TRAX-CHEMxt: Towards the Homogeneous Chemical Stage of Radiation Damage.

The indirect effect of radiation plays an important role in radio-induced biological damages. Monte Carlo codes have been widely used in recent years to study the chemical evolution of particle tracks. However, due to the large computational efforts required, their applicability is typically limited to simulations in pure water targets and to temporal scales up to the µs. In this work, a new extension of TRAX-CHEM is presented, namely TRAX-CHEMxt, able to predict the chemical yields at longer times, with the capability of exploring the homogeneous biochemical stage. Based on the species coordinates produced around one track, the set of reaction-diffusion equations is solved numerically with a computationally light approach based on concentration distributions. In the overlapping time scale (500 ns-1 µs), a very good agreement to standard TRAX-CHEM is found, with deviations below 6% for different beam qualities and oxygenations. Moreover, an improvement in the computational speed by more than three orders of magnitude is achieved. The results of this work are also compared with those from another Monte Carlo-based algorithm and a fully homogeneous code (Kinetiscope). TRAX-CHEMxt will allow for studying the variation in chemical endpoints at longer timescales with the introduction, as the next step, of biomolecules, for more realistic assessments of biological response under different radiation and environmental conditions.

Impact of Target Oxygenation on the Chemical Track Evolution of Ion and Electron Radiation.

The radiosensitivity of biological systems is strongly affected by the system oxygenation. On the nanoscopic scale and molecular level, this effect is considered to be strongly related to the indirect damage of radiation. Even though particle track radiolysis has been the object of several studies, still little is known about the nanoscopic impact of target oxygenation on the radical yields. Here we present an extension of the chemical module of the Monte Carlo particle track structure code TRAX, taking into account the presence of dissolved molecular oxygen in the target material. The impact of the target oxygenation level on the chemical track evolution and the yields of all the relevant chemical species are studied in water under different irradiation conditions: different linear energy transfer (LET) values, different oxygenation levels, and different particle types. Especially for low LET radiation, a large production of two highly toxic species ( HO 2 • and O 2 • - ), which is not produced in anoxic conditions, is predicted and quantified in oxygenated solutions. The remarkable correlation between the HO 2 • and O 2 • - production yield and the oxygen enhancement ratio observed in biological systems suggests a direct or indirect involvement of HO 2 • and O 2 • - in the oxygen sensitization effect. The results are in agreement with available experimental data and previous computational approaches. An analysis of the oxygen depletion rate in different radiation conditions is also reported. The radiosensitivity of biological systems is strongly affected by the system oxygenation. On the nanoscopic scale and molecular level, this effect is considered to be strongly related to the indirect damage of radiation. Even though particle track radiolysis has been the object of several studies, still little is known about the nanoscopic impact of target oxygenation on the radical yields. Here we present an extension of the chemical module of the Monte Carlo particle track structure code TRAX, taking into account the presence of dissolved molecular oxygen in the target material. The impact of the target oxygenation level on the chemical track evolution and the yields of all the relevant chemical species are studied in water under different irradiation conditions: different linear energy transfer (LET) values, different oxygenation levels, and different particle types. Especially for low LET radiation, a large production of two highly toxic species ( HO 2 • and O 2 • - ), which is not produced in anoxic conditions, is predicted and quantified in oxygenated solutions. The remarkable correlation between the HO 2 • and O 2 • - production yield and the oxygen enhancement ratio observed in biological systems suggests a direct or indirect involvement of HO 2 • and O 2 • - in the oxygen sensitization effect. The results are in agreement with available experimental data and previous computational approaches. An analysis of the oxygen depletion rate in different radiation conditions is also reported.

3D high resolution clonogenic survival measurement of xrs-5 cells in low-dose region of carbon ion plans.

