Purpose Animal models that accurately reflect human responses to radiation injury are needed for advanced mechanistic investigation and development of effective therapeutics. The rabbit is an established animal model accepted by the FDA for studies of cardiovascular disease, lipid metabolism, the development of anticoagulants, testing of bone implants, and the development of treatments for infectious diseases such as HIV. The purpose of this study was to investigate the New Zealand White (NZW) Rabbit model as a model of acute radiation exposure because of its established similarity to human vascular, immune, and coagulation responses. Materials and methods Two sequential studies were performed in a total of 81 male NZW rabbits, 16-20 weeks of age. All animals underwent clinical observations and peripheral blood analyses following a single dose of 0, 6, 7, 8, 8.5, 9, or 10 Gy of total body irradiation via a 6 MV Linear accelerator photon source on day 0. Animals were treated with timed release fentanyl patch (days 0-30), subcutaneous hydration (day 1, Study 2 only), and oral sulfamethoxazole/trimethoprim 30 mg/kg once daily (days 3-30) and were followed for 30 days or to time of mortality. Results Study 1 revealed the estimated LD30, -50, -70, and -90 with 95% confidence intervals (CI) at 30 days to be 6.7 (CI: 5.9-7.4), 7.3 (CI: 6.7-7.8), 7.9 (CI: 7.3-8.4), and 8.8 (CI: 7.9-9.7) Gy, respectively. In study 2, a survey of blood coagulation and biochemical parameters were performed over time and necropsy. Complete blood counts taken from animals exposed to 7, 8, or 10 Gy, demonstrated dose-dependent depletion of lymphocytes, neutrophils, and platelets. Platelet counts recovered to baseline levels in survivors by day 30, whereas lymphocyte and neutrophil counts did not. Decedent animals demonstrated grade 3 or 4 neutropenia and lymphopenia at time of death; 64% of the decedents experienced a 30% or greater drop in hematocrit. Decedent animals demonstrated more than 100% increases from serum baseline levels of blood urea nitrogen, creatinine, aspartate aminotransferase, and triglyceride levels at the time of death whereas survivors on average demonstrated modest or no elevation. Conclusion This NZW rabbit model demonstrates dose-dependent depletion of hematopoietic parameters. The LD50/30 of 7.8 Gy (95% CI: 6.6-8.4) with supportive care appears to be close to the ranges reported for rhesus macaques (5.25-7.44 Gy) and humans (6-8 Gy) with supportive care. These findings support the utility of the NZW rabbit model for further mechanistic investigation of acute radiation exposure and medical countermeasure testing.

The development of effective biomarkers for detecting the magnitude of radiation exposure and resiliency of host response is crucial to identifying appropriate treatment strategies after radiation exposure. We hypothesized that the gastrointestinal resident bacteria would demonstrate predictable, dose-dependent changes after radiation exposure across two large animal models of acute radiation syndrome. Here, Göttingen minipigs (GMP) (n = 50) and rhesus macaques (n = 48) were exposed to five dose levels (resulting in mortality rates of 33-100% and 25-68.7%, respectively). Fecal samples taken prior to and after irradiation (day 0 for GMP; day 0, 3 and 14 for macaques) were used for 16S rRNA gene sequence amplicon high-throughput sequencing. Baseline gut microbiota profiles were dissimilar between GMP and macaques, however, radiation appeared to have similar effect at the phylum level, resulting in Bacteroidetes decrease and Firmicutes increase in both models. The abundance of the main Bacteroidetes genus ( Bacteroides for GMP, Prevotella for macaques) was profoundly decreased by irradiation. Intracellular symbionts [Elusimicrobia in GMP, Treponema (Spirochaetes) in macaques] consistently increased after irradiation, suggesting their use as potential biomarkers of intestinal injury, and potential negative effect on health. Prevotella, Lactobacillus, Clostridium XIVa, Oscillibacter and Elusimicrobium/ Treponema abundances were found to be very significantly correlated with radiation intensity. Furthermore, Prevotella, Enterorhabdus and Ruminococcus and Enterorhabdus maintenance was strongly associated with survival in GMP, while Prevotella, Oscillibacter and Treponema were strongly associated with survival and Streptococcus with death in macaques. Overall, we found that a wide range of gut bacterial genera known to be abundant in the human gut microbiota are excellent biomarkers of radiation intensity and resilience in animal models, and that detrimental effects can be monitored, and potentially prevented, by targeting selected genera.

