Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

The mRNA Vaccine Revolution: COVID-19 Has Launched the Future of Vaccinology.

During the COVID-19 pandemic, mRNA (mRNA) vaccines emerged as leading vaccine candidates in a record time. Nonreplicating mRNA (NRM) and self-amplifying mRNA (SAM) technologies have been developed into high-performing and clinically viable vaccines against a range of infectious agents, notably SARS-CoV-2. mRNA vaccines demonstrate efficient in vivo delivery, long-lasting stability, and nonexistent risk of infection. The stability and translational efficiency of in vitro transcription (IVT)-mRNA can be further increased by modulating its structural elements. In this review, we present a comprehensive overview of the recent advances, key applications, and future challenges in the field of mRNA-based vaccinology.

Focal drug-resistant temporal lobe epilepsy associated with an ipsilateral anterior choroidal artery aneurysm: illustrative case.

BACKGROUND: The occurrence of both an intracranial aneurysm and epilepsy, especially drug-resistant epilepsy (DRE), is rare. Although the overall incidence of aneurysms associated with DRE is unclear, it is thought to be particularly infrequent in the pediatric population. Surgical ligation of the offending aneurysm has been reported in conjunction with resolving seizure activity, although few cases have cited a combined approach of aneurysm ligation and resection of an epileptogenic focus. OBSERVATIONS: We present the case of a 14-year-old female patient with drug-resistant temporal lobe epilepsy and an ipsilateral supraclinoid internal carotid artery aneurysm. Seizure semiology, electroencephalography monitoring, and magnetic resonance imaging all indicated a left temporal epileptogenic focus, in addition to an incidental aneurysm. The authors recommended a combined surgery involving resection of the temporal lesion and surgical clip ligation of the aneurysm. Near-total resection and successful ligation were achieved, and the patient has remained seizure free since surgery at 1 year postoperatively. LESSONS: In patients with focal DRE and an adjacent intracranial aneurysm, a combined surgical approach involving both resection and surgical ligation can be used. Several surgical timing and neuroanesthetic considerations should be made to ensure the overall safety and efficacy of this procedure.

In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery.

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

Comparison of adaptive radiotherapy techniques for external radiation therapy of canine bladder cancer.

Daily bladder variations make it difficult to utilize standard radiotherapy as a primary treatment option for muscle-invasive bladder cancer. Our purpose was to develop a model comparing dose distributions of image-guided and adaptive radiotherapy (ART) techniques for canine bladder cancer. Images were obtained retrospectively from cone-beam computed tomography (CBCT) scans used for daily positioning of four dogs undergoing fractionated image-guided radiotherapy (IGRT). Four different treatment plans were modeled for each dog, and dosimetric data were compared. Two plans were developed using planning target volumes based on planning computed tomography (CT) bladder volume. These plans then used bony anatomy or soft tissue anatomy for daily positioning and dosimetric modeling. The third plan type was a hybrid IGRT and ART technique utilizing a library of premade anisotropic planning target volumes using bladder wall motion data and selection of a "plan-of-the-day" determined from positioning CBCT bladder volumes. The fourth plan was an ART technique that constructed a new planning target volume each day based on daily bladder volume as determined by pretreatment CBCT. Dose volume histograms were generated for each plan type and dose distribution for the bladder and rectum were compared between plan types. Irradiated rectal volume decreased and irradiated bladder volume increased as plan conformality increased. ART provided the greatest rectal sparing, with lowest irradiated rectal volume (P < 0.001), and largest bladder volume receiving 95% of the prescription dose (P < 0.001). In our model, adaptive radiotherapy techniques for canine bladder cancer showed significant reduction in rectal volume irradiated when compared to nonadaptive techniques, while maintaining appropriate bladder coverage.

Comparison of planning techniques when air/fluid is present using the strut-adjusted volume implant (SAVI) for HDR-based accelerated partial breast irradiation.