PURPOSE: In this study, we performed biological verification measurements of cell survival of a 12C ion irradiation plan employing a high-resolution 3D culture setup. This allowed, in particular, to access the cell inactivation in the low-dose regions close to the target area. MATERIALS AND METHODS: We established the protocol for a 3D culture setup where xrs-5 cells were grown inside a layered matrigel structure in 384-well plates. Their radiosensitivity to conventional and 12C ion radiation was evaluated by irradiating them either with 250 kV X-rays at GSI or with monoenergetic 12C beams of 110 MeV/u at MIT, and compared with those of monolayers. A treatment plan for a rectangular target was prepared using the GSI research treatment planning system TRiP98. xrs-5 cells were seeded in the matrigel-based setup and irradiated in dose fall-off regions using active scanning 12C ion beams. In addition, film dosimetry utilizing radiochromic EBT3 film has been performed to assess the field homogeneity downstream of 384-well V-bottom plates with or without additional agarose coating of the well plate bottom. RESULTS: Dose response curves following X-ray and 12C ion irradiation had linear shape and showed a significant decrease in survival fraction at even moderate doses. Survival measurements in the low-dose regions of the plan for the extended target showed good agreement to the predicted survival fraction. The irradiated film profiles yielded a flat dose distribution without apparent artifacts or inhomogeneities for well plates both with and without agarose coating, confirming the suitability of the experimental setup. CONCLUSIONS: We conclude that the V-bottom 384-well plates in combination with the radiation-sensitive xrs-5 cell line constitute a suitable radiobiological verification tool which can be used especially for low doses. Furthermore, the measured survival of xrs-5 cells show a good agreement with the expected survival in the low-dose out-of-field regions, both laterally and downstream of the target.

May oxygen depletion explain the FLASH effect? A chemical track structure analysis.

BACKGROUND AND PURPOSE: Recent observations in animal models show that ultra-high dose rate ("FLASH") radiation treatment significantly reduces normal tissue toxicity maintaining an equivalent tumor control. The dependence of this "FLASH" effect on target oxygenation has led to the assumption that oxygen "depletion" could be its major driving force. MATERIALS AND METHODS: In a bottom-up approach starting from the chemical track evolution of 1 MeV electrons in oxygenated water simulated with the TRAX-CHEM Monte Carlo code, we determine the oxygen consumption and radiolytic reactive oxygen species production following a short radiation pulse. Based on these values, the effective dose weighted by oxygen enhancement ratio (OER) or the in vitro cell survival under dynamic oxygen pressure is calculated and compared to that of conventional exposures, at constant OER. RESULTS: We find an excellent agreement of our Monte Carlo predictions with the experimental value for radiolytic oxygen removal from oxygenated water. However, the application of the present model to published radiobiological experiment conditions shows that oxygen depletion can only have a negligible impact on radiosensitivity through oxygen enhancement, especially at typical experimental oxygenations where a FLASH effect has been observed. CONCLUSION: We show that the magnitude and dependence of the "oxygen depletion" hypothesis are not consistent with the observed biological effects of FLASH irradiation. While oxygenation plays an undoubted role in mediating the FLASH effect, we conclude that state-of-the-art radiation chemistry models do not support oxygen depletion and radiation-induced transient hypoxia as the main mechanism.

Carbohydrate-carbohydrate interaction prominence in 3D supramolecular self-assembly.

Self-association in water of biologically significant carbohydrate molecules is a controversial topic due to the strong solvation of these molecules in this solvent and the difficulty to experimentally detect these very weak intermolecular forces by biophysical techniques. Herein we report the tremendous ability of amphiphilic carbohydrate molecules to form complex three-dimensional architectures. We have experimentally observed the 3D self-assembly into multilayers of disaccharide neoglycolipid dimers on graphite by means of noncontact AFM and we have also theoretically modeled the interaction between two dimers in order to learn about the structure and composition of these layers. A simple bilayer structure as observed for many amphiphilic lipids was discarded by the experiments. Instead, based on the good agreement between experiments and calculations, we propose that multilayer formation takes place through the assembly of building blocks consisting of two dimers each. The fundamental key in the formation of this supramolecular structure is the complementarity between the van der Waals surfaces of the amphiphilic carbohydrate molecules, a result which differs from the most common idea that H-bonding interactions are prominent in carbohydrate-mediated interactions.

Multi-dimensional dosimetric verification of stereotactic radiotherapy for uveal melanoma using radiochromic EBT film.

