Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography.

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.

Frugal and Translatable [15O]O2 Production for Human Inhalation with Direct Delivery from the Cyclotron to a Hybrid PET/MR.

Oxygen-15 (ß+, t1/2 = 122 s) radiolabeled diatomic oxygen, in conjunction with positron emission tomography, is the gold standard to quantitatively measure the metabolic rate of oxygen consumption in the living human brain. We present herein a protocol for safe and effective delivery of [15O]O2 over 200 m to a human subject for inhalation. A frugal quality control testing procedure was devised and validated. This protocol can act as a blueprint for other sites seeking to implement similar imaging programs.

A Protocol for Simultaneous In Vivo Imaging of Cardiac and Neuroinflammation in Dystrophin-Deficient MDX Mice Using [18F]FEPPA PET.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss-notably within muscles and the central neurons system. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies have improved life expectancy; however, this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, better assessment of dystrophic heart and brain pathophysiology is needed. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration; however, neuroinflammation's role is largely unknown in DMD despite being prevalent in other neurodegenerative diseases. Here, we present an inflammatory marker translocator protein (TSPO) positron emission tomography (PET) protocol for in vivo concomitant assessment of immune cell response in hearts and brains of a dystrophin-deficient mouse model [mdx:utrn(+/-)]. Preliminary analysis of whole-body PET imaging using the TSPO radiotracer, [18F]FEPPA in four mdx:utrn(+/-) and six wildtype mice are presented with ex vivo TSPO-immunofluorescence tissue staining. The mdx:utrn(+/-) mice showed significant elevations in heart and brain [18F]FEPPA activity, which correlated with increased ex vivo fluorescence intensity, highlighting the potential of TSPO-PET to simultaneously assess presence of cardiac and neuroinflammation in dystrophic heart and brain, as well as in several organs within a DMD model.

Orthopaedic Surgery Residency: Perspective of Applicants and Program Directors on Medical Student Virtual Experiences.

INTRODUCTION: Orthopaedic Residency Directors advised against visiting subinternship rotations in the peak of the pandemic. To adapt, programs offered multiple virtual experiences. The purpose of this study was to evaluate programs and applicants perceptions regarding the value of virtual experiences during the 2020 to 2021 application cycle and their utility in future application cycles. METHODS: A survey was disseminated to 31 residency programs gathering data about virtual experiences offered in this cycle. A second survey was disseminated to interns who have successfully matched at those programs to identify how interns think to have benefited from the experiences. RESULTS: Twenty-eight programs completed the survey (90% response rate). One hundred eight new interns completed the survey (70% response rate). Virtual information sessions and resident socials were the highest attended (94% and 92%, respectively). Interns and leadership agreed that virtual rotations provided students with a good understanding of program culture and what the programs offer educationally. Neither the leadership nor the interns would recommend replacing in-person aways with virtual experiences. CONCLUSION: Virtual experiences helped bridge the gap after away rotations were canceled. Alongside in-person aways, virtual experiences are likely to play a role in future cycles. However, virtual experiences remain incomparable to in-person away rotations and are not recommended as a replacement.

Effect of Postoperative Immobilization Time on PROMIS Scores and Clinical outcomes in Ankle Fracture Patients.

