Near monochromatic X-rays for digital slot-scan mammography: initial findings.

X-ray spectra are composed of a broad bremsspectrum and anode-characteristic emission lines. In mammography typically molybdenum (Mo), rhodium (Rh) or tungsten (W) anodes are used in combination with Mo, Rh or aluminium filters. Only the photons with energies between 17 and 22 keV of the resulting spectrum are suitable for the soft tissue imaging needed for mammography. The aim of this article is to present first results obtained with a monochromator module mounted at the exit of the X-ray tube of a conventional clinical mammography unit. The experimental setup consists of a Siemens Mammomat 300, an X-ray monochromator module and a linear array detector for image acquisition. The technique is similar to the slot-scan technique known from digital mammography. The experimental machine allows to obtain images both with polychromatic and monochromatic X-rays. Initial evaluation of the system was performed by examination of a contrast-detail phantom (CD-MAM-phantom, Nijmegen, The Netherlands). Images done with the new monochromatic technique were compared to images of the phantom done with polychromatic spectra, with film-screen mammography as well as with digital mammography. The new technique with monochromatic slot-scan mammography resulted in correct identification of 93% of the phantom. Digital slot-scan mammography with polychromatic beam resulted in correct identification of 87%, digital full-field mammography in 83% and conventional film-screen mammography in 70% of the phantom. The results suggest that monochromatization has a potential for improving image quality or decreasing dose in X-ray mammography.

Gadolinium neutron capture therapy (GdNCT) of melanoma cells and solid tumors with the magnetic resonance imaging contrast agent Gadobutrol.

RATIONALE AND OBJECTIVES: The therapeutic gain of neutron capture therapy with a neutral macrocyclic gadolinium (Gd) complex (Gadobutrol) was evaluated through in vitro and in vivo studies in a beam of low-energy neutrons. METHODS: Neutron irradiation for both the in vitro and in vivo studies was performed in a beam of low-energy neutrons produced by the research reactor of the Hahn-Meitner-Institut, Berlin. Malignant melanoma cells of human origin were irradiated in the presence or absence of Gadobutrol. In vivo irradiation was performed on tumor-bearing nude mice. The tumor site was irradiated subsequent to intratumoral injection of Gadobutrol and compared with irradiation in the absence of the Gd complex. RESULTS: In vitro studies showed a Gd-dependent delay of cell proliferation as a consequence of neutron irradiation. In animals, intratumoral administration of the Gd complex at a dose of 1.2 mmol Gd/kg before neutron irradiation results in a significant delay in tumor growth with respect to the control groups. CONCLUSIONS: In vitro and in vivo studies showed a therapeutic benefit with the neutral Gd complex and suggest Gd-containing magnetic resonance contrast media are potential candidates for neutron capture therapy. The Gd dose used in the irradiation experiments was four times the presently accepted high dose in clinical magnetic resonance imaging.

Targeting of ultrasmall superparamagnetic iron oxide (USPIO) particles to tumor cells in vivo by using transferrin receptor pathways.

Human transferrin was covalently coupled to ultrasmall superparamagnetic iron oxide (USPIO) particles, and the transferrin-USPIO obtained was investigated in vivo in experimental SMT/2A tumor-bearing rats (rat mammary carcinoma). Physicochemical characterization showed an overall size of 36 nm (DLS) with a core size of 5 nm (TEM). Relaxivities were R1 = 23.6 and R2 = 52.1 liter/mmol.s (0.47 T). Bound transferrin was 280 micrograms/mg of iron. Pharmacokinetic investigations revealed a half-life of 17 min in normal rats. The MR evaluation of tumor signal intensity over time showed a 40% (range 25-55%) signal reduction 150 min after injection with the reduction persisting for at least 8 h. Control experiments using the parent USPIO compound or USPIO labeled with a nonspecific human serum albumin (HSA-USPIO) showed a change of only 10% (range 5-15%) in tumor signal intensity over time. The results demonstrate that a combination of the USPIO relaxivity properties with the specificity of transferrin-mediated endocytosis allows in vivo detection of tumors by MR imaging.

Magnetic iron oxide particles coated with carboxydextran for parenteral administration and liver contrasting. Pre-clinical profile of SH U555A.