The presence of air/fluid surrounding implantable devices used for partial breast irradiation may significantly impact dose coverage to at-risk tissue. Of the 67 total patients retrospectively evaluated for this study, 32 (48%) had greater than 1 cc volume of air/fluid extending outside of the strut-adjusted volume implant (SAVI) device surface and were selected for comparison of planning approaches. The planning approaches utilized two different definitions of PTV_EVAL. One definition of a PTV_EVAL (PTV_EVALSAVI) was based on expanding 1 cm beyond the SAVI device only while accounting for the air/fluid using the NSABP Protocol B-39/RTOG Protocol 0413. The second PTV_EVAL definition (PTV_EVALCAV) was based on expanding 1 cm beyond the cavity (SAVI device plus air/fluid volume). The results indicate use of the B-39 formalism to account for air/fluid displacing the PTV_EVAL may overestimate the dose coverage to the at-risk tissue, especially for large contiguous volumes of air/fluid. Using the SAVI device to optimize dose covering the PTV_EVALCAV volume surrounding the cavity improves dosimetric coverage to at-risk tissue by 11.3% and 8.7% for V100 and V90, respectively, while the average V150 and V200 indices for PTV_EVALCAV increased by 9.1 cc and 5.0cc, respectively, and the average maximum rib and skin doses increased by 11.1% and 6.1%, respectively. The maximum skin dose, rib dose, V150, and V200 all met the planning objectives despite any increase in these parameters.

Orthotopic model of canine osteosarcoma in athymic rats for evaluation of stereotactic radiotherapy.

OBJECTIVE: To develop an orthotopic model of canine osteosarcoma in athymic rats as a model for evaluating the effects of stereotactic radiotherapy (SRT) on osteosarcoma cells. ANIMALS: 26 athymic nude rats. PROCEDURES: 3 experiments were performed. In the first 2 experiments, rats were injected with 1 × 10(6) Abrams canine osteosarcoma cells into the proximal aspect of the tibia (n = 12) or distal aspect of the femur (6). Tumor engraftment and progression were monitored weekly via radiography, luciferase imaging, and measurement of urine pyridinoline concentration for 5 weeks and histologic evaluation after euthanasia. In the third experiment, 8 rats underwent canine osteosarcoma cell injection into the distal aspect of the femur and SRT was administered to the affected area in three 12-Gy fractions delivered on consecutive days (total radiation dose, 36 Gy). Percentage tumor necrosis and urinary pyridinoline concentrations were used to assess local tumor control. The short-term effect of SRT on skin was also evaluated. RESULTS: Tumors developed in 10 of 12 tibial sites and all 14 femoral sites. Administration of SRT to rats with femoral osteosarcoma was feasible and successful. Mean tumor necrosis of 95% was achieved histologically, and minimal adverse skin effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The orthotopic model of canine osteosarcoma in rats developed in this study was suitable for evaluating the effects of local tumor control and can be used in future studies to evaluate optimization of SRT duration, dose, and fractionation schemes. The model could also allow evaluation of other treatments in combination with SRT, such as chemotherapy or bisphosphonate, radioprotectant, or parathyroid hormone treatment.

Evaluation of canine prostate intrafractional motion using serial cone beam computed tomography imaging.

This study used kilovoltage (kV) cone beam computed tomography (CBCT) imaging to characterize canine intrafractional prostate motion during hypofractionated stereotactic radiotherapy treatment. Serial CBCT images taken just prior to initiating treatment, and at several times during the treatment session, were acquired throughout the course of treatment for canine patients. All patients were immobilized in dorsal recumbency while using an air-inflated rectal balloon. For each treatment session, rigid registration of intrafraction CBCT images with the interfraction CBCT used for setup verification was performed. Contours of the prostate and urethra were drawn on each CBCT image set and the center of mass for each structure was evaluated as a function of time. A total of seven canine patients was included in the study, resulting in 41 CBCT images collected during a total of 12 treatment sessions. Over 70% of our data were collected for CBCTs taken between 20 and 51 min after final patient setup was complete. The mean intrafraction movement in a single direction for the prostate and urethra was ≤0.14 mm and ≤0.22 mm, respectively. The maximum intrafraction movement for the prostate and urethra was ≤ 1.60 mm and ≤ 2.00 mm, respectively. The maximum variability in intrafraction movement for the prostate and urethra, as defined by two standard deviations, was ≤1.40 mm and ≤1.50 mm, respectively. Minimal intrafraction variability using appropriate patient positioning and rectal balloon, combined with kV CBCT image-guided radiation therapy tools to account for interfraction changes, permit accurate and precise targeting of structures of interest.

Repeatability of a planning target volume expansion protocol for radiation therapy of regional lymph nodes in canine and feline patients with head tumors.

