Gadolinium in human glioblastoma cells for gadolinium neutron capture therapy.

157Gd is a potential agent for neutron capture cancer therapy (GdNCT). We directly observed the microdistribution of Gd in cultured human glioblastoma cells exposed to Gd-diethylenetriaminepentaacetic acid (Gd-DTPA). We demonstrated, with three independent techniques, that Gd-DTPA penetrates the plasma membrane, and we observed no deleterious effect on cell survival. A systematic microchemical analysis revealed a higher Gd accumulation in cell nuclei compared with cytoplasm. This is significant for prospective GdNCT because the proximity of Gd to DNA increases the cell-killing potential of the short-range, high-energy electrons emitted during the neutron capture reaction. We also exposed Gd-containing cells to thermal neutrons and demonstrated the GdNC reaction effectiveness in inducing cell death. These results in vitro stimulated in vivo Gd-DTPA uptake studies, currently underway, in human glioblastoma patients.

Formation of sphalerite (ZnS) deposits in natural biofilms of sulfate-reducing bacteria.

Abundant, micrometer-scale, spherical aggregates of 2- to 5-nanometer-diameter sphalerite (ZnS) particles formed within natural biofilms dominated by relatively aerotolerant sulfate-reducing bacteria of the family Desulfobacteriaceae. The biofilm zinc concentration is about 10(6) times that of associated groundwater (0.09 to 1.1 parts per million zinc). Sphalerite also concentrates arsenic (0.01 weight %) and selenium (0.004 weight %). The almost monomineralic product results from buffering of sulfide concentrations at low values by sphalerite precipitation. These results show how microbes control metal concentrations in groundwater- and wetland-based remediation systems and suggest biological routes for formation of some low-temperature ZnS deposits.

Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH.

Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

UV-ozone ashing of cells and tissues for spatially resolved trace element analysis.

UV/ozone ashing of thin tissue sections and cell cultures is a simple technique to enhance relative elemental concentrations, while maintaining their spatial location at the sub-micron level. This approach may enhance the capability of spatially resolved analysis techniques to detect the distribution of trace elements in biological matrices. We present results from light microscopy and x-ray spectromicroscopy studies of tissues and cells demonstrating that the micro-structure is very well conserved. We show the signal enhancement resulting from the removal of carbon, which allows otherwise undetectable gadolinium to be mapped in cancer tissue for a novel neutron capture therapy.

Charging phenomena in PEEM imaging and spectroscopy.

Spectromicroscopy with the imaging technique of X-ray photoelectron emission microscopy (X-PEEM) is a microchemical analytical tool installed in many synchrotron radiation laboratories, and which is finding application in diverse fields of research. The method of sample analysis, X-ray absorption spectroscopy, does not encounter the same problems as X-ray photoemission spectroscopy when sample charging occurs, hence even good insulators may often be analyzed without any apparent artifacts in images or spectra. We show, however, that charging effects cannot be neglected. We model the effect of surface charge formation on the secondary electron yield from uniform samples to demonstrate that surface charge primarily reduces the yield of electrons which may contribute to the detected signal. We illustrate that on non-uniform insulating samples, localized centers of charge may substantially affect microscope imaging and resolution as the electrostatic field close to the surface is distorted. Finally, in certain circumstances non-uniform surface charge may lead to unexpected lineshapes in X-ray absorption spectra causing, in some extreme cases, negative spectra. These negative spectra are explained, and several strategies are reviewed to minimize the impact of sample charging when analyzing poorly conducting samples of any nature.

Cell ashing for trace element analysis: A new approach based on ultraviolet/ozone.

We studied a new approach to cell ashing based on illuminating the specimens with a low-pressure mercury discharge lamp. We analyzed with synchrotron spectromicroscopy its effects on different physiological elements in neurobiological specimens. Our results demonstrate that carbon is removed, whereas phosphorus, calcium, potassium, and sulfur are retained and their relative concentrations are enhanced. Applied to trace elements, this technique will enhance their practical detectability.

High sensitivity quantitative analysis of cobalt uptake in rat cerebellar granule cells with and without excitatory amino acids.

We quantified the effect of the excitatory amino acids kainate and glutamate on the uptake of cobalt in primary rat cerebellar granule neurons, by using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). We quantitatively demonstrated that Co2+ uptake, although enhanced by glutamate and kainate also takes place in the absence of excitatory amino acids. We also found that cobalt uptake is not significantly altered by the presence of glutamate receptor competitive or noncompetitive antagonists, indicating that cobalt uptake in granule neurons does not require glutamate receptor stimulation. Our results suggest, therefore, that Co2+ may enter the cell by passive diffusion through the plasma membrane.

The effect of ashing on cells: spectromicroscopy of physiological elements.

We analyzed the effects of cold oxygen plasma ashing of neurobiological specimens on different elements with synchrotron spectromicroscopy. Our results demonstrate that while carbon is almost completely removed, phosphorus, calcium, potassium, sulfur, and, to some extent, nitrogen are retained and their relative concentration is enhanced.

Neurone decapping characterization by atomic force microscopy: a topological systematic analysis.

We tested a new approach to cell decapping on rat cerebellar neurones, and observed its effects on cell topography by atomic force microscopy (AFM). The results clearly demonstrate the effectiveness of our decapping approach, and also the ability of AFM to reveal fine details of the decapped cells. Specifically, varying the conditions and duration of the decapping process modifies the extent of the decapping. Such a method can be used to investigate the cytoplasm with surface sensitive techniques.