RATIONALE AND OBJECTIVES: To evaluate the physical and pharmacological profiles of SH U555A, a suspension of magnetic iron oxide particles that is designed to enhance the visualization of liver tumors and metastases. MATERIAL AND METHODS: Chemical and physical methods were used to characterize the size and structure of these magnetic iron oxide particles in aqueous solution. The biodistribution and pharmacokinetics of the particles were studied in mice, rats and dogs. The imaging efficacy of the particles was demonstrated by MR imaging in rat liver tumors RESULTS: The SH U555A particles consist of low-molecular-weight carboxydextran-coated iron oxides predominantly of the gamma-Fe2O3 form with a hydrodynamic diameter ranging from 57-59 nm and strong T2 relaxivity of 164 liters x mmol(-1) x s(-1) (water, 0.47 T). In rats the particles exhibited a dose-dependent half-life of between 2 and 3 days in the liver at a dose of 20 micromol Fe/kg and a shorter half-life at lower doses. No major side effects were found. In a rat tumor model the tumor-to-liver contrast was markedly improved after i.v. administration of SH U555A. At a dose of 14 micromol Fe/kg the half-maximal contrast-effect was obtained even in nonoptimized T1-weighted spin-echo images. CONCLUSION: SH U555A is a superparamagnetic MR contrast agent for i.v. administration and has substantial potential for the demarcation of liver tumors.

Fluorescence energy transfer on erythrocyte membranes.

Stationary and time-dependent fluorescence were measured for a donor/ acceptor (DA) pair bound to membrane proteins of bovine erythrocyte ghosts. The donor N-(p-(2-benzoxazolyl)phenyl)-maleimide (BMI) and the acceptor fluram bind to SH- and NH2-residues, respectively. The fluorescence spectra and the time-dependent emission were consistent with radiationless fluorescence energy transfer (RET). Band3 protein is the only membrane spanning protein with accessible SH-groups for the coupling of BMI molecules, and therefore only acceptor binding sites on the same band3 protein were counted by the RET measurements performed. A density of RET-effective acceptor binding sites c = 0.072 nm-2 was calculated on the basis of the two-dimensional Förster-kinetics.

Neutron capture therapy with gadopentetate dimeglumine: experiments on tumor-bearing rats.

RATIONALE AND OBJECTIVES: The therapeutic effect of neutron capture therapy with the gadolinium (Gd) complex gadopentetate dimeglumine was studied in vivo in rats using a beam of thermal neutrons. METHODS: Rats with Jensen sarcomas 10-15 mm in diameter in their right thigh were irradiated with a thermal neutron beam that had fluences of 3.6 x 10(11) (20 min) or 5.4 x 10(11) n/cm2 (30 min) in the absence and presence of 5,500 or 13,750 ppm gadopentetate dimeglumine. Gadopentetate dimeglumine was administered directly into the tumor prior to neutron irradiation. Four groups of rats were studied: two groups of nonirradiated controls (Gd-n- and Gd+n-) and two irradiated groups (Gd-n+ and Gd+n+). In the follow-up period, we measured the subjects' clinical status and tumor size as a function of time postirradiation. RESULTS: In both control groups (Gd-n- and Gd+n-), the tumor progressively grew. Pure irradiation by thermal neutrons in the Gd-n+ group resulted in a transient inhibition of tumor growth with total regressions of 15%. Intratumoral administration of 13,750 ppm gadolinium per gram of tumor and subsequent neutron irradiation (the Gd+n+ group; fluence = 3.6.10(11) n/cm2) significantly increased the tumoricidal effects (i.e., decrease of tumor growth up to a complete regression of the tumors in about 80%). Treatment-specific differences between the groups were confirmed by histologic observations. CONCLUSION: The intratumoral administration of the hydrophilic magnetic resonance imaging contrast medium gadopentetate dimeglumine prior to irradiation with thermal neutrons leads to a therapeutic gain (i.e., reduction) on experimental Jensen sarcomas.

Investigation of mechanisms influencing the accumulation of ultrasmall superparamagnetic iron oxide particles in lymph nodes.

RATIONALE AND OBJECTIVES: The current study was designed to investigate the lymph node accumulation mechanisms of dextran-coated ultrasmall superparamagnetic iron oxide particles (USPIO) in rats. METHODS: The iron deposition in the lymph nodes after intravenous administration of USPIO at a dose of 200 mumol Fe/kg was measured in vitro by inductively coupled plasma atomic emission spectrometer in control rats, in rats after depletion of complement C3, after induction of antidextran antibodies, and with prior ligation of afferent lymphatic vessels. The results were correlated with baseline iron concentration and histology. RESULTS: A significant increase in iron concentration but unequal distribution between central and peripheral nodes occurred after administration of USPIO in rats. Much less accumulation was observed in guinea pigs. In rats, the nodal uptake of USPIO was not impaired by depletion of complement C3 using cobra venom factor. The central lymph nodes (mesenteric nodes) showed significantly more accumulation of iron particles in the presence of antidextran antibodies induced by dextran preimmunization. Afferent lymphatic vessel ligation did not effect iron particle accumulation. CONCLUSIONS: Accumulation of USPIO in lymph nodes is largely species-dependent but is independent of afferent lymphatic flow and of C3 complement opsonization in plasma. However, regional distribution of particles can be influenced by preimmunization using dextran.

