encapsulated water‐soluble compounds during fusion of Semliki Forest virus (SFV) or Sindbis virus with liposomes at low pH. Under conditions allowing complete fusion of the liposomes, a limited fluorescence dequenching of liposome‐encapsulated calcein was observed, particularly for SFV. Also, radioactively labeled inulin or sucrose were largely retained. Freezing and thawing of the viruses in the absence of sucrose resulted in an enhanced leakiness of fusion. These results support the notion that the alphavirus fusion event per se is non‐leaky and may well involve a discrete hemifusion intermediate.

Delivery of Foreign Substances to Cells Mediated by Fusion-Active Reconstituted Influenza Virus Envelopes (Virosomes)

AbstractThis paper presents a survey of the properties and applications of reconstituted influenza virus envelopes (virosomes). Influenza virosomes can be reconstituted from the original viral membrane lipids and spike glycoproteins, after solubilization of intact virus with octaethyleneglycol monododecyl ether (C12E8) and removal of this detergent with a hydrophobic resin (BioBeads SM-2). These virosomes are functionally active, i.e their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral hemagglutinin (HA). By virtue of their fusion activity, virosomes represent a powerful carrier system for cellular delivery of foreign substances, encapsulated in their aqueous interior or co-reconstituted in their membranes. Delivery of an encapsulated, water-soluble, compound is illustrated with data on the toxin gelonin. Protein synthesis in BHK-21 cells in culture is efficiently inhibited when gelonin-containin...

Five-year follow-up of oral functioning and quality of life in patients with oral cancer with implant-retained mandibular overdentures

The purpose of this prospective study was to assess the quality of life (QOL) and oral functioning of patients with oral cancer up to 5 years after prosthodontic rehabilitation with mandibular implant‐retained overdentures.

Targeting influenza virosomes to ovarian carcinoma cells

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR‐3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)‐derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab′ fragments of mAb 323/A3 (anti‐epithelial glycoprotein‐2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR‐3 cells. These antibody‐redirected virosomes fused with membranes of OVCAR‐3 cells in a pH‐dependent fashion.