Frame-based stereotactic radiosurgery (SRS) requires fixation of an invasive head ring to ensure accurate targeting. Minimizing waiting time with a fixed head ring is important for patient comfort and satisfaction. We report a practical preplanning solution for the Brainlab iPlan treatment planning system that reduces waiting time by expediting the planning process on treatment day. A water-filled anthropomorphic head phantom was used to acquire a surrogate CT image set for preplanning and fused with patient's MRI, which was obtained before the day of treatment. Once an acceptable preplan was obtained, it was saved as a plan template and the phantom image set was removed from the Brainlab database to prevent any confusion and mix-up of image sets. On the treatment day, the patient's CT and MRI were fused, and the customized beam settings of the preplan template were then applied and optimized. Up to 10-fold of reduction in treatment plan time was demonstrated by bench testing with multiple planners and a variety of cases. Loading the plan template and fine-tuning the preconfigured beam settings took only a small fraction of the preplan time to restore the conformity and dose falloff comparable to those of the preplan. For instance, preplan time was 2 hr for a two-isocenter case, whereas, it took less than 20 min for a less experienced planner to plan it on the day of treat-ment using the preplan method. The SRS preplanning technique implemented in this study for the Brainlab iPlan treatment planning system offers an opportunity to explore possible beam configurations thoroughly, optimize planning parameters, resolve gantry angle clearance issues, and communicate and address challenges with physicians before the treatment day. Preplanning has been proven to improve plan quality and to improve efficiency in our clinic, especially for multiple-isocenter and dosimetrically challenging cases.

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PURPOSE:Low temporal latency between a gating ON/OFF signal and the LINAC beam ON/OFF during respiratory gating is critical for patient safety. Here the authors describe a novel method to precisely measure gating lag times at high temporal resolutions.METHODS:A respiratory gating simulator with an oscillating platform was modified to include a linear potentiometer for position measurement. A photon diode was placed at linear accelerator isocenter for beam output measurement. The output signals of the potentiometer and diode were recorded simultaneously at 2500 Hz with an analog to digital converter for four different commercial respiratory gating systems. The ON and OFF of the beam signal were located and compared to the expected gating window for both phase and position based gating and the temporal lag times extracted.RESULTS:For phase based gating, a real-time position management (RPM) infrared marker tracking system with a single camera and a RPM system with a stereoscopic camera were measured to have mean gate ON/OFF lag times of 98/90 and 86/44 ms, respectively. For position based gating, an AlignRT 3D surface system and a Calypso magnetic fiducial tracking system were measured to have mean gate ON/OFF lag times of 356/529 and 209/60 ms, respectively.CONCLUSIONS:Temporal resolution of the method was high enough to allow characterization of individual gate cycles and was primary limited by the sampling speed of the data recording device. Significant variation of mean gate ON/OFF lag time was found between different gating systems. For certain gating devices, individual gating cycle lag times can vary significantly.

We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a(-/-)) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a(-/-) mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt(+/-) mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a(-/-) mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a(-/-) mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.

Radiation-induced pulmonary fibrosis (RIF) is a severe complication of thoracic radiotherapy that limits its dose, intensity, and duration. The contribution of the endocannabinoid signaling system in pulmonary fibrogenesis is not known. Using a well-established mouse model of RIF, we assessed the involvement of cannabinoid receptor-1 (CB1) in the onset and progression of pulmonary fibrosis. Female C57BL/6 mice and CB1 knockout mice generated on C57BL/6 background received 20 Gy (2 Gy/min) single-dose thoracic irradiation that resulted in pulmonary fibrosis and animal death within 15 to 18 weeks. Some C57BL/6 animals received the CB1 peripherally restricted antagonist AM6545 at 1 mg/kg intraperitoneally three times per week. Animal survival and parameters of pulmonary inflammation and fibrosis were evaluated. Thoracic irradiation (20 Gy) was associated with marked pulmonary inflammation and fibrosis in mice and high mortality within 15 to 18 weeks after exposure. Genetic deletion or pharmacological inhibition of CB1 receptors with a peripheral CB1 antagonist AM6545 markedly attenuated or delayed the lung inflammation and fibrosis and increased animal survival. Our results show that CB1 signaling plays a key pathological role in the development of radiation-induced pulmonary inflammation and fibrosis, and peripherally restricted CB1 antagonists may represent a novel therapeutic approach against this devastating complication of radiotherapy/irradiation.

