Protective Activity of Inactivated Rabies Vaccine Using Flagellin-Based Adjuvant.

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

Carbon Ions for Hypoxic Tumors: Are We Making the Most of Them?

Hypoxia, which is associated with abnormal vessel growth, is a characteristic feature of many solid tumors that increases their metastatic potential and resistance to radiotherapy. Carbon-ion radiation therapy, either alone or in combination with other treatments, is one of the most promising treatments for hypoxic tumors because the oxygen enhancement ratio decreases with increasing particle LET. Nevertheless, current clinical practice does not yet fully benefit from the use of carbon ions to tackle hypoxia. Here, we provide an overview of the existing experimental and clinical evidence supporting the efficacy of C-ion radiotherapy in overcoming hypoxia-induced radioresistance, followed by a discussion of the strategies proposed to enhance it, including different approaches to maximize LET in the tumors.

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.

Potential benefits of using radioactive ion beams for range margin reduction in carbon ion therapy.

Sharp dose gradients and high biological effectiveness make ions such as 12C an ideal tool to treat deep-seated tumors, however, at the same time, sensitive to errors in the range prediction. Tumor safety margins mitigate these uncertainties, but during the irradiation they lead to unavoidable damage to the surrounding healthy tissue. To fully exploit the Bragg peak benefits, a large effort is put into establishing precise range verification methods. Despite positron emission tomography being widely in use for this purpose in 12C therapy, the low count rates, biological washout, and broad activity distribution still limit its precision. Instead, radioactive beams used directly for treatment would yield an improved signal and a closer match with the dose fall-off, potentially enabling precise in vivo beam range monitoring. We have performed a treatment planning study to estimate the possible impact of the reduced range uncertainties, enabled by radioactive 11C ions treatments, on sparing critical organs in tumor proximity. Compared to 12C treatments, (i) annihilation maps for 11C ions can reflect sub- millimeter shifts in dose distributions in the patient, (ii) outcomes of treatment planning with 11C significantly improve and (iii) less severe toxicities for serial and parallel critical organs can be expected.

Depth dose measurements in water for 11C and 10C beams with therapy relevant energies.

Owing to the favorable depth-dose distribution and the radiobiological properties of heavy ion radiation, ion beam therapy shows an improved success/toxicity ratio compared to conventional radiotherapy. The sharp dose gradients and very high doses in the Bragg peak region, which represent the larger physical advantage of ion beam therapy, make it also extremely sensitive to range uncertainties. The use of ß +-radioactive ion beams would be ideal for simultaneous treatment and accurate online range monitoring through PET imaging. Since all the unfragmented primary ions are potentially contributing to the PET signal, these beams offer an improved image quality while preserving the physical and radiobiological advantages of the stable counterparts. The challenging production of radioactive ion beams and the difficulties in reaching high intensities, have discouraged their clinical application. In this context, the project Biomedical Applications of Radioactive ion Beams (BARB) started at GSI (Helmholtzzentrum für Schwerionenforschung GmbH) with the main goal to assess the technical feasibility and investigate possible advantages of radioactive ion beams on the pre-clinical level. During the first experimental campaign 11C and 10C beams were produced and isotopically separated with the FRagment Separator (FRS) at GSI. The ß +-radioactive ion beams were produced with a beam purity of 99% for all the beam investigated (except one case where it was 94%) and intensities potentially sufficient to treat a small animal tumors within few minutes of irradiation time, ∼ 106 particle per spill for the 10C and ∼ 107 particle per spill for the 11C beam, respectively. The impact of different ion optical parameters on the depth dose distribution was studied with a precision water column system. In this work, the measured depth dose distributions are presented together with results from Monte Carlo simulations using the FLUKA software.

Towards harmonizing clinical linear energy transfer (LET) reporting in proton radiotherapy: a European multi-centric study.