Since 1997, linac based stereotactic radiotherapy (SRT) of uveal melanoma has been continuously developed at the Department of Radiotherapy, Medical University Vienna. The aim of the present study was (i) to test a new type of radiochromic film (Gafchromic EBT) for dosimetric verification of class solutions for these treatments and (ii) to verify treatment plan acceptance criteria, which are based on gamma values statisitcs. An EPSON Expression 1680 Pro flat bed scanner was utilized for film reading. To establish a calibration curve, films were cut in squares of 2 x 2 cm2, positioned at 5 cm depth in a solid water phantom and were irradiated with different dose levels (0.5 and 5 Gy) in a 5 x 5 cm2 field at 6 MV. A previously developed solid phantom (polystyrene) was used with overall dimensions corresponding to an average human head. EBT films were placed at four different depths (10, 20, 25 and 30 mm) and all films were irradiated simultaneously. Four different treatment plans were verified that resemble typical clinical situations. These plans differed in irradiation technique (conformal mMLC or circular arc SRT) and in tumour size (PTV of 1 or 2.5 cm3). In-house developed software was applied to calculate gamma (gamma) index values and to perform several statistical operations (e.g. gamma-area histograms). At depths of 10 mm gamma1%, (gamma-value where 1% of the points have an equal or higher value in the region of interest) were between 1-3 and maximum gamma > 1 (% of gamma-values > 1 in the region of interest) areas were almost 30%. At larger depths, i.e. more close to the isocenter, gamma 1% was < 1 and gamma > 1 areas were mostly < 5%. Average gamma values were about 0.5. Besides the compromised accuracy in the buildup region, previously defined IMRT acceptance criteria [Stock et al., Phys. Med Biol. 50 (2005) 399-411] could be applied as well to SRT. Radiochromic EBT films, in combination with a flat-bed scanner, were found to be an ideal multidimensional dosimetric tool for treatment plan quality assurance. EBT films are a suitable and reliable dosimetric tool that could replace traditionally used radiographic films. The presented acceptance criteria for SRT treatment plans might be used as a benchmarking data-set for other stereotactic applications and/or other equipment (planning system and delivery hardware) combinations.

Persistence of radiation-induced aberrations in patients after radiotherapy with C-ions and IMRT.

BACKGROUND AND PURPOSE: Chromosomal aberrations in peripheral blood lymphocytes are a biomarker for radiation exposure and are associated with an increased risk for malignancies. To determine the long-term cytogenetic effect of radiotherapy, we analyzed the persistence of different aberration types up to 2.5 years after the treatment. MATERIALS AND METHODS: Cytogenetic damage was analyzed in lymphocytes from 14 patients that had undergone C-ion boost + IMRT treatment for prostate cancer. Samples were taken immediately, 1 year and 2.5 years after therapy. Aberrations were scored using the multiplex fluorescence in situ hybridization technique and grouped according to their transmissibility to daughter cells. RESULTS: Dicentric chromosomes (non-transmissible) and translocations (transmissible) were induced with equal frequencies. In the follow-up period, the translocation yield remained unchanged while the yield of dicentrics decreased to ≈40% of the initial value (p = 0.011 and p = 0.001 for 1 and 2.5 years after compared to end of therapy). In 2 patients clonal aberrations were observed; however they were also found in samples taken before therapy and thus were not radiotherapy induced. CONCLUSION: The shift in the aberrations spectrum towards a higher fraction of translocations indicates the exposure of hematopoietic stem and progenitor cells underlining the importance of a careful sparing of bone marrow during radiotherapy to minimize the risk for secondary cancers.

Validation of an automated system for aliquoting of HIV-1 Env-pseudotyped virus stocks.

The standardized assessments of HIV-specific immune responses are of main interest in the preclinical and clinical stage of HIV-1 vaccine development. In this regard, HIV-1 Env-pseudotyped viruses play a central role for the evaluation of neutralizing antibody profiles and are produced according to Good Clinical Laboratory Practice- (GCLP-) compliant manual and automated procedures. To further improve and complete the automated production cycle an automated system for aliquoting HIV-1 pseudovirus stocks has been implemented. The automation platform consists of a modified Tecan-based system including a robot platform for handling racks containing 48 cryovials, a Decapper, a tubing pump and a safety device consisting of ultrasound sensors for online liquid level detection of each individual cryovial. With the aim to aliquot the HIV-1 pseudoviruses in an automated manner under GCLP-compliant conditions a validation plan was developed where the acceptance criteria-accuracy, precision as well as the specificity and robustness-were defined and summarized. By passing the validation experiments described in this article the automated system for aliquoting has been successfully validated. This allows the standardized and operator independent distribution of small-scale and bulk amounts of HIV-1 pseudovirus stocks with a precise and reproducible outcome to support upcoming clinical vaccine trials.

Dosimetric characterization of GafChromic EBT film and its implication on film dosimetry quality assurance.

The suitability of radiochromic EBT film was studied for high-precision clinical quality assurance (QA) by identifying the dose response for a wide range of irradiation parameters typically modified in highly-conformal treatment techniques. In addition, uncertainties associated with varying irradiation conditions were determined. EBT can be used for dose assessment of absorbed dose levels as well as relative dosimetry when compared to absolute absorbed dose calibrated using ionization chamber results. For comparison, a silver halide film (Kodak EDR-2) representing the current standard in film dosimetry was included. As an initial step a measurement protocol yielding accurate and precise results was established for a flatbed transparency scanner (Epson Expression 1680 Pro) that was utilized as a film reading instrument. The light transmission measured by the scanner was found to depend on the position of the film on the scanner plate. For three film pieces irradiated with doses of 0 Gy, approximately 1 Gy and approximately 7 Gy, the pixel values measured in portrait or landscape mode differed by 4.7%, 6.2% and 10.0%, respectively. A study of 200 film pieces revealed an excellent sheet-to-sheet uniformity. On a long time scale, the optical development of irradiated EBT film consisted of a slow but steady increase of absorbance which was not observed to cease during 4 months. Sensitometric curves of EBT films obtained under reference conditions (SSD = 95 cm, FS = 5 x 5 cm(2), d = 5 cm) for 6, 10 and 25 MV photon beams did not show any energy dependence. The average separation between all curves was only 0.7%. The variation of the depth d (range 2-25 cm) in the phantom did not affect the dose response of EBT film. Also the influence of the radiation field size (range 3 x 3-40 x 40 cm(2)) on the sensitometric curve was not significant. For EDR-2 films maximum differences between the calibration curves reached 7-8% for X6MV and X25MV. Radiochromic EBT film, in combination with a flatbed scanner, presents a versatile system for high-precision dosimetry in two dimensions, provided that the intrinsic behaviour of the film reading device is taken into account. EBT film itself presents substantial improvements on formerly available models of radiographic and a radiochromic film and its dosimetric characteristics allow us to measure absorbed dose levels in a large variety of situations with a single calibration curve.

Modelling low energy electron and positron tracks for biomedical applications.

PURPOSE: To incorporate the effects of low energy electrons and positrons into radiation interaction models. MATERIALS AND METHODS: The simulation method proposed here was based on experimental and theoretical cross section data and energy loss spectra we have previously derived. After a summary of the main techniques used to obtain reliable input data, the basis of a Low Energy Particle Track Simulation (LEPTS) procedure was established. The programme is specifically designed to describe electron and positron interactions below 10 keV, down to thermal energies. RESULTS: Single electron and positron tracks in water are presented and the possibility of using these results to develop tools for nanodosimetry is discussed. CONCLUSIONS: Standard approximations based on high incident energies, such as the Born-Bethe theory, are not suitable to simulate electron and positron tracks below 10 keV. Prior to the inclusion of low-energy effects in a radiation model, an appropriate study is required to determine both the interaction cross sections and the energy loss spectra.

An automated HIV-1 Env-pseudotyped virus production for global HIV vaccine trials.

BACKGROUND: Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. METHODOLOGY/PRINCIPAL FINDINGS: The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO(2) incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. CONCLUSIONS: An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.

Supramolecular self-assembled arrangements of maltose glyconanoparticles.

Our group previously reported the preparation of water-soluble Au-Fe(x)O(y) nanoparticles functionalized with a maltose neoglycoconjugate. A fraction soluble in methanol was also separated and originated a new supramolecular polymeric aggregate. We report here the full characterization of this novel material by transmission electron microscopy (TEM), fluorescence emission, and atomic force microscopy. By means of noncontact dynamic atomic force microscopy, we have been able to obtain information about the organization of the organic components of the polymers, which eluded TEM analysis. We have observed that polymers packed in units about 65 nm in length and 40 nm in width on Au surfaces. The nanoparticles seem to be encapsulated by the organic material. We propose interactions between the sugar residues and the amphiphilic character of the maltose neoglycoconjugate (with a lipophilic undecane spacer) as responsible for the origin of these amazing supramolecular arrangements.