Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation.

PURPOSE: The authors' aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF). METHODS: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10-100 keV), an x-ray beam (100 kVp), and (125)I and (103)Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared. RESULTS: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2) using 30 nm AuNPs, would require 5.1 ± 0.2, 9 ± 1, 10 ± 1, 10 ± 1 mg Au/g tissue in the nucleus, in the cytoplasm, on the cell surface, or in the extracellular space, respectively. Using 50 nm AuNPs, the required amount decreases to 3.1 ± 0.3, 8 ± 1, 9 ± 1, 9 ± 1 mg Au/g tissue, respectively. CONCLUSIONS: NDEF is a new metric that can predict the radiation enhancement of AuNPs for various experimental conditions. Cell model, the subcellular location and size of AuNPs, and the number of AuNPs per cell, as well as the x-ray photon energy all have effects on NDEFs. Larger AuNPs in the nucleus of cluster cells exposed to x-rays of 15 or 40 keV maximize NDEFs.

Ultrasonic backscatter coefficient quantitative estimates from Chinese hamster ovary cell pellet biophantoms.

A cell pellet biophantom technique is introduced, and applied to the ultrasonic backscatter coefficient (BSC) estimate using Chinese hamster ovary (CHO) cells. Also introduced is a concentric sphere scattering model because of its geometrical similarities to cells with a nucleus. BSC comparisons were made between the concentric sphere model and other well-understood models for mathematical verification purposes. BSC estimates from CHO cell pellet biophantoms of known number density were performed with 40 and 80 MHz focused transducers (overall bandwidth: 26-105 MHz). These biophantoms were histologically processed and then evaluated for cell viability. Cell pellet BSC estimates were in agreement with the concentric sphere model. Fitting the model to the BSC data yielded quantitative values for the outer sphere and inner sphere. The radius of the cell model was 6.8 ± 0.7 µm; the impedance of the cytoplasm model was 1.63 ± 0.03 Mrayl and the impedance of the nuclear model was 1.55 ± 0.09 Mrayl. The concentric sphere model appears as a new tool for providing quantitative information on cell structures and will tend to have a fundamental role in the classification of biological tissues.

Three-dimensional reconstructive analysis of intracytoplasmic lumina found in ependymomas.

Three-dimensional reconstructive analyses revealed that the intracytoplasmic lumina found in ependymomas were actually formed by subsidence of an extracellular membrane, resembling a volcano. This finding was compatible with cytologic and electron microscopic findings. In addition, there were many tiny thorns resembling a holly leaf on the extracellular membrane, such that cilia and microvilli on the cellular membrane discontinued cell-to-cell tight junctions.

The structure of the cytoplasm of lens fibers as determined by conical tomography.

Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50-100 nm) onto a single plane (the "projection artifact"). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the "soluble" crystallins with low ionic strength buffers to make "ghosts." Tomographic series in conical geometry were collected at 55 degrees tilts and by 5 degrees rotations until completing a 360 degrees turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a "clustered matrix." The matrix was comprised of thin ( approximately 6 nm diameter) filaments bent sharply at 110-120 degrees angles and studded with cube-shaped particles (the "beaded" filaments). In cortical fibers, the particles measured a=14+/-2, b=13+/-2 and c=10+/-2.4 nm (n=30, mean+/-SD) and were spaced at distances measuring 27.5+/-2.4 nm apart (n=8, mean+/-SD), center-to-center. The matrix was formed as "beaded" filaments, bound to clusters of "soluble" proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of "beaded" filaments that organize the "soluble" crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation.

Isostructural fluorescent and radioactive probes for monitoring neural stem and progenitor cell transplants.

