The microPET R4 scanner is a dedicated positron emission tomograph (PET) for studies of rodents. A number of scanner parameters such as spatial resolution, sensitivity, scatter, and count rate performance were determined in this work, which showed that the microPET R4 is a suitable PET scanner for small animals like mice and rats. In the center of the field of view (FOV) a maximal sensitivity of 43.66 cps/kBq for a centered point source was calculated from a measurement with a germanium-68 line source within an energy widow of 250-750 keV. A spatial resolution of 1.85 mm full-width at half-maximum (FWHM) in the axial direction and 1.66 mm FWHM in the transaxial direction was measured in the center with a 1-mm-diameter sodium-22 point source. Within the inner 20 mm of the FOV the volumetric resolution is better than 15.6 micro l, corresponding to a linear resolution of less than 2.5 mm in all three dimensions. Images of a high-resolution phantom and from mice and rat studies illustrate the good performance of the scanner. A maximal noise equivalent count rate (NECR) was reached at 174 kcps for a mouse phantom and at 93 kcps for a rat phantom (energy window 250-750 keV). Scatter fractions were measured between 0.30 and 0.42 for an energy window of 250-750 keV and phantom diameters similar to mice and rats. A comparison with the microPET P4 model for primates illustrates the gain in sensitivity due to a smaller detector ring diameter but also the changes in NECR.

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate–dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.0...

PurposeIn recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [18F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo.MethodsWe studied the uptake of [18F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [18F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections.ResultsAll rats showed significant [18F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [18F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo.ConclusionThe TSPO radioligand [18F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [18F]DPA-714 as an alternative radiotracer to image human glioma.