Effect of concentration of SH U 555A labeled human melanoma cells on MR spin echo and gradient echo signal decay at 0.2, 1.5, and 3T.

In the current study the effect of increasing concentrations of superparamagnetic iron oxide labeled cells on the MRI signal decay at magnetic field strengths of 0.2, 1.5, and 3 T was evaluated. The spin echo and gradient echo cellular transverse relaxivity was systematically studied for various concentrations (N = 1, 5, 10, 20, 40, and 80 cells/microl(gel)) of homogeneously suspended SH U 555A labeled SK-Mel28 human melanoma cells. For all field strengths investigated a linear relationship between cellular transverse relaxation enhancement and cell concentration was found. In the spin echo case, the cellular relaxivities [i.e., d(deltaR2)/dN] were determined to 0.12 s(-1) (cell/microl)(-1) at 0.2 T, 0.16 s(-1) (cell/microl)(-1) at 1.5 T, and 0.17 s(-1) (cell/microl) at 3 T. In the gradient echo case, the calculated cellular relaxivities (i.e., d(deltaR2*)/dN) were 0.51 s(-1) (cell/microl)(-1) at 0.2 T, 0.69 s(-1) (cell/microl)(-1) at 1.5 T, and 0.71 s(-1) (cell/microl)(-1) at 3 T. The proposed preparation technique has proven to be a simple and reliable approach to quantify effects of magnetically labeled cells in vitro. On the basis of this quantification well suited tissue specific models can be derived.

[A preparation technique for quantitative investigation of SPIO-containing solutions and SPIO-labelled cells by MRI]. / Eine Präparationstechnik zur quantitativen Untersuchung SPIO-haltiger Lösungen und SPIO-markierter Zellen in der MRT.

UNLABELLED: PURPOSE. This work aims to present a preparation technique for ex-vivo MR examination of SPIO (superparamagnetic iron oxide) containing solutions or SPIO labeled cells. Accumulations of SPIO particles and labeled cells were prepared in different concentrations using agar gel phantoms. Signal extinction around accumulations of magnetic material was examined systematically by gradient echo sequences with variable echo times and spatial resolution. The correlation between local iron concentration and diameter of signal extinction in MR gradient echo images was investigated. METHODS: Resovist, (SHU 555A) was used as superparamagnetic contrast medium. Different concentrations of SPIO-containing solutions (0.75 - 15 mg Fe/10 ml) and magnetically labeled SK-Mel28 cells (25,000-1,000,000 cells/10 ml) were accommodated inside a defined volume in an agar matrix. Diameters of signal void were assessed in dependence on local iron concentration, echo time (5-25 ms) and isotropic spatial resolution (length of voxel 0.25 - 0.60 mm). Measurements were performed on a clinical MR whole body scanner (3 Tesla) using a spoiled gradient echo sequence (FLASH). RESULTS: For the present experimental conditions sensitivity to detect the magnetic label was maximized using TE 25 ms. In contrast, the area of signal cancellation was minimized using TE 5 ms and isotropic resolution of 0.25 mm. In the latter case the image indicated the area of magnetic material most precisely. Diameter of signal cancellation was a logarithmic function on local iron concentration. In the presented set-up detection of concentrations as low as 0.75 mg Fe/10 ml in SPIO-containing solution or 1.25 mg Fe/10 ml in SPIO-labeled SK-Mel28 cells was certainly possible. CONCLUSION: The proposed preparation strategy with a well defined spatial distribution of the magnetic material in an agar gel phantom produced reliable results and appears clearly superior compared to set-ups with randomly distributed material in glass tubes. The diameter of the signal extinction in gradient echo images was significantly affected by the choice of echo time and spatial resolution. The calibration of signal cancellation versus iron concentrations may be valuable to assess SPIO concentrations and possibly numbers of labeled cells under specific conditions in vitro or even in vivo.

[Liposome-mediated transfection of thrombomodulin in vascular smooth muscle cells--a gene therapy concept to inhibit recurrent stenosis]. / Liposomal vermittelte Transfektion von Thrombomodulin in vaskuläre glatte Muskelzellen - ein Gentherapie-Konzept zur Inhibition der Rezidiv-Stenose?

PURPOSE: Thrombomodulin (TM), an integral endothelial receptor, is known for its anticoagulant functions. Moreover, there is evidence of growth-modulating effects of this cell surface -protein. The aim of our study was to establish by in vitro transfection a stable cell line of vascular smooth muscle cells with overexpression of TM for further investigations concerning the influence of TM on cellular proliferation and its potential role during the formation of restenosis. METHODS: Aortic smooth muscle cells of the rat were transfected with cDNA of mouse TM or one of its three mutants (M1, M2, M4) by a liposome-mediated technique. The expression of mouse TM mRNA in the selected clones was proven with the help of RT-PCR. Changes of cell proliferation were determined by proliferation kinetics over 24 days. The quantification of the total protein TM was made by Western blots. RESULTS: In 44 of 100 cases the RT-PCR confirmed a successful transfection of mouse-TM. The clones with transfected TM, M1 or M2 showed an inhibited cell growth, whereas M4 demonstrated an increased proliferation compared with controls. The comparison of amounts of total TM with cell growth of individual clones resulted in a negative correlation between proliferation and TM-expression (coefficient of correlation for TM -0.87, for M1 -0.59). CONCLUSIONS: It is possible to reproduce stable cell-lines of vascular smooth muscle cells with overexpression of TM by the presented model of in vitro transfection. Thus, a basis exists for detailed examinations of growth-regulating mechanisms by TM.