Background: Ankle fractures are among the most common injuries treated by orthopaedic surgeons, yet little guidance exists in postoperative protocols for ankle fractures concerning time of immobilization. Here, we aim to investigate the association between early mobilization and patient-reported outcomes. Our null hypothesis was that no difference in Patient-Reported Outcomes Measurement Information System (PROMIS) scores would be identified in patients when comparing the effect of time of immobilization. Methods: A retrospective review identified ankle fractures that underwent surgical fixation between 2015 and 2020 at a level 1 trauma center and its associated facilities. One hundred nineteen patients from 9 providers met inclusion criteria for our final analysis. Forty-seven patients were immobilized for <6 weeks (early) and 68 patients were immobilized for ≥6 weeks (late). Our primary outcome measures included the PROMIS questionnaire, time of immobilization, and time to full weightbearing. Our secondary outcome measures included time to return to work, wound complications (infection, delayed healing), and complications associated with fracture fixation (loss of reduction, delayed union, reoperation, hardware failure). Repeated measures analysis of variance as well as linear mixed outcome regression were used to predict each of the PROMIS outcomes of anxiety, depression, physical function, and pain interference. Each model included the predictors of age, sex, race, body mass index (BMI), diabetes, rheumatoid arthritis, smoking status, payor, provider, time to radiographic union, time to return to work, time to full weightbearing, and early vs late immobilized groups. Results: We found no differences in PROMIS scores between mobilization groups even when controlling for possible confounders such as age, BMI, rheumatoid arthritis, smoking status, and diabetes mellitus (P > .05). Furthermore, we found no differences in complications associated with fracture fixation (P > .05). Across our cohort, lower physical function scores were associated with higher BMI, increasing age, and longer time to return to work/play (P < .05). Our analysis further showed that depression, anxiety, pain interference, and physical function levels improve as a function of time (P < .05). Higher BMI was also noted to have a significant impact on PROMIS depression and anxiety when controlling for other variables. African Americans had greater pain interference scores (P < .05). Conclusion: Our study suggests that early mobilization in a walker boot after operative treatment of ankle fractures is a safe alternative to casting in non-neuropathic patients. When considering operative treatment of ankle fractures, factors such as increasing age and BMI are likely to negatively affect postoperative anxiety, physical function, and depression PROMIS scores regardless of immobilization time. Level of Evidence: Level III, retrospective cohort study.

Complementary early-phase magnetic particle imaging and late-phase positron emission tomography reporter imaging of mesenchymal stem cells in vivo.

Stem cell-based therapies have demonstrated significant potential in clinical applications for many debilitating diseases. The ability to non-invasively and dynamically track the location and viability of stem cells post administration could provide important information on individual patient response and/or side effects. Multi-modal cell tracking provides complementary information that can offset the limitations of a single imaging modality to yield a more comprehensive picture of cell fate. In this study, mesenchymal stem cells (MSCs) were engineered to express human sodium iodide symporter (NIS), a clinically relevant positron emission tomography (PET) reporter gene, as well as labeled with superparamagnetic iron oxide nanoparticles (SPIOs) to allow for detection with magnetic particle imaging (MPI). MSCs were additionally engineered with a preclinical bioluminescence imaging (BLI) reporter gene for comparison of BLI cell viability data to both MPI and PET data over time. MSCs were implanted into the hind limbs of immunocompromised mice and imaging with MPI, BLI and PET was performed over a 30-day period. MPI showed sensitive detection that steadily declined over the 30-day period, while BLI showed initial decreases followed by later rapid increases in signal. The PET signal of MSCs was significantly higher than the background at later timepoints. Early-phase imaging (day 0-9 post MSC injections) showed correlation between MPI and BLI data (R2 = 0.671), while PET and BLI showed strong correlation for late-phase (day 10-30 post MSC injections) imaging timepoints (R2 = 0.9817). We report the first use of combined MPI and PET for cell tracking and show the complementary benefits of MPI for sensitive detection of MSCs early after implantation and PET for longer-term measurements of cell viability.

Development of a minimally invasive simultaneous estimation method for quantifying translocator protein binding with [18F]FEPPA positron emission tomography.

