C. C. Fleischer

Methodology for quantifying and visualizing cell homing/accumulation by combined positron emission tomography and magnetic resonance imaging

A method for precise and sensitive organ specific quantification of 124I (124IdUR) activities in small animals has been developed. The method is based upon high precision co-registration between PET and MR imagery, utilizing an innovative mouse fixation system with external point sources visible in both modalities. The methodology is generic, and thus can easily be extended to other types of small animal studies. The methodology comprises PET/MR rigid co-registration utilising external fiducial markers, measurement of activities within small 3D volumes manually drawn in MR data and extracted from the co-aligned PET data, and subsequently corrected for Partial Volume Effect.

A Database Solution for Laboratory Information Management

In our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. The database represents a computer‐based replacement for the laboratory notebooks used in the majority of research laboratories worldwide. In addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. Using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. The system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. Like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. In each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. Thus, the database is essentially five databases linked by a hierarchy of one‐to‐many relations, organizing information in a folder‐like structure. Importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. The limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts ‘somewhere’. The often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. Thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline.

Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T cells

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen‐specific T‐cell response. As the frequency of antigen‐specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen‐pulsed dendritic cells (DCs) to increase the number of antigen‐specific, interferon‐γ (IFN‐γ)‐producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV‐seropositive and ‐seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV‐seropositive donors was 23–86% lower compared to the corresponding fresh blood samples. Antigen‐pulsed DCs could not improve the sensitivity of the intracellular cytokine‐detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate‐pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN‐γ‐producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.

Monitoring in vivo T cell traficking to tumors by positron emission tomography and magnetic resonance imaging

2571 Background: Tracking adoptively transfered cells is an essential prerequisite for devising better protocols for cellular therapy. Thus, a highly sensitive method for non-invasive visualization of labeled lymphocytes in vivo by combined PET and magnetic resonance imaging (MRI) has been developed and applied for tracking adoptively transfered tumor-specific T cells in a mouse model system. METHODS C57BL/6J mice carrying subcutaneous tumors of the ovalbumin (OVA)-expressing malignant melanoma cell line B16-OVA were adoptively transfered with OVA-specific CD8+ T cells labeled with 124-iodine-conjugated deoxyuridine (124-IdU). Five days after transfer of T cells, mice were killed and subjected to PET and MR imaging. Using a newly developed method for co-registration of the two image modalities, the anatomical localization of the transfered cells was visualized and the amount of radioactivity in various anatomical locations were very accurately determined. RESULTS Results showed a clear homing of the transfered cells to the tumors. In two independent experiments comprising 12 and 13 evaluable mice, respectively, we found a mean value of 0.909±0.468 Bq and 0.926±0.553 Bq in the tumors, and only 0.182±0.479 Bq and 0.026±0.480 Bq in the corresponding contralateral control volumes. The difference in activity between the tumor regions and the control regions was statistically highly significant with 2p-values of 0.002 and 0.006 for the two experiments. CONCLUSIONS This method may represent a significant advancement for studies of adoptively transfered specific T cells, and could potentially be developed for diagnostic purposes. Moreover, since these studies show that tumor-specific T cells home to subcutaneous tumors in substantial numbers, we suggest that these migrating cells could be employed in a new form of therapy as carriers of toxic substances to tumors. [Table: see text].

The kinetics of accumulation of adoptively transferred ovalbumin-specific T cells in a transgenic, ovalbumin-expressing murine tumor

We have studied the recruitment of adoptively transferred tumor-specific CD8-positive T cells in a mouse model system. Subcutaneous tumors of the transgenic, ovalbuminexpressing murine melanoma cell line B16-OVA were established in mice, which subsequently received intravenous transfer of ovalbumin-specific T cells, derived from the mouse strain OT-I, transgenic for a T cell receptor recognizing an ovalbumin-derived peptide, SIINFEKL.