A construct for tagging neurospheres and monitoring cell transplantations was developed using a new technology for producing luminescent and radiolabeled probes that have identical structures. The HIV1-Tat basic domain derivatives NAcGRKKRRQRRR(SAACQ)G (SAACQ-1) and [NAcGRKKRRQRRR(Re(CO)3SAACQ)G]+ (ReSAACQ-1) were prepared in excellent yields using the single amino acid chelate-quinoline (SAACQ) ligand and its Re(I) complex and conventional automated peptide synthesis methods. The distribution of the luminescent Re probe, using epifluorescence microscopy, showed that it localized primarily in the cell nucleus with a significant degree of association on the nuclear envelope. A smaller amount was found to be dispersed in the cytoplasm. The 99m Tc analogue was then prepared in 43+/-7% (n=12) yield and very high effective specific activity. Following incubation, average uptake of the probe in neurospheres ranged between 10 and 20 Bq/cell. As determined by colorimetric assays, viability for cells labeled with high effective specific activity 99m TcSAACQ-1 was 97+/-4% at 2 h postlabeling and 85+/-25% at 24 h postlabeling for incubation activities ranging from 245 to 8900 Bq/cell. DNA analysis showed that at these levels, there was no significant difference between the extent of DNA damage in the treated cells versus control cells. A series of preliminary SPECT/CT studies of transplants in mice were performed, which showed that the strategy is convenient and feasible and that it is possible to routinely assess procedures noninvasively and determine the number of cells transplanted.

Ultrastructural localization of intraneuronal Abeta-peptide in Alzheimer disease brains.

Autopsied brain tissue from Alzheimer's disease patients and old non-demented controls was studied after immunocytochemistry with the 4G8 monoclonal antibody that recognizes amyloid-beta peptides. Intraneuronal 4G8-positive reaction product was detected in all of the studied brains. The same brain regions in the Alzheimer's disease samples consistently showed both more immunopositive neurons and more stained reaction product per neuron than those from the non-demented brains. Ultrastructurally, the immunopositive reaction product accumulated in clusters of cytoplasmic elements that had a lipofuscin-like appearance, showed a fibrogranular content and were also closely apposed to lipid droplets located either on their periphery or within them. The most strongly 4G8-immunopositive elements had diffuse limits with their fibrogranular content free in the cytoplasm, whereas elements either without or showing only light immunoreaction had a limiting membrane. All immunopositive neurons displayed a similar pattern of clumping heterochromatin. The hypothetical neurotoxic role of intraneuronal amyloid-beta peptides free in the cytoplasm is discussed as is the possible relationship between the amyloid-beta peptides recognized by the 4G8 antibody and the lipid droplets which would presumably contain esterified cholesterol.

"Indeterminate" cystic lesion of the kidney partially lined by small cells with clear cytoplasm--malignant or benign?

Complex renal cysts, which present radiographically as "indeterminate for malignancy" (Bosniak category III), can prove challenging both pathologically and clinically. We report a case of a renal cyst that, by standard radiographic and histologic criteria, should have been diagnosed as a malignant cystic renal cell carcinoma. However, the cytogenetic profile appeared more closely consistent with cystic renal adenoma or low-grade papillary renal cell carcinoma--tumors with limited metastatic potential. We postulate that other, similarly complex, renal cysts might also be more precisely defined by meticulous histopathologic examination, supported by cytogenetic study.

99mTc annexin V imaging of neonatal hypoxic brain injury.

BACKGROUND AND PURPOSE: Delayed cell loss in neonates after cerebral hypoxic-ischemic injury (HII) is believed to be a major cause of cerebral palsy. In this study, we used radiolabeled annexin V, a marker of delayed cell loss (apoptosis), to image neonatal rabbits suffering from HII. METHODS: Twenty-two neonatal New Zealand White rabbits had ligation of the right common carotid artery with reduction of inspired oxygen concentration to induce HII. Experimental animals (n=17) were exposed to hypoxia until an ipsilateral hemispheric decrease in the average diffusion coefficient occurred. After reversal of hypoxia and normalization of average diffusion coefficient values, experimental animals were injected with (99m)Tc annexin V. Radionuclide images were recorded 2 hours later. RESULTS: Experimental animals showed no MR evidence of blood-brain barrier breakdown or perfusion abnormalities after hypoxia. Annexin images demonstrated multifocal brain uptake in both hemispheres of experimental but not control animals. Histology of the brains from experimental animals demonstrated scattered pyknotic cortical and hippocampal neurons with cytoplasmic vacuolization of glial cells without evidence of apoptotic nuclei by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Double staining with markers of cell type and exogenous annexin V revealed that annexin V was localized in the cytoplasm of scattered neurons and astrocytes in experimental and, less commonly, control brains in the presence of an intact blood-brain barrier. CONCLUSIONS: Apoptosis may develop after HII even in brains that appear normal on diffusion-weighted and perfusion MR. These data suggest a role of radiolabeled annexin V screening of neonates at risk for the development of cerebral palsy.