[Dose dependent effects of the combined beta-gamma-emitter 188Rhenium on the growth of human vessel wall cells]. / Dosisabhängige Auswirkungen des kombinierten beta-gamma-Emitters 188Rhenium auf humane Endothelzellen und humane vaskuläre glatte Muskelzellen.

PURPOSE: To evaluate the dose of (188)Re that completely suppresses growth and clonogenic activity of human aortic smooth muscle cells (haSMC) since these cells are mainly responsible for restenosis occurring after PTA. For comparison, growth and clonogenic activity of endothelial cells (EC) were investigated with corresponding doses. MATERIALS AND METHODS: Two days after plating, haSMC and EC were incubated with (188)Re for five days. The doses applied ranged from 4 to 16 Gy. Cell growth was observed for a period of 20 days (EC) or 30 days (haSMC), respectively. Clonogenic activity was monitored over a period of 20 days for both cell lines. RESULTS: Irradiation caused dose-depend-ent inhibition of cell growth and clonogenic activity both in haSMC and in EC. HaSMC growth was completely blocked with 8 Gy, while EC still showed some proliferation even with 16 Gy. The clonal activity of haSMC was also completely blocked with 8 Gy while EC still showed little clonal activity even with 16 Gy. CONCLUSION: Cell growth of both haSMC and EC can be effectively suppressed in a dose-dependent manner. Only haSMC showed a complete growth arrest with 8 Gy while EC were able to proliferate even with 16 Gy. HaSMC colony formation was completely suppressed after application of 8 Gy, while the EC still showed colony formation activity with 16 Gy. (188)Re has some advantageous properties for intravascular irradiation in comparison to other radionuclides making it an interesting radionuclide for stent coating to prevent restenosis.

[Microvascular denudation of the femoral artery of the mouse as a model for restenosis]. / Mikrovaskuläre Denudation der Arteria femoralis der Maus--ein Restenosemodell für die Experimentelle Radiologie.

OBJECTIVE: To present technique and results of a microvascular denudation of the common femoral artery of the mouse as a model for inducing intimal hyperplasia in interventional radiology. MATERIALS AND METHODS: Under general anesthesia introduced by intraperitoneal injection, 14 B6129F1 hybrid mice (7 females and 7 males) at a mean age of 12.1 +/- 1.8 weeks and a mean weight of 28 +/- 2.8 grams had a groin incision of the vascular bundle directly distal to the inguinal ligament in preparation of placing a vascular clamp. Thereafter, the femoral artery was dissected distal to the origin of the epigastric artery and a loop prepared for a ligation proximal to the planned arteriotomy. Through an arteriotomy performed free-hand with a pair of micro scissors, a 0.010" (= 0.25 mm) guidewire was introduced into the vessel and advanced to the aortic bifurcation. The guide-wire was moved back and forth three times. The same procedure was performed on the other side as sham-operation, i.e., without introduction and passage of a guidewire. The resulting changes of the vessel wall were evaluated by histology and morphometry. RESULTS: Four weeks after intervention, the mean intima-to-media-ratio (IMR) was 1.80 +/- 0.28. A significant difference was observed between the sexes, with an IMR of 1.41 +/- 0.29 in females and an IMR of 2.24 +/- 0.45 in males (p = 0.0173). The neointima led to an overall luminal loss of 50.2% +/- 8.3% without significant sex difference (p = 0.09), but the average luminal loss was still more severe in females, amounting to 43.9% in comparison to 56.1% in males. This technique induces a significant neointima formation in a reproducible manner. The internal elastic membrane was preserved in all vessels. CONCLUSION: This technique is an excellent model to examine the differences between genetically modified mice to clarify the role of putative key molecules in the pathophysiology of restenosis.

[Oxidation of indole and its derivatives by heme-independent chloroperoxidases]. / Okislenie indola i ego proizvodnykh gem-nezavisimymi khlorperoksidazami.

Indole, indolylacetic acid, and tryptophan were oxidized by cloroperoxidases isolated from strains of Streptomyces lividans and Pseudomonas pyrrocinia. Indigo (indoxyl), isatin, and anthranilic acid (intermediate products of oxidative degradation of indole and indole derivatives) were extracted from the reaction medium.

Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene.

For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens Tü24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tü24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%.