IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.     

Determining the most important cellular characteristics for fracture healing using design of experiments methods

Computational models are employed as tools to investigate possible mechanoregulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The objective of this study was to determine the most important cellular characteristics of a mechanoregulatory model describing both cell phenotype-specific and mechanobiological processes that are active during bone healing using a statistical approach. The computational model included an adaptive two-dimensional finite element model of a fractured long bone. Three different outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at early, mid and late stages of healing and (3) the total time until complete healing. For the statistical analysis, first a resolution IV fractional factorial design (L₆₄) was used to identify the most significant factors. Thereafter, a three-level Taguchi orthogonal array (L₂₇) was employed to study the curvature (non-linearity) of the 10 identified most important parameters. The results show that the ability of the model to predict the sequences of normal fracture healing was predominantly influenced by the rate of matrix production of bone, followed by cartilage degradation (replacement). The amount of bone formation at early stages was solely dependent on matrix production of bone and the proliferation rate of osteoblasts. However, the amount of bone formation at mid and late phases had the rate of matrix production of cartilage as the most influential parameter. The time to complete healing was primarily dependent on the rate of cartilage degradation during endochondral ossification, followed by the rate of cartilage formation. The analyses of the curvature revealed a linear response for parameters related to bone, where higher rates of formation were more beneficial to healing. In contrast, parameters related to fibrous tissue and cartilage showed optimum levels. Some fibrous connective tissue- and cartilage formation was beneficial to bone healing, but too much of either tissue delayed bone formation. The identified significant parameters and processes are further confirmed by in vivo animal experiments in the literature. This study illustrates the potential of design of experiments methods for evaluating computational mechanobiological model parameters and suggests that further experiments should preferably focus at establishing values of parameters related to cartilage formation and degradation.     

Novel Biocatalysts by Identification and Design

Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution.     

The consequences of uncertainties in land use, climate and vegetation responses on the terrestrial carbon

The IPCC SRES narratives were implemented in IMAGE 2.2 to evaluate thefuture condition of the climate system (including the biosphere). A series of scenario experiments was used to assess possible ranges in emissions and concentrations of greenhouse gases, climate change and impacts. These experiments focussed on the role of the terrestrial carbon cycle. The experiments show that the SRES narratives dominate human emissions and not natural processes. In contrary, atmospheric CO2 concentration strongly differs between the experiments. Atmospheric CO2 concentrations range for A1B from 714 to 1009 ppmv CO2 in 2100. The spread of this range is comparable with the full SRES range as implemented in IMAGE 2.2 (515-895 μmol/mol CO2). The most important negative and positive feedback processes in IMAGE 2.2 on the build-up of CO2 concentrations are CO2 fertilisation and soil respiration respectively. Indirect effects of these processes furtherchange land-use patterns, deforestation rates and alter the natural C fluxes. The cumulative effects of these changes have a pronounced influence on the final CO2 concentrations. Our scenario experiments highlight the importance of a proper parameterisation of feedback processes, C-cycle and land use in determining the future states of the climate system.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

Rapid Fishery Assessment by Market Survey (RFAMS) – An Improved Rapid-Assessment Approach to Characterising Fish Landings in Developing Countries

The complex multi-gear, multi-species tropical fisheries in developing countries are poorly understood and characterising the landings from these fisheries is often impossible using conventional approaches. A rapid assessment method for characterising landings at fish markets, using an index of abundance and estimated weight within taxonomic groups, is described. This approach was developed for contexts where there are no detailed data collection protocols, and where consistent data collection across a wide range of fisheries types and geographic areas is required, regardless of the size of the site and scale of the landings. This methodology, which was demonstrated at seven fish landing sites/fish markets in southern Indonesia between July 2008 and January 2011, provides a rapid assessment of the abundance and diversity in the wild catch over a wide variety of taxonomic groups. The approach has wider application for species-rich fisheries in developing countries where there is an urgent need for better data collection protocols, monitoring future changes in market demographics, and evaluating health of fisheries.     

Specific Labeling of Stem Cell Activity in Human Colorectal Organoids Using an ASCL2-Responsive Minigene

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Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space.

We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

     

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) isoforms (2b and 3) as well as four Ca ²⁺ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP₃ binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca ²⁺ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P₃R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP₃R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP₃R-1 and InsP₃R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP₃R-1 and InsP₃R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP₃R isoforms, the two SERCA isoforms and the four Ca²⁺ binding proteins investigated. This heterogeneity may underlie specialization of the Ca²⁺ stores and the subsequent initiation of intracellular Ca²⁺ signals.