Targeted Molecular Imaging

Molecular imaging aims to visualize the cellular and molecular processes occurring in living tissues, and for the imaging of specific molecules in vivo, the development of reporter probes and dedicated imaging equipment is most important. Reporter genes can be used to monitor the delivery and magnitude of therapeutic gene transfer, and the time variation involved. Imaging technologies such as micro-PET, SPECT, MRI and CT, as well as optical imaging systems, are able to non-invasively detect, measure, and report the simultaneous expression of multiple meaningful genes. It is believed that recent advances in reporter probes, imaging technologies and gene transfer strategies will enhance the effectiveness of gene therapy trials

Hydrolysis of maltoheptaose in flow through silicon wafer microreactors containing immobilised alpha-amylase and glycoamylase

In this study,a silicon micro immobilised enzyme reactor (mu IMER) has been applied for hydrolysis of maltoheptaose as a model maltodextrin and starch using immobilised otamylase (from Aspergillus oryzae) and glycoamylase (from Aspergillus niger). The influence of several parameters was investigated such as immobilisation chemistry, buffer constituents, pH, temperature, flow rate and substrate concentration. The conversion efficiency profile of the substrate was measured and the long-term stability of the reactor was tested. For separation and detection of the formed hydrolysis products, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used. The results show that the mu IMERs can also be used for hydrolysis of starch and also additionally be connected directly on-line with, e.g., liquid chromatography, making it possible to perform on-line characterisation and analysis of starch hydrolysis products

Priming of T cells with Ad-transduced DC followed by expansion with peptide-pulsed DC significantly enhances the induction of tumor-specific CD8+ T cells : implications for an efficient vaccination strategy

In recent years, vaccination strategies using antigen-presenting cells (APC) have been under investigation. Antigen delivery using genetic immunization through ex vivo transduction of dendritic cells (DC) is supposed to enhance the induction of antitumor responses in humans by activating a broad range of peptide-specific CD8+ T cells. In this study, we compared the potential of adenoviral (Ad)-transduced versus peptide-pulsed DC to induce melanoma-antigen (Ag)-specific T-cell responses in vitro. Whereas gp100-peptide-pulsed DC induced long-lasting specific CD8+ T-cell responses against single peptides, Ad-transduced DC induced broad and strong, specific immunity against various peptides of the gp100-Ag. Surprisingly, several restimulations led to decreasing gp100-specific and in parallel to increasing anti-adenoviral T-cell responses. Nevertheless, those anti-adenoviral T-cell responses provided an %22adjuvant%22 effect by inducing an early release of high amounts of IL-2/IFN-gamma, therewith enhancing CTL induction in the initiation phase. Based on these data, we suggest a prime/boost vaccination strategy in melanoma patients--combining the use of Ad-DC and peptide-pulsed DC--to obtain efficient and long-term antitumor T-cell responses

Efficient transduction of mature CD83+ dendritic cells using recombinant adenovirus suppressed T cell stimulatory capacity

We have developed a culture method for the foreign serum-free generation of highly immunostimulatory, CD83+ human dendritic cells (DC). In this study, we evaluated the feasibility and consequences of endogenously expressing antigens in mature DC using adenoviral vectors. Transduction of DC with Ad-EGFP demonstrated endogenous fluorescence in 50-85% of CD83+ DC. Ad-transduced DC stimulated the proliferation of allogeneic CD8+ and CD4+ T cells at low DC: T cell ratios. However, at high DC: T cell ratios the stimulatory capacity of Ad-transduced DC was suppressed. This immunosuppressive effect was confirmed by demonstrating that the stimulatory function of untreated DC could be suppressed in a dose-dependent manner by addition of Ad-transduced DC. Furthermore, transwell experiments suggested that direct cell contact was required. Taken together, our results demonstrate the feasibility of efficiently expressing antigens in CD83+ DC using adenoviruses. However, immunosuppressive effects must be considered and carefully studied before Ad-transduced DC are employed for clinical trials. Gene Therapy (2000) 7, 249-254

XIAP promotes melanoma growth by inducing tumour neutrophil infiltration

Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer

A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection

Dendritic cells (DCs) elicit potent anti-tumoral T-cell responses in vitro and in vivo. However, different types of DC have yet to be compared for their capacity to induce anti-tumor responses in vivo at different developmental stages. Herein, we correlated the efficiencies of different types of monocyte-derived DC as vaccines on the resulting anti-tumor immune responses in vivo. Immature and mature DCs were separately pulsed with a peptide derived from tyrosinase, MelanA/MART-1 or MAGE-1 and a recall antigen. Both DC populations were injected every 2 weeks in different lymph nodes of the same patient. Immune responses were monitored before, during and after vaccination. Mature DCs induced increased recall antigen-specific CD4( ) T-cell responses in 7/8 patients, while immature DCs did so in only 3/8. Expansion of peptide-specific IFN-gamma-producing CD8( ) T cells was observed in 5/7 patients vaccinated with mature DCs but in only 1/7 using immature DCs. However, these functional data did not correlate with the tetramer staining. Herein, immature DCs also showed expansion of peptide-specific T cells. In 2/4 patients vaccinated with mature DCs, we observed induction of peptide-specific cytotoxic T cells, as monitored by chromium-release assays, whereas immature DCs failed to induce peptide-specific cytotoxic T cells in the same patients. Instead, FCS-cultured immature DCs induced FCS-specific IgE responses in 1 patient. Our data demonstrate that this novel vaccination protocol is an efficient approach to compare different immunization strategies within the same patient. Thus, our data define FCS-free cultured mature DCs as superior inducers of T-cell responses in melanoma patients

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA)