NOD/scid IL-2Rgnull mice: a preclinical model system to evaluate human dendritic cell-based vaccine strategies in vivo

<p>Abstract</p> <p>Background</p> <p>To date very few systems have been described for preclinical investigations of human cellular therapeutics <it>in vivo</it>. However, the ability to carry out comparisons of new cellular vaccines <it>in vivo </it>would be of substantial interest for design of clinical studies. Here we describe a humanized mouse model to assess the efficacy of various human dendritic cell (DC) preparations. Two reconstitution regimes of NOD/scid IL2Rg^null(NSG) mice with adult human peripheral blood mononuclear cells (PBMC) were evaluated for engraftment using 4-week and 9-week schedules. This led to selection of a simple and rapid protocol for engraftment and vaccine evaluation that encompassed 4 weeks.</p> <p>Methods</p> <p>NSG recipients of human PBMC were engrafted over 14 days and then vaccinated twice with autologous DC via intravenous injection. Three DC vaccine formulations were compared that varied generation time <it>in vitro </it>(3 days versus 7 days) and signals for maturation (with or without Toll-like receptor (TLR)3 and TLR7/8 agonists) using MART-1 as a surrogate antigen, by electroporating mature DC with <it>in vitro </it>transcribed RNA encoding full length protein. After two weekly vaccinations, the splenocyte populations containing human lymphocytes were recovered 7 days later and assessed for MART-1-specific immune responses using MHC-multimer-binding assays and functional assessment of specific killing of melanoma tumor cell lines.</p> <p>Results</p> <p>Human monocyte-derived DC generated <it>in vitro </it>in 3 days induced better MART-1-specific immune responses in the autologous donor T cells present in the humanized NSG mice. Moreover, consistent with our <it>in vitro </it>observations, vaccination using mature DC activated with TLR3 and TLR7/8 agonists resulted in enhanced immune responses <it>in vivo</it>. These findings led to a ranking of the DC vaccine effects <it>in vivo </it>that reflected the hierarchy previously found for these mature DC variations <it>in vitro</it>.</p> <p>Conclusions</p> <p>This humanized mouse model system enables comparisons among different DC vaccine types to be rapidly assessed <it>in vivo</it>. In addition, <it>ex vivo </it>analyses of human CD3⁺T cells recovered from the spleens of these mice are also possible, including studies on lymphocyte subsets, Th1/Th2 polarization, presence of regulatory T cells and the impact of DC vaccination on their functions.</p

Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

<p>Abstract</p> <p>Background</p> <p>Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) <it>in vitro</it>. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.</p> <p>Methods</p> <p>In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.</p> <p>Results</p> <p>Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of <it>in vitro </it>transcribed (<it>ivt</it>)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of <it>ivt</it>RNA.</p> <p>Conclusions</p> <p>This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or <it>ivt</it>RNA as sources of specific antigen.</p

Soil Salinity and pH Drive Soil Bacterial Community Composition and Diversity Along a Lateritic Slope in the Avon River Critical Zone Observatory, Western Australia

Soils are crucial in regulating ecosystem processes, such as nutrient cycling, and supporting plant growth. To a large extent, these functions are carried out by highly diverse and dynamic soil microbiomes that are in turn governed by numerous environmental factors including weathering profile and vegetation. In this study, we investigate geophysical and vegetation effects on the microbial communities of iron-rich lateritic soils in the highly weathered landscapes of Western Australia (WA). The study site was a lateritic hillslope in southwestern Australia, where gradual erosion of the duricrust has resulted in the exposure of the different weathering zones. High-throughput amplicon sequencing of the 16S rRNA gene was used to investigate soil bacterial community diversity, composition and functioning. We predicted that shifts in the microbial community would reflect variations in certain edaphic properties associated with the different layers of the lateritic profile and vegetation cover. Our results supported this hypothesis, with electrical conductivity, pH and clay content having the strongest correlation with beta diversity, and many of the differentially abundant taxa belonging to the phyla Actinobacteria and Proteobacteria. Soil water repellence, which is associated with Eucalyptus vegetation, also affected beta diversity. This enhanced understanding of the natural system could help to improve future crop management in WA since the physicochemical properties of the agricultural soils in this region are inherited from laterites via the weathering and pedogenesis processes

Lamination of Separators to Electrodes using Electrospinning.

This study shows the feasibility of the Electrospinning method as a process step for advanced and fast production Li ion cells. Lamination is a key technology for Lithium-ion battery production. It bares different advantages, such as a fast production line speed by fixing the separator to the electrodes. Unfortunately, this technology is inapplicable for separator and electrode formulations not based on thermoplastic binders. Using Electrospinning, this disadvantage can be overcome. In our study, beaded PVDF polymer nanofibres were spun onto a fibre-reinforced, inorganic-filled separator. This modified separator was then laminated onto a NMC111-cathode using a temperature profile of 110/110/120°C within the laminator. After Lamination, the separator was pulled-off again and placed in a SEM to see the adhesive behaviour of the applied polymer. The information gathered with SEM clearly shows a successful lamination of the separator to the electrode

Sustainable Surface Modification of Polyetheretherketone (PEEK) Implants by Hydroxyapatite/Silica Coating—An In Vivo Animal Study

Polyetheretherketone (PEEK) has the potential to overcome some of the disadvantages of titanium interbody implants in anterior cervical and discectomy and fusion (ACDF). However, PEEK shows an inferior biological behavior regarding osseointegration and bioactivity. Therefore, the aim of the study was to create a bioactive surface coating on PEEK implants with a unique nanopore structure enabling the generation of a long-lasting interfacial composite layer between coating material and implant. Seventy-two PEEK implants—each thirty-six pure PEEK implants (PI) and thirty-six PEEK implants with a sprayed coating consisting of nanocrystalline hydroxyapatite (ncHA) embedded in a silica matrix and interfacial composite layer (SPI)—were inserted in the femoral condyles of adult rats using a split-side model. After 2, 4 and 8 weeks, the femur bones were harvested. Half of the femur bones were used in histological and histomorphometrical analyses. Additionally, pull-out tests were performed in the second half. Postoperative healing was uneventful for all animals, and no postoperative complications were observed. Considerable crestal and medullary bone remodeling could be found around all implants, with faster bone formation around the SPI and fewer regions with fibrous tissue barriers between implant and bone. Histomorphometrical analyses showed a higher bone to implant contact (BIC) in SPI after 4 and 8 weeks (p &lt; 0.05). Pull-out tests revealed higher pull-out forces in SPI at all time points (p &lt; 0.01). The presented findings demonstrate that a combination of a bioactive coating and the permanent chemical and structural modified interfacial composite layer can improve bone formation at the implant surface by creating a sustainable bone-implant interface. This might be a promising way to overcome the bioinert surface property of PEEK-based implants

Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70

<p>Abstract</p> <p>Background</p> <p>For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4⁺T helper 1 cells, which then support the development of CD8⁺cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development.</p> <p>Methods</p> <p>After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C).</p> <p>Results</p> <p>Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation.</p> <p>Conclusion</p> <p>Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.</p