Monte Carlo study of a highly efficient gas ionization detector for megavoltage imaging and image-guided radiotherapy.

The imaging characteristics of an arc-shaped xenon gas ionization chamber for the purpose of megavoltage CT imaging were investigated. The detector consists of several hundred 320 microm thick gas cavities separated by thin tungsten plates of the same thickness. Dose response, efficiency and resolution parameters were calculated using Monte Carlo simulations. The calculations were compared to measurements taken in a 4 MV photon beam, assuming that the measured signal in the chambers corresponds to the therein absorbed dose. The measured response profiles for narrow and broad incident photon beams could be well reproduced with the Monte Carlo calculations. They show, that the quantum efficiency is 29.2% and the detective quantum efficiency at zero frequency DQE(0) is 20.4% for the detector arc placed in focus with the photon source. For a detector placed out of focus, these numbers even increase. The efficiency of this kind of radiation detector for megavoltage radiation therefore surpasses the reported efficiency of existing detector technologies. The resolution of the detector is quantified with calculated and measured line spread functions. The corresponding modulation transfer functions were determined for different thicknesses of the tungsten plates. They show that the resolution is only slightly dependent on the plate thickness but is predominantly determined by the cell size of the detector. The optimal plate thickness is determined by a tradeoff between quantum efficiency, total signal generation and resolution. Thicker plates are more efficient but the total signal and the resolution decrease with plate thickness. In conclusion, a gas ionization chamber of the described type is a highly efficient megavoltage radiation detector, allowing to obtain CT images with very little dose for a sufficient image quality for anatomy verification. This kind of detector might serve as a model for a future generation of highly efficient radiation detectors.

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells.

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

Calibration of a tomotherapeutic MVCT system.

Megavoltage CT provides the ability to image the patient before, during or after a radiotherapy treatment. This allows one to verify not only the placement of a patient's external boundary, but also the locations of internal anatomy. In addition, the reconstructed MVCT values are potentially useful for treatment planning inhomogeneity corrections and dose reconstruction. To this end, dosimetric calibration of the University of Wisconsin Tomotherapy Benchtop MVCT system was investigated. It was found that MVCT values correlate extremely well with electron density and that unlike kilovoltage CT, this correlation is well maintained for higher atomic number materials. Improvements of the order of 1% in the dosimetric calculations of high atomic number materials should be possible by deriving input images from MVCT as opposed to kVCT, and calibrating in terms of electron density, as opposed to physical density.

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Differential regulation of translation and eIF4E phosphorylation during human thymocyte maturation.

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+ CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+ CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.

A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction.

A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.

Induction of the autoantigen proliferating cell nuclear antigen in T lymphocytes by a mycobacterial antigen.

Mycobacteria have been implicated in the pathogenesis of autoimmunity. To determine the potential effect of mycobacterial antigens on peripheral blood mononuclear cells (PBMC), we analyzed PBMC incubated with the acetone-precipitable fraction of Mycobacterium tuberculosis (APMT) for changes in cellular protein expression. Two-dimensional gel analysis showed induction of a 36-kD polypeptide identified as proliferating cell nuclear antigen (PCNA), a known autoantigen, after incubation with AP-MT. PCNA plays a role in cell proliferation and is expressed as a late growth regulated factor. However, its synthesis in response to AP-MT was induced as an early event. The early induction of PCNA was regulated at a posttranscriptional level and was restricted to T cells. Treatment of PBMC with known T cell mitogens, namely PHA, anti-CD3 antibodies, and staphylococcal superantigens failed to induce an early PCNA increase. The distinct characteristics of the AP-MT effect on PCNA expression suggest a separate mechanism of induction in response to AP-MT, compared with the late increase observed in response to mitogens. The induction of PCNA in response to mycobacterial antigens may represent a pathogenically relevant mechanism in autoimmunity.

Effects of hydrogen chloride on respiratory response and pulmonary function of the baboon.

The effects of hydrogen chloride (HCI) inhalation on respiratory response during exposure and on pulmonary function during the 3 mo following exposure were studied in the baboon. Each of 4 groups of three anesthetized animals was exposed in a head-only mode for 15 min to air or one of three HCI concentrations (500, 5000, or 10,000 ppm). The acute respiratory response consisted of a concentration-related increase in frequency and minute volume, with a marked decrease in blood PaO2 at the two highest concentrations. The exposures did not cause significant alterations in any of the pulmonary function parameters measured at 3 d and 3 mo postexposure. Thus, nonhuman primates were able to survive short exposures to high concentrations of HCI without any significant effects on pulmonary function during the 3 mo after exposure. Furthermore, comparison of the response of primates and rodents suggests that the human is much less sensitive to the effects of HCI than the mouse.

Male reproductive assessment of the rubber accelerator N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide in rats.

The rubber accelerator N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide (OTOS) was evaluated to determine its potential to cause reproductive effects. No evidence of a compound-related effect on mating, fertility, gestation length, number of implants or live births, pup growth, and survival was observed using Sprague-Dawley rats. Furthermore, light and electron microscopy of the testes from the high-dose males failed to reveal any morphological changes compared to the controls. Groups of 12 male Sprague-Dawley rats were continuously administered diets containing 0, 60, 200, or 600 ppm OTOS for 12 weeks. Following 56 days of exposure, the males were subsequently cohoused nightly with two females for a maximum of 21 days. During this mating period, males continued to receive control and OTOS-containing diets; however, feeders were removed for nightly cohabitation. Although a 4 to 8% reduction in body weight was observed in the 600 ppm animals, statistical significance was reached only at the end of the first week of treatment. In the 60 and 200 ppm males body weights generally were slightly elevated compared to the control, with the 60 ppm body weights showing statistically significant differences during Weeks 5 to 7 of exposure.

The effects of long-term dietary administration of the rubber accelerator, N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide, to rats.

Continuous administration of diets containing 0, 20, 60, 200, or 600 ppm of N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide to groups of male and female Sprague-Dawley rats (60/sex/group) for over 2 years resulted in oncogenic and toxic effects which generally were limited to the high-dose (600 ppm) group. The most significant findings of this study were in the urinary system of the high-dose males and females. Postmortem examinations revealed a compound-related increase in both benign and malignant urothelial tumors in these animals but not in animals at lower doses. These observations in the high-dose animals paralleled an increase in kidney weights and non-neoplastic urinary tract abnormalities. In both sexes of the high-dose group, body weights were significantly lower and the incidence of rales was significantly higher than those of control animals throughout the course of the study. While some statistically significant changes in hematology, chemical chemistry, and urinalysis values were noted, no compound-related effect on these parameters was evident.

Mutagenic evaluations of four rubber accelerators in a battery of in vitro mutagenic assays.

The mutagenic/carcinogenic potential of four commercial accelerators were evaluated using a battery of in vitro assays. All of these compounds were mutagenic in one or more assays. Positive responses were noted in the Escherichia coli pol A+/pol A- DNA repair, mouse lymphoma L5178Y TK+/- forward mutation, BALB/3T3 cell transformation, and CHO cell chromosome aberration assays. In contrast to previous studies of accelerators, no mutagenic response was observed in the E coli WP2 uvrA- assay or in any of the Salmonella typhimurium strains tested. These studies have indicated that rubber accelerators should be regarded as potential human health hazards and that further in vitro and in vivo studies are needed to assess the potential genetic hazards of this large class of chemicals.