In a mass casualty radiation event situation, individualized therapy may overwhelm available resources and feasibility issues suggest a need for the development of population-based strategies. To investigate the efficacy of a population-based strategy, Chinese macaques (n = 46) underwent total-body irradiation and received preemptive antibiotics, IV hydration on predetermined postirradiation days and were then compared to macaques (n = 48) that received subject-based care in which blood transfusions, IV hydration, nutritional supplementation and antibiotic supportive measures were provided. Estimated radiation doses for LD30/60, LD50/60 and LD70/60 of animals with subject-based care: 6.83 Gy (6.21, 7.59), 7.44 Gy (6.99, 7.88) and 8.05 Gy (7.46, 8.64), respectively, and for population-based care: 5.61 Gy (5.28, 6.17), 6.62 Gy (6.13, 7.18) and 7.63 Gy (7.21, 8.20), respectively. Analysis of four time periods, 0-9, 10-15, 16-25 and 26-60 days postirradiation, identified significant mortality differences during the period of 10-15 days. A subset analysis of higher radiation doses (6.75-7.20 Gy, n = 32) indicated hydration, nutrition and septic status were not significantly different between treatments. Whole blood transfusion treatment, administered only in subject-supportive care, was associated with significantly higher platelet and absolute neutrophil counts. Median platelet counts greater than 5,670 cells/μl and absolute neutrophil counts greater than 26 cells/μl during this period correlated with survival. We observed that the population-based treatment increased the LD50/60 compared to nontreatment (6.62 Gy vs. 4.92 Gy) and may be further optimized during days 10-15, where strategic blood transfusions or other strategies to achieve increases in neutrophil and platelet counts may further increase survival rates in subjects exposed to high doses of radiation.

OBJECTIVES:In head and neck squamous cell carcinoma (HNSCC), docetaxel, cisplatin and 5-fluorouracil (TPF) has become an accepted induction chemotherapy regimen. However, carboplatin-paclitaxel (CT) regimens have shown comparable outcomes. Here, we compared the outcomes of patients treated with either TPF or CT as induction chemotherapy followed by definitive chemoradiation. PATIENTS AND METHODS:We performed a single-institution retrospective analysis of patients with Stage III-IV HNSCC. From a database of 803 patients, we identified 143 patients treated with TPF or CT induction chemotherapy between 1999 and 2012. RESULTS:53 patients and 90 patients received TPF or CT induction chemotherapy, respectively. The median follow-up was 18.9 months. The 1 year locoregional control was 80.5% for CT compared to 55.5% for TPF (HR 0.32, P=.0002). The 1 year progression free survival was 73.2% for CT compared to 60.7% for TPF (HR 0.57; P=.02). On multivariable analysis, CT remained significant for LRC (HR 0.28; P=0.04). TPF induction chemotherapy was associated with worse renal toxicity as measured by peak creatinine increases during induction chemotherapy (P=0.001). TPF was also associated with a trend toward more chemotherapy dose reductions or changes in systemic agents during concurrent chemoradiation (43.4% for TPF vs. 27.8% for CT; P=0.06). CONCLUSIONS:Compared to TPF induction chemotherapy, CT induction chemotherapy had at least similar if not better LRC and PFS in patients while having less renal toxicity. Thus, CT induction chemotherapy may benefit patients with locally advanced HNSCC by facilitating adequate chemoradiation regimens that enhanced disease control.

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Purpose: To evaluate the effect of using global vs local gamma analysis on planar dose IMRT QA . Methods: Seven Eclipse IMRT plans, with nine fields each, for head and neck and pelvic cancer were evaluated; planar doses from verification plans calculated on a flat phantom were compared with measurements obtained using a 2D detector array (Matrixx). An in‐house developed software was used to evaluate the measured doses using the local gamma scheme where percent dose difference is calculated relative to dose at each point, as well as the global gamma in which percent dose difference is calculated relative to the maximum dose in the measurement plane. Percent of pixels that passed (gamma >1) were compared and points that failed were investigated. Results: With 3%/3mm gamma criteria and 10% threshold dose, overall passing rate with local gamma was 4.9% (+/− 1.6%) smaller on the average than global gamma rates. Maximum difference in the passing rate was as high as 16%, with an average of 10.3% (+/−3.6%) for all fields; the variation was more pronounced for pelvic plans with simultaneous integrated boost which involved multiple planning target volumes with different prescribed doses. Points failing the local gamma criteria were mostly in lower dose (i.e., about 40–60% of maximum planar dose) and low gradient regions. Conclusions: For planar dose IMRT QA, global gamma analysis gives higher passing rates where points in lower dose and low gradient regions pass the global dose criterion with higher actual percent dose differences between measured and planned doses. Higher percent dose deviations in lower dose regions might possibly be due to deficiencies in IMRT commissioning and/or planning which could have been missed using the global gamma analysis. Users need to be aware of the limitations of the techniques used in the dosimetric analysis of IMRT plans.