Contrast-enhanced MR imaging of liver and spleen: first experience in humans with a new superparamagnetic iron oxide.

The aim of this prospective study was to obtain the first human safety and magnetic resonance (MR) imaging results with a new formulation of superparamagnetic iron oxide (SPIO) (SHU 555 A). The SPIO was tested at four iron doses, from 5 to 40 mumol/kg. Laboratory tests and clinical measurements were done in 32 healthy volunteers for up to 3 weeks after administration. MR imaging at 1.5 T was performed before and 8 hours to 14 days after fast intravenous injection (500 mumol Fe/min) of the SPIO (six subjects per dose). Results of this phase I study demonstrate that SHU 555 A at a concentration of 0.5 mol Fe/L was well tolerated. A dose-dependent minor increase in activated partial thromboplastin time, which remained within the normal range, was seen. All doses of SPIO caused a signal loss in both liver and spleen (P < .05) with a spin-echo sequence (TR = 2,300 msec, TE = 45 msec). The signal losses in the liver 8 hours after contrast agent injection were 58%, 79%, 82%, and 87% for the 5, 10, 20, and 40 mumol Fe/kg doses, respectively. The corresponding signal losses in the spleen were 23%, 45%, 65%, and 78%, respectively. The doses that reduced signal intensity by half were 3.1 mumol Fe/kg for the liver and 12.8 mumol Fe/kg for the spleen. The results suggest that the new SPIO formulation is a safe and efficient MR contrast agent.

MR lymphography using iron oxide particles. Detection of lymph node metastases in the VX2 rabbit tumor model.

MR images of the iliac lymph nodes of 25 VX2 carcinoma-bearing rabbits and of 5 tumor-free rabbits were obtained at 1.5 T before and after endolymphatic administration of superparamagnetic iron oxide particles (SPIO) at a dose of 1 mumol Fe per extremity. Imaging results were correlated with histology. In unenhanced images intranodal metastases were not detectable with any of the pulse sequences used and the signal intensities of tumor-free and metastatic lymph nodes did not differ significantly. After administration of the contrast medium, a significant signal loss (p < or = 0.05) occurred in the healthy lymph node tissue, whereas the signal intensity of lymph node metastases remained unchanged. In SPIO enhanced images, the threshold size for detection of lymph node metastases was 2 mm. Metastatic involvement was detected in 28 of the 30 tumorous lymph nodes with the SE 2000/15 sequence but in a smaller number of lymph nodes with the sequences SE 500/22 (n = 27) and 2000/65 (n = 21).

[Magnet resonance spectroscopy of tumor-bearing rat livers: magnetite particles as an aid in volume selection]. / Magnetresonanzspektroskopie der tumortragenden Rattenleber: Magnetitpartikel als Hilfe bei der Volumenselektion.

Magnetite, an RES-specific contrast medium, has been used in animal experiments and in a few patients for MRI of the liver. We examined the use of magnetite particles for 31P-MR spectroscopy using a phantom, and perfused tumour-bearing rat livers and liver tumours in living rats. As expected, there is homogeneous uptake of the magnetite in normal liver leading to extinction of the signal when one uses suitable spectroscopic parameters. Since the ferrite particles do not penetrate non-hepatic tissue, such as metastases, a signal remains uniquely from the tumour and this can be used, for instance, for following the effect of cytostatic therapy. The use of magnetite produces a selective effect and interference from normal liver is thereby avoided. Multiple lesions with irregular configuration can be examined simultaneously by this method. It remains to be seen how useful the application of magnetite will be for avoiding motion artifacts during spectroscopy.

Pathways of water through erythrocyte membranes. Routes along defect structures.

A new model for the passive (diffusional) permeation of water and for the transfer of protons across membranes from mammalian erythrocytes is presented. The proton transfer is anion-coupled through the action of the band3 protein. Inhibition studies reveal that the pathways for the anion- and water-exchange are independent. Dynamic grain boundaries and defect structures in thermodynamic equilibrium (steady state in the general case) with the membrane are considered to be the major routes for the water permeation. Modulators of membrane functions may either act only locally or may lead to a reordering of the membrane. The specific inhibitor of the anion-exchange, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), is considered to induce only local effects by closing the anion-transfer gate. The more unspecific binding of the mercurial p-chloromercuribenzenesulfonic acid (pCMBS) should change the membrane aggregational states. Thus pCMBS can reduce the water permeation and at the same time stimulate the passive proton translocation as is experimentally observed.

First direct observation of the water exchange across the membrane of a single-cell green alga on a cellular level.