For canine and feline patients with head tumors, simultaneous irradiation of the primary tumor and mandibular and retropharyngeal lymph nodes (LNs) is often indicated. The purpose of this study was to assess the repeatability of a planning target volume (PTV) expansion protocol for these LNs. Two CT image sets from 44 dogs and 37 cats that underwent radiation therapy for head tumors were compared to determine LN repositioning accuracy and precision; planning-CT (for radiation therapy planning) and cone-beam CT (at the time of actual treatment sessions). Eleven percent of dogs and 65% of cats received treatment to their LNs. In dogs, the mandibular LNs were positioned more caudally (P = 0.0002) and the right mandibular and right retropharyngeal LNs were positioned more to the left side of the patient (P = 0.00015 and P = 0.003, respectively). In cats, left mandibular LN was positioned higher (toward roof) than the planning-CT (P = 0.028). In conclusion, when the patient immobilization devices and bony anatomy matching are used to align the primary head target and these LNs are treated simultaneously, an asymmetrical PTV expansion that ranges 4-9 mm (dogs) and 2-4 mm (cats), depending on the directions of couch movement, should be used to include the LNs within the PTV at least 95% of the time.

Characterization of differences in calculated and actual measured skin doses to canine limbs during stereotactic radiosurgery using Gafchromic film.

Accurate calculation of absorbed dose to the skin, especially the superficial and radiosensitive basal cell layer, is difficult for many reasons including, but not limited to, the build-up effect of megavoltage photons, tangential beam effects, mixed energy scatter from support devices, and dose interpolation caused by a finite resolution calculation matrix. Stereotactic body radiotherapy (SBRT) has been developed as an alternative limb salvage treatment option at Colorado State University Veterinary Teaching Hospital for dogs with extremity bone tumors. Optimal dose delivery to the tumor during SBRT treatment can be limited by uncertainty in skin dose calculation. The aim of this study was to characterize the difference between measured and calculated radiation dose by the Varian Eclipse (Varian Medical Systems, Palo Alto, CA) AAA treatment planning algorithm (for 1-mm, 2-mm, and 5-mm calculation voxel dimensions) as a function of distance from the skin surface. The study used Gafchromic EBT film (International Specialty Products, Wayne, NJ), FilmQA analysis software, a limb phantom constructed from plastic water™ (fluke Biomedical, Everett, WA) and a canine cadaver forelimb. The limb phantom was exposed to 6-MV treatments consisting of a single-beam, a pair of parallel opposed beams, and a 7-beam coplanar treatment plan. The canine forelimb was exposed to the 7-beam coplanar plan. Radiation dose to the forelimb skin at the surface and at depths of 1.65 mm and 1.35 mm below the skin surface were also measured with the Gafchromic film. The calculation algorithm estimated the dose well at depths beyond buildup for all calculation voxel sizes. The calculation algorithm underestimated the dose in portions of the buildup region of tissue for all comparisons, with the most significant differences observed in the 5-mm calculation voxel and the least difference in the 1-mm voxel. Results indicate a significant difference between measured and calculated data extending to average depths of 2.5 mm, 3.4 mm, and 10 mm for the 1-mm, 2-mm, and 5-mm dimension calculation matrices, respectively. These results emphasize the importance of selecting as small a treatment planning software calculation matrix dimension as is practically possible and of taking a conservative approach for skin treatment planning objectives. One suggested conservative approach is accomplished by defining the skin organ as the outermost 2-3 mm of the body such that the high dose tail of the skin organ dose-volume histogram curve represents dose on the deep side of the skin where the algorithm is more accurate.

Use of cone-beam computed tomography to characterize daily urinary bladder variations during fractionated radiotherapy for canine bladder cancer.

Urinary bladder cancer is difficult to treat accurately with fractionated radiation therapy (RT) due to daily positional changes of the bladder and surrounding soft-tissue structures. We quantified the daily motion experienced by the canine bladder with patients in dorsal vs. sternal vs. lateral recumbency. We also described the dose distribution for three different planning target volume expansions (5, 10, and 15 mm) for each of the three positions to ensure adequate bladder dose and minimize irradiation of nearby healthy tissues. Analysis was based on data from retrospective daily cone-beam computed tomography (CT) (CBCT) images obtained for positioning of canine patients undergoing routine RT. Organs of interest were contoured on each CBCT data set and the images, along with the contours, were registered to the original planning CT. All measurements were made relative to the planning CT and dosimetric data for the organs of interest was determined using a dose volume histogram generated from sample parallel-opposed beam configuration. There was a wide range in bladder position throughout treatment. The least amount of bladder variation and the lowest rectal dose was with dogs in lateral recumbency. It was also determined that a margin of 10 mm would allow for sufficient dose to be delivered to the bladder while minimizing rectal dose.