Atomic Force microscopy of neuron networks.

We imaged uncoated neuron networks by an atomic force microscope in the repulsive regime of contact mode. Images of granule cells and their axons have been clearly revealed with details smaller than 20 nm. The good stability of the sample and the mechanical reproducibility of the microscope allowed the imaging of a neuron culture area of several square microns. By combining tens of images, we were able to reconstruct a highly defined neuronal network. Furthermore, the images were very reproducible over repeated scanning acquisition, demonstrating the mechanical and thermal stability of the instrument-sample system.

Application of photoelectron spectromicroscopy to a systematic study of toxic and natural elements in neurons.

A systematic photoelectron spectromicroscopy study is presented of the spatial distribution of a toxic element, aluminium, iron or chromium, in neuron cultures, after exposure to a solution of the element. The study was performed by the X-ray secondary-emission microscopy (XSEM) version of photoelectron Spectromicroscopy. The distribution of the elements was investigated with two complementary approaches: digital subtraction imaging and individual X-ray absorption spectra from microscopic areas. The results coherently indicate different localization patterns for different elements, and, in particular, extreme localization of aluminium to a few rare cells identifiable as Purkinje neurons. In the case of iron-exposed specimens, the distribution analysis was extended to naturally present phosphorus, and used to estimate the XSEM sensitivity.

Aluminium in rat cerebellar primary cultures: glial cells and GABAergic neurones.

Experimental evidence of the preferential uptake of aluminium by GABAergic neurones and glial cells was provided by synchrotron spectromicroscopy studies. We observed rat cerebellar cultures enriched for GABAergic neurones or glial cells exposed to aluminium ions, detecting the presence and identifying the chemical status of aluminium on cell structures.

A multicentric seroepidemiological survey of HTLV-I/II in Italy.

BACKGROUND: Several studies carried out in the USA and in Europe have shown the presence of HTLV-I/II antibodies in subjects belonging to high-risk groups for HIV infection as well as blood donors. Concern about the presence of HTLV-I/II markers in the normal population, as well as the efficient transmission of HTLV-I/II by whole blood or infected blood cells have led several countries to include screening for anti-HTLV-I/II among the mandatory serological testing of blood donors. OBJECTIVE: In order to assess the risk of HTLV-I/II infection related to blood transfusions, a multicentric survey for antibodies against HTLV-I and HTLV-II was carried out involving 10 Italian sites during the spring of 1991. STUDY DESIGN: Serum specimens were collected from 14,598 blood donors, 1,411 injecting drug users, 1,015 thalassemics, 142 hemophiliacs and 138 hemodialysis patients. HTLV antibodies were detected by a screening EIA which combines a viral lysate with a recombinant HTLV-I env protein (p21e). The serological confirmation was performed by a semi-automated dot-blot immunoassay that detects gag p19 and p24 and env p21e specific antibodies, while the discrimination of HTLV-I and HTLV-II reactivities was carried out by EIAs employing synthetic peptides of the ENV region specific for each virus. RESULTS: The seroprevalence of confirmed positives was 0.034% among blood donors and 3.61% among IDUs, while no sample of the other categories could be confirmed, although several were indeterminate and one thalassemic reacted against HTLV-I on peptide testing. HTLV-I reactivity was observed in one blood donor, while all 38 of the 51 confirmed seropositive IDU's reacted only to the HTLV-II synthetic peptide. CONCLUSIONS: These data confirm a high prevalence of HTLV-II among Italian IDUs and show an HTLV-I/II seroprevalence among blood donors very similar to that which was found in the USA volunteer blood donors. A surveillance program among blood donors seems advisable in order to establish the possible need of a mandatory screening for HTLV-I/II.

Fibrinogen plasma levels as a marker of thrombin activation in diabetes.

This study attempted to verify the existence of a correlation between fibrinogen, a major cardiovascular risk factor in diabetes, and indexes of thrombin generation and action, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer (D-D), in a group of diabetic subjects compared with a matched control group. Forty insulin-dependent diabetes mellitus patients and 30 matched healthy control subjects participated in this study. The subjects were tested for the following parameters: fibrinogen, prothrombin F1 + 2, D-D, fasting glycemia, and HbA1c. In addition, 5 diabetic subjects who maintained stable fibrinogen plasma levels > 300 mg/dl for at least 6 months before the study were treated with 12,500 U/day subcutaneous heparin for 7 days. Diabetic subjects showed increased levels of fibrinogen, prothrombin F1 + 2, and D-D plasma levels. Simple linear regression analysis detected a positive correlation between fibrinogen and prothrombin F1 + 2, D-D, and glycosylated HbA1c. In the five diabetic subjects treated with heparin fibrinogen, prothrombin F1 + 2 and D-D levels decreased at the end of the treatment. All these parameters returned to baseline after 7 days of washout. These data indicate that fibrinogen plasma levels are correlated to parameters of thrombin activation in plasma in diabetic patients and suggest that high fibrinogen plasma levels might be a risk marker for cardiovascular disease in diabetes because it is an expression of an existing thrombophilia.

Aluminium in rat cerebellar neural cultures.

A systematic microchemical analysis of unstained and uncoated neurone cultures was performed with synchrotron radiation photoemission spectromicroscopy after exposure to an aluminium solution. Clear evidence was found for localized aluminium uptake in a few cells. Their possible identification based on morphology is discussed.

Time-resolved fluorescence of S-100a protein in the absence and presence of calcium and phospholipids.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.