Radiocarbon based assessment of soil organic matter contribution to soil respiration in a pine stand of the Campine region, Belgium

The contribution of decomposing soil organic carbon (SOC) to total annual soil respiration (SR) was evaluated by radiocarbon measurements at a Scots pine stand growing on a plaggen soil in the Belgian Campine region. Two approaches were used to estimate the contribution of different C pools to SR. In the first approach, the variations in ¹⁴C content of soil CO₂ efflux were monitored during one year (2003) and compared to the atmospheric and SOC ¹⁴C signatures to determine the contribution of ??fast?? (root respiration and fast decomposing SOC) and ??slow?? cycling C pools to total SR. In the second approach an estimate of the total heterotrophic soil respiration (Rh), comprising the slow cycling C and the heterotrophic part of the fast-cycling C pools, was derived applying a box model based on the amount of the bulk SOC pool and its ¹⁴C-derived mean residence time (MRT). The quantification of the Rh and the decomposition rate of the slow-cycling SOC allows to indirectly determining the contribution of the heterotrophic C that decompose within a year. Measurements of total SR performed in the field allowed assessing the contribution of the different C pools to total soil C efflux. On an annual basis, the fast-cycling C was the main contributor to SR, about 85%, while the contribution of the slow-cycling C (with MRT >1 yr) to total SR was 15%. Total annual Rh was 36% of total SR, which is in the lower range reported for temperate coniferous forests. The comparison of Rh with other estimates for the same site (47?C50% of total SR) suggest a possible underestimation of the C flux from the mineral soil. In fact, the ??very old?? C contained in the plaggen horizon strongly affects the signature of the mostly young C leaving the soil. In conclusion, our results indicate that the contribution of SOC decomposition to total soil CO₂ flux in this forest is less than 40%, and at least half of it comes from organic compounds less than 1 year old.     

Development and Characterization of an Antibody-Labeled Super-Paramagnetic Iron Oxide Contrast Agent Targeting Prostate Cancer Cells for Magnetic Resonance Imaging

In this study we developed, characterized and validated in vitro a functional superparagmagnetic iron-oxide based magnetic resonance contrast agent by conjugating a commercially available iron oxide nanoparticle, Molday ION Rhodamine-B Carboxyl (MIRB), with a deimmunized mouse monoclonal antibody (muJ591) targeting prostate-specific membrane antigen (PSMA). This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI (Achieva, Philips) showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.     

Chimeric viral vectors--the best of both worlds?

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The vector systems currently available have not allowed the simultaneous provision of both of these goals. Several groups are now developing chimeric viral vector systems that incorporate the favorable attributes of two different viral vectors. These chimeric vectors might allow the goals for specific gene therapy applications to be realized.     

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.     

Genetic incorporation of human metallothionein into the adenovirus protein IX for non-invasive SPECT imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.