Skyshine radiation resulting from 6 MV and 10 MV photon beams from a medical accelerator.

Skyshine radiation scattered in the atmosphere above a radiation therapy accelerator facility can result in measurable dose rates at locations near the facility on the ground and at roof level. A Reuter Stokes RSS-120 pressurized ion chamber was used to measure exposure rates in the vicinity of a Varian Trilogy Linear Accelerator at the Colorado State University Veterinary Medical Center. The linear accelerator was used to deliver bremsstrahlung photons from 6 MeV and 10 MeV electron beams with several combinations of field sizes and gantry angles. An equation for modeling skyshine radiation in the vicinity of medical accelerators was published by the National Council on Radiation Protection and Measurements in 2005. However, this model did not provide a good fit to the observed dose rates at ground level or on the roof. A more accurate method of estimating skyshine may be to measure the exposure rate of the radiation exiting the roof of the facility and to scale the results using the graphs presented in this paper.

Tuning the magnetic resonance imaging properties of positive contrast agent nanoparticles by surface modification with RAFT polymers.

A novel surface modification technique was employed to produce a polymer modified positive contrast agent nanoparticle through attachment of well-defined homopolymers synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. A range of RAFT homopolymers including poly[N-(2-hydroxypropyl)methacrylamide], poly(N-isopropylacrylamide), polystyrene, poly(2-(dimethylamino)ethyl acrylate), poly(((poly)ethylene glycol) methyl ether acrylate), and poly(acrylic acid) were synthesized and subsequently used to modify the surface of gadolinium (Gd) metal-organic framework (MOF) nanoparticles. Employment of a trithiocarbonate RAFT agent allowed for reduction of the polymer end groups under basic conditions to thiolates, providing a means of homopolymer attachment through vacant orbitals on the Gd3+ ions at the surface of the Gd MOF nanoparticles. Magnetic resonance imaging (MRI) confirmed the relaxivity rates of these novel polymer modified structures were easily tuned by changes in the molecular weight and chemical structures of the polymers. When a hydrophilic polymer was used for modification of the Gd MOF nanoparticles, an increase in molecular weight of the polymer provided a respective increase in the longitudinal relaxivity. These relaxivity values were significantly higher than both the unmodified Gd MOF nanoparticles and the clinically employed contrast agents, Magnevist and Multihance, which confirmed the construct's ability to be utilized as a positive contrast nanoparticle agent in MRI. Further characterization confirmed that increased hydrophobicity of the polymer coating on the Gd MOF nanoparticles yielded minimal changes in the longitudinal relaxivity properties but large increases in the transverse relaxivity properties in the MRI.

Assessment of a radiotherapy patient cranial immobilization device using daily on-board kilovoltage imaging.

The purpose of this study was to utilize state-of-the-art on-board digital kilovoltage (kV) imaging to determine the systematic and random set-up errors of an immobilization device designed for canine and feline cranial radiotherapy treatments. The immobilization device is comprised of a custom made support bridge, bite block, vacuum-based foam mold and a modified thermoplastic mask attached to a commercially available head rest designed for human radiotherapy treatments. The immobilization device was indexed to a Varian exact couch-top designed for image guided radiation therapy (IGRT). Daily orthogonal kV images were compared to Eclipse treatment planning digitally reconstructed radiographs (DRRs). The orthogonal kV images and DRRs were directly compared online utilizing the Varian on-board imaging (OBI) system with set-up corrections mmediately and remotely transferred to the treatment couch prior to treatment delivery. Off-line review of 124 patient treatments indicates systematic errors consisting of +0.18 mm vertical, +0.39mm longitudinal and -0.08 mm lateral. The random errors corresponding to 2 standard deviations (95% CI) consist of 4.02 mm vertical, 2.97 mm longitudinal and 2.53 mm lateral and represent conservative CTV to PTV margins if kV OBI is not available. Use of daily kV OBI along with the cranial immobilization device permits reduction of the CTV to PTV margins to approximately 2.0 mm.

Protein added to a sports drink improves fluid retention.