BACKGROUND: Clinical data suggest that the relative biological effectiveness (RBE) in proton therapy (PT) varies with linear energy transfer (LET). However, LET calculations are neither standardized nor available in clinical routine. Here, the status of LET calculations among European PT institutions and their comparability are assessed. MATERIALS AND METHODS: Eight European PT institutions used suitable treatment planning systems with their center-specific beam model to create treatment plans in a water phantom covering different field arrangements and fulfilling commonly agreed dose objectives. They employed their locally established LET simulation environments and procedures to determine the corresponding LET distributions. Dose distributions D1.1 and DRBE assuming constant and variable RBE, respectively, and LET were compared among the institutions. Inter-center variability was assessed based on dose- and LET-volume-histogram parameters. RESULTS: Treatment plans from six institutions fulfilled all clinical goals and were eligible for common analysis. D1.1 distributions in the target volume were comparable among PT institutions. However, corresponding LET values varied substantially between institutions for all field arrangements, primarily due to differences in LET averaging technique and considered secondary particle spectra. Consequently, DRBE using non-harmonized LET calculations increased inter-center dose variations substantially compared to D1.1 and significantly in mean dose to the target volume of perpendicular and opposing field arrangements (p < 0.05). Harmonizing LET reporting (dose-averaging, all protons, LET to water or to unit density tissue) reduced the inter-center variability in LET to the order of 10-15% within and outside the target volume for all beam arrangements. Consequentially, inter-institutional variability in DRBE decreased to that observed for D1.1. CONCLUSION: Harmonizing the reported LET among PT centers is feasible and allows for consistent multi-centric analysis and reporting of tumor control and toxicity in view of a variable RBE. It may serve as basis for harmonized variable RBE dose prescription in PT.

Ultra-High Dose Rate (FLASH) Carbon Ion Irradiation: Dosimetry and First Cell Experiments.

PURPOSE: To establish a beam monitoring and dosimetry system to enable the FLASH dose rate carbon ion irradiation and investigate, at different oxygen concentrations, the in vitro biological response in comparison to the conventional dose rate. METHODS AND MATERIALS: CHO-K1 cell response to irradiation at different dose rates and at different levels of oxygenation was studied using clonogenic assay. The Heidelberg Ion-Beam Therapy Center (HIT) synchrotron, after technical improvements, was adjusted to extract ≥5 × 108 12C ions within approximately 150 milliseconds. The beam monitors were filled with helium. RESULTS: The FLASH irradiation with beam scanning yields a dose of 7.5 Gy (homogeneity of ±5%) for a 280 MeV/u beam in a volume of at least 8 mm in diameter and a corresponding dose rate of 70 Gy/s (±20%). The dose repetition accuracy is better than 2%, the systematic uncertainty is better than 2%. Clonogenic assay demonstrates a significant FLASH sparing effect which is strongly oxygenation-dependent and mostly pronounced at 0.5% O2 but absent at 0% and 21% O2. CONCLUSION: The FLASH dose rates >40 Gy/s were achieved with carbon beams. Cell survival analysis revealed FLASH dose rate sparing in hypoxia (0.5%-4% O2).

Radioactive Beams for Image-Guided Particle Therapy: The BARB Experiment at GSI.

Several techniques are under development for image-guidance in particle therapy. Positron (ß+) emission tomography (PET) is in use since many years, because accelerated ions generate positron-emitting isotopes by nuclear fragmentation in the human body. In heavy ion therapy, a major part of the PET signals is produced by ß+-emitters generated via projectile fragmentation. A much higher intensity for the PET signal can be obtained using ß+-radioactive beams directly for treatment. This idea has always been hampered by the low intensity of the secondary beams, produced by fragmentation of the primary, stable beams. With the intensity upgrade of the SIS-18 synchrotron and the isotopic separation with the fragment separator FRS in the FAIR-phase-0 in Darmstadt, it is now possible to reach radioactive ion beams with sufficient intensity to treat a tumor in small animals. This was the motivation of the BARB (Biomedical Applications of Radioactive ion Beams) experiment that is ongoing at GSI in Darmstadt. This paper will present the plans and instruments developed by the BARB collaboration for testing the use of radioactive beams in cancer therapy.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI's hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.