The isotopic water exchange across the membrane of a single-cell alga is made visible by optical differences of H2O and 2H2O. In the near infrared (NIR) (1000 to 2500 nm) H2O shows pronounced absorption bands while 2H2O is almost transparent. Results from in vivo experiments on the diffusive water permeation across the membrane of the spherical freshwater alga Eremosphaera viridis are presented. The evaluation of the isotope-exchange kinetics allows the calculation of the permeability coefficient, Pd, and the approximation of the intracellular diffusion constant, D. The extension of H2O/2H2O-exchange measurements to two dimensions opens new ways to study transport pathways up to the spatial resolution of a microscope. First NIR video images demonstrate the capability of the method.

Effect of preparative procedures on ghostcells from bovine erythrocytes.

Several methods for the preparation of "white ghosts" or "resealed ghosts" were described in the recent literature. This article compares three methods to prepare white, resealed ghosts from bovine erythrocytes based on the principle of hypotonic lysis. The methods described differ by the removal of hemoglobin from the empty cells. The main difference between the standard centrifugation, the gelfiltration and the hollow-fibre diafiltration is the mechanical stress on the leaky membranes after swelling in hypotonic media. Mean cellular volumes, rates of potassium-efflux and the access of impermeable dyes to cytoplasmatic proteins are criteria to differentiate between ghostcell-populations.

Fusion of negatively charged liposomes with clathrin-uncoated vesicles.

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles.

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.

On the water and proton permeabilities across membranes from erythrocyte ghosts.

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.

Isotopic light scattering of lipid vesicles. Water permeation and effect of alpha-tocopherol.

The influence of the index of refraction of the solvent on light scattering properties of lecithin bilayer vesicles is described. Large vesicles (diameter 300 nm) are considered where one lamella separates the intravesicular compartment from the external medium. Stationary and transient cases are distinguished with special emphasis on the isotopic substitution of the solvent, i.e. H2O vs. D2O. Theoretical calculations based on the Mie theory of light-scattering are in accord with results from experiments. The two stationary cases considered serve to calibrate the numerical calculations and illustrate the capability of the method. Transient experiments allow the determination of permeation rates; in particular the D2O/H2O permeability coefficients can be obtained. Single component vesicular lecithin bilayers and ones containing tocopherol are compared. In the crystalline state the incorporation of tocopherol increases the fluidity of the lipid bilayer in parallel with the water permeation rate.

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering.

A light-scattering technique used to measure the water permeability across closed biomembranes is described, which is based on the different indices of refraction of D2O and H2O. This transient technique is compared with a similar method using D2O-sensitive fluorophores in the intravesicular space. The results of both techniques are equivalent although the signal-to-noise ratio favors the light-scattering or turbidity experiment. The light-scattering method is only applicable to larger particles (no point-scatterers) and is easily extended to biological objects. Data on the H2O/D2O exchange across membranes of ghosts from human erythrocytes suggest two mechanisms: the D2O and H2O permeation through the membrane and a slower D2O-induced conformational change of membraneous proteins.

On the exchange of H2O/D2O molecules across membranes of erythrocyte ghosts from patients with Huntington's disease and from normal individuals.

A recently developed technique is used to study the permeation of water through membranes of human erythrocyte ghosts. The method, based on the difference of the indices of refraction of H2O and D2O, is briefly described. Comparative measurements on erythrocyte ghosts from normal donors and from patients with Huntington's disease were performed. The calculated permeability coefficients for the two groups were almost identical and did not allow differentiation between the controls and patients with Huntington's disease.

Fluorescence of intramolecular and intermolecular interactions of aminonaphthyl-sulfonate with nucleotides.

Fluorescence studies of the intramolecular and intermolecular interactions between aminonaphthylsulfonate and nucleotides of uracil or adenine are described. The fluorescence originates solely from the naphthyl moiety and is intramolecularly quenched by the base, uracil being more effective than adenine. The enzymatic splitting of the molecule into a nucleoside monophosphate and the pyrophosphate product of the aminonaphthylsulfonate removes the intramolecular quenching and, especially in the case of uracil, a drastic increase of the fluorescence intensity results. The intact molecule exists predominantly in the folded form except in cases where electrostatic repulsion exceeds the stacking attraction. This is borne out by the pH dependence and the existence of a pronounced solvent-isotope effect of the fluorescence quantum yield for the uracil derivative at basic pH. At pH values above the pK of the enol proton of the uracil base the fluorescent properties of the intact and phosphodiesterase-digested molecules are very similar. The intermolecular interactions between 1-aminonaphthalene-5-sulfonate with AMP and UMP can be explained on the basis of dynamic quenching (collisional quenching) without any significant participation of ground-state complexes (static quenching). The interaction of the pyrophosphate adduct of 1-aminonaphthalene-5-sulfonate with UMP can best be explained by invoking two interacting nucleotide species: the free nucleotide and a sodium-nucleotide complex.