The purpose of this study was to compare fluid retention of carbohydrate plus protein, a carbohydrate-only, and water following 2.5% body weight (BW) loss. Thirteen subjects dehydrated to 2.5% of BW, then ingested a CHO (6%) plus protein drink (1.5%; CP), a 6% CHO drink, or water (WA) at a volume equal to BW loss during a 3-h recovery. Fluid retention was significantly greater for CP (88 +/- 4.7%) than CHO (75 +/- 14.6%), which was greater than WA (53 +/- 16.1%). Serum and urine osmolalities were greater for CP (284.7 +/- 5.0; 569.4 +/- 291.4 mOsm/kg) than CHO (282.6 +/- 5.2; 472.9 +/- 291.5 mOsm/kg) which were greater than WA (280.6 +/- 5.9, 303.7 +/- 251.5 mOsm/kg). Results indicate that fluid retention for CP was 15% greater than CHO and 40% greater than WA. Water ingestion led to a dilution of the serum and resulted in only 53% fluid retention.

Chest wall dose in MammoSite breast brachytherapy: radiobiologic estimations of late complication risk based on dose-volume considerations.

PURPOSE: To estimate the risk of late effects in women treated with MammoSite brachytherapy (MBT), the balloon catheters of which were placed near the ribs. METHODS AND MATERIALS: Upon reviewing 93 plans, 16 patients (17%) treated with MBT were considered to have received a high chest wall dose (>or=120% isodose line in contact with a rib). A dose-volume histogram was generated for this rib, and its distance from the MBT balloon measured. Using the linear quadratic equation, the equivalent dose, delivered in 10 fractions, to the dose that causes a 5% and 50% risk of rib late effects at 5 years using 2Gy per fraction, was calculated to be 37 and 44Gy, respectively. The rib volume receiving greater than or equal to these doses (V37 and V44) was correlated to the balloon-to-rib distance. Chest wall signs, symptoms, and radiologic findings for all 16 patients were recorded. RESULTS: The median balloon-to-rib distance was 4.8mm. The median values of V37 and V44 were 13.5% and 3.3%, respectively. All patients with a V37>or=15% and V44>or=5% had a minimum balloon-to-rib distance of <5mm. Two patients reported treatment-related chest wall tenderness (both had balloons placed <5mm from the chest wall), but neither presented with radiologic complications. CONCLUSIONS: Sixteen patients considered to receive relatively high chest wall doses had less than one-third of their primary rib volume being exposed to the estimated TD 5/5 and TD 50/5 doses. Therefore, we estimate the risk of late effects in women treated with MBT, the balloon catheters of which placed near the ribs were negligible, and believe that MBT remains a safe and effective treatment for selected patients with early stage breast cancer.

Performance characterization of megavoltage computed tomography imaging on a helical tomotherapy unit.

Helical tomotherapy is an innovative means of delivering IGRT and IMRT using a device that combines features of a linear accelerator and a helical computed tomography (CT) scanner. The HI-ART II can generate CT images from the same megavoltage x-ray beam it uses for treatment. These megavoltage CT (MVCT) images offer verification of the patient position prior to and potentially during radiation therapy. Since the unit uses the actual treatment beam as the x-ray source for image acquisition, no surrogate telemetry systems are required to register image space to treatment space. The disadvantage to using the treatment beam for imaging, however, is that the physics of radiation interactions in the megavoltage energy range may force compromises between the dose delivered and the image quality in comparison to diagnostic CT scanners. The performance of the system is therefore characterized in terms of objective measures of noise, uniformity, contrast, and spatial resolution as a function of the dose delivered by the MVCT beam. The uniformity and spatial resolutions of MVCT images generated by the HI-ART II are comparable to that of diagnostic CT images. Furthermore, the MVCT scan contrast is linear with respect to the electron density of material imaged. MVCT images do not have the same performance characteristics as state-of-the art diagnostic CT scanners when one objectively examines noise and low-contrast resolution. These inferior results may be explained, at least partially, by the low doses delivered by our unit; the dose is 1.1 cGy in a 20 cm diameter cylindrical phantom. In spite of the poorer low-contrast resolution, these relatively low-dose MVCT scans provide sufficient contrast to delineate many soft-tissue structures. Hence, these images are useful not only for verifying the patient's position at the time of therapy, but they are also sufficient for delineating many anatomic structures. In conjunction with the ability to recalculate radiotherapy doses on these images, this enables dose guidance as well as image guidance of radiotherapy treatments.