Nuclear localization of 111In after intravenous injection of [111In-DTPA-D-Phe1]-octreotide in patients with neuroendocrine tumors.

UNLABELLED: Treatment with tumor-targeting substances is currently being evaluated in clinical trials. For patients with neuroendocrine tumors expressing somatostatin receptors, the 111In-labeled somatostatin analog [diethylenetriaminepentaacetic acid (DTPA)-DPhe1]-octreotide has been used with promising results. To further investigate the clinical effect of the injected conjugate, we analyzed the cellular distribution of 111In by ultrastructural autoradiography. METHODS: Seven patients with somatostatin receptor-expressing midgut carcinoid tumors scheduled for abdominal surgery were investigated by somatostatin receptor scintigraphy. During operation, tumor tissue samples and samples of normal intestine were collected, fixed, and processed for electron microscopy. A thin layer of film emulsion was applied on sections and after the exposure film was developed. The cellular distribution of silver precipitations indicating the presence of isotope was evaluated. RESULTS: Cell surface receptor binding and internalization of [111In-DTPA-D-Phe1]-octreotide in the tumor cells was easily revealed by silver precipitations in the film. Multiple silver grains were seen at the plasma membrane, in the cytoplasmic area among secretory granules and vesicular compartments, and in the perinuclear area. Silver grains were also regularly located in the nucleus. For all patients, the silver precipitation patterns from 111In decay were identical in all examined cells from removed tumors, and in most cells 111In could be seen in the nucleus. The specificity of the silver reaction products is supported by the observation that enterocytes in intestinal tissue specimens from near the tumor did not show any silver grains and no background labeling was seen in the plastic. CONCLUSION: After internalization through the somatostatin receptor system, 111In is translocated to the perinuclear area and into the nucleus. Whether the nuclide is still conjugated to the intact somatostatin analog or to part of it cannot be evaluated in this study. Despite the short irradiation range of 111In, the nuclear localization can explain its clinical effectiveness. The results from this study suggest that [111In-DTPA-D-Phe1]-octreotide may act as a powerful tumor cell-targeting substance.

Structural peculiarities of the tissue formed in modelling of the articular surface.

A model of articular cavity with smooth cover was produced by influence of shape-forming element on regenerating bone tissue. Morphological, ultrastructural, histochemical, and biochemical peculiarities of the tissue of the formed articular surface are described.

Nickel binding and uptake in thymocytes and peripheral blood lymphocytes of nickel-allergic and control subjects.

In the present investigation, the binding and uptake of 63NiCl2 was studied in human thymocytes and in peripheral blood lymphocytes from nickel-allergic and control subjects. A direct binding of the isotope to the lymphocyte membranes was shown by autoradiography. This binding was not more marked in peripheral blood lymphocytes of nickel-allergic subjects than in those of control subjects or in thymocytes. As judged by liquid scintillation, there was an uptake of isotope both into the nucleus and cytoplasm of the cells, being low compared to the medium activity. The uptake into blood lymphocyte nuclei of nickel-allergic subjects was significantly (p less than 0.05) higher than into those of the control group. The present findings indicate a direct action of nickel on lymphocytes, which might be of importance for DNA synthesis stimulation.

[Radioactive labeling of cytoplasmic hormone receptors. Determination and clinical significance of estrogen and progesterone receptors]. / Radioaktive Markierung des zytoplasmatischen Hormonrezeptors. Bestimmung und klinische Bedeutung der Ostrogen- und Progesteron-Rezeptoren.

Neither autoradiography nor in vitro labeling of thin sections of tumor tissue can be used for the quantitative determination of radioactive labeled cytoplasmic hormone receptor. The acknowledged method for his purpose is the DCC (Dextran Coated Charcoal) method. Hormone receptor estimations are routinely used to predict those patients with hormone-dependent tumors who will respond to endocrine therapy. Some 80% of patients with positive estrogen and progesterone receptor show objective remissions after endocrine therapy. Receptor determinations are carried out routinely in patients with mammary, uterine or ovarian carcinoma to identify those subjects who would benefit most from endocrine therapy.