A novel intensity-modulated radiation therapy (IMRT) phantom for use in three-dimensional in vitro cell experiments, based on a commercially available system (CIRS Inc., Norfolk, VA), was designed and fabricated. The water-equivalent plastic phantom can, with a set of water-equivalent plastic inserts, enclose 1-3 multi-well tissue culture plates. Dosimetry within the phantom was assessed using thermoluminescence dosimeters (TLDs) and film. The phantom was loaded with three tissue culture plates, and an array of TLDs or a set of three films was placed underneath each plate within the phantom, and then irradiated using an IMRT plan created for it. Measured doses from each dosimeter were compared to those acquired from the treatment planning system. The percent differences between TLD measurements and the corresponding points in the treatment plan ranged from 1.3% to 2.9%, differences which did not show statistical significance. Average point-by-point percent dose differences for each film plane ranged from 1.6% to 3.1%. The percentage dose difference for which 95% of the points in the film matched those corresponding to the calculated dose plane to within 3.0% ranged from 2.8% to 4.2%. The good agreement between predicted and measured dose shows that the phantom is a useful and efficient tool for three-dimensional in vitro cell experiments.

PURPOSE:To experimentally validate how temporal modification of the applied dose pattern within a single fraction of radiation therapy affects cell survival. METHOD AND MATERIALS:Using the linear-quadratic model, we have previously demonstrated that the greatest difference in cell survival results from comparing a temporal dose pattern delivering the highest doses during the middle of a fraction and the lowest at the beginning and end ("Triangle") to one with the lowest doses at the middle and the highest at the beginning and end ("V-shaped"). Also, these differences would be greatest in situations with low alpha/beta and large dose/fraction and fraction length. Two low (WiDr, PC-3) and one high (SQ-20B) alpha/beta cell lines were irradiated in six-well plates with 900 cGy over 20 min (900 cGy/20 min), one each with a Triangle and V-shaped dose pattern. WiDr cells were subjected to the same experiments with first 180 cGy/20 min, then 900 cGy/5 min. Cell survival was assessed using the clonogenic assay. RESULTS:At 900 cGy/20 min, irradiation with a V-shaped pattern resulted in an increased survival compared with use of a Triangle pattern of 21.2% for WiDr (p < 0.01), 18.6% for PC-3 (p < 0.025), and 4.7% for SQ-20B cells (p > 0.05). For WiDr cells at 180 cGy/20 min, this increase reduced to 2.7% (p > 0.05) and to -0.8% (p > 0.05) at 900 cGy/5 min. CONCLUSIONS:These results verify the assertions of the modeling study in vitro, and imply that the temporal pattern of applied dose should be considered in treatment planning and delivery.

Total body irradiation (TBI) has been used as a form of systemic therapy for the treatment of hematological malignancies and as a pre-conditioning regimen prior to bone marrow transplant. However, standard techniques are associated with both acute and chronic toxicities due to the large volumes of normal tissue irradiated. Intensity modulated radiation therapy (IMRT), with its ability to conform the high dose region to the shape of the target, offers a method to reduce radiation related sequelae and potentially allow for the delivery of higher than conventional doses. The goal of this study is to investigate the feasibility of a linac-based approach to intensity modulated total marrow irradiation (IM-TMI) and to discuss the challenges associated with its clinical implementation. The successful development of linac-based IM-TMI would represent advancement in the field of radiotherapy and potentially reduce the incidence and severity of complications associated with current TBI techniques.

Graft versus host disease (GVHD), mediated by donor T cells, is a significant source of morbidity and mortality following allogeneic stem cell transplantation. Mesenchymal stem cells (MSC) can successfully treat ongoing graft versus host disease, presumably due to their ability to suppress donor T cell proliferation. Little is known about the potential of MSC to prevent GVHD. Here we show that bone marrow-isolated MSC can suppress the development of GVHD if given after donor T cell recognition of antigen. IFN-gamma was required to initiate MSC efficacy. Recipients of IFN-gamma(-/-) T cells did not respond to MSC treatment and succumbed to GVHD. MSC, pre-treated with IFN-gamma, became immediately active and could suppress GVHD more efficiently than a fivefold-greater number of MSC that were not activated. When given at the time of bone marrow transplantation, activated MSC could prevent GVHD mortality (100% survival, p=0.006). MSC activation was dependent on the magnitude of IFN-gamma exposure, with increased IFN-gamma exposure leading to increased MSC suppression of GVHD. Activated MSC present a new strategy for preventing GVHD using fewer MSC.