BACKGROUND: The purpose of this study was to assess the feasibility of using a minimally invasive simultaneous estimation method (SIME) to quantify the binding of the 18-kDa translocator protein tracer [18F]FEPPA. Arterial sampling was avoided by extracting an image-derived input function (IDIF) that was metabolite-corrected using venous blood samples. The possibility of reducing scan duration to 90 min from the recommended 2-3 h was investigated by assuming a uniform non-displaceable distribution volume (VND) to simplify the SIME fitting. RESULTS: SIME was applied to retrospective data from healthy volunteers and was comprised of both high-affinity binders (HABs) and mixed-affinity binders (MABs). Estimates of global VND and regional total distribution volume (VT) from SIME were not significantly different from values obtained using a two-tissue compartment model (2CTM). Regional VT estimates were greater for HABs compared to MABs for both the 2TCM and SIME, while the SIME estimates had lower inter-subject variability (41 ± 17% reduction). Binding potential (BPND) values calculated from regional VT and brain-wide VND estimates were also greater for HABs, and reducing the scan time from 120 to 90 min had no significant effect on BPND. The feasibility of using venous metabolite correction was evaluated in a large animal model involving a simultaneous collection of arterial and venous samples. Strong linear correlations were found between venous and arterial measurements of the blood-to-plasma ratio and the remaining [18F]FEPPA fraction. Lastly, estimates of BPND and the specific distribution volume (i.e., VS = VT - VND) from a separate group of healthy volunteers (90 min scan time, venous-scaled IDIFs) agreed with estimates from the retrospective data for both genotypes. CONCLUSIONS: The results of this study demonstrate that accurate estimates of regional VT, BPND and VS can be obtained by applying SIME to [18F]FEPPA data. Furthermore, the application of SIME enabled the scan time to be reduced to 90 min, and the approach worked well with IDIFs that were scaled and metabolite-corrected using venous blood samples.

The functional and structural associations of aberrant microglial activity in major depressive disorder.

BACKGROUND: Major depressive disorder (MDD) is a debilitating mental illness that has been linked to increases in markers of inflammation, as well as to changes in brain functional and structural connectivity, particularly between the insula and the subgenual anterior cingulate cortex (sgACC). In this study, we directly related inflammation and dysconnectivity in treatment-resistant MDD by concurrently measuring the following: microglial activity with [18F]N-2-(fluoroethoxyl)benzyl-N-(4phenoxypyridin-3-yl)acetamide ([18F]FEPPA) positron emission tomography (PET); the severity of MDD; and functional or structural connectivity among insula or sgACC nodes. METHODS: Twelve patients with treatment-resistant MDD (8 female, 4 male; mean age ± standard deviation 54.9 ± 4.5 years and 23 healthy controls (11 female, 12 male; 60.3 ± 8.5 years) completed a hybrid [18F]FEPPA PET and MRI acquisition. From these, we extracted relative standardized uptake values for [18F]FEPPA activity and Pearson r-to-z scores representing functional connectivity from our regions of interest. We extracted diffusion tensor imaging metrics from the cingulum bundle, a key white matter bundle in MDD. We performed regressions to relate microglial activity with functional connectivity, structural connectivity and scores on the 17-item Hamilton Depression Rating Scale. RESULTS: We found significantly increased [18F]FEPPA uptake in the left sgACC in patients with treatment-resistant MDD compared to healthy controls. Patients with MDD also had a reduction in connectivity between the sgACC and the insula. The [18F]FEPPA uptake in the left sgACC was significantly related to functional connectivity with the insula, and to the structural connectivity of the cingulum bundle. [18F]FEPPA uptake also predicted scores on the Hamilton Depression Rating Scale.Limitations: A relatively small sample size, lack of functional task data and concomitant medication use may have affected our findings. CONCLUSION: We present preliminary evidence linking a network-level dysfunction relevant to the pathophysiology of depression and related to increased microglial activity in MDD.

Low cost and open source purification apparatus for GMP [13N]Ammonia production.

Nitrogen-13 labeled ammonia ([13N]NH3) has been used for myocardial perfusion imaging with Positron Emission Tomography for decades. Recent increases to regulatory oversight have led to stricter adherence to Good Manufacturing Practice (GMP) when producing this short half-life (9.97 min) radiopharmaceutical. This has increased production costs. Our cyclotron facility initially developed a manual GMP production method, but it was prone to human error. With increased costs in mind, we developed and validated an Arduino-based device to purifying [13N]NH3 for clinical use. Construction, programming, and GMP validation results are discussed. The automated method was found to produce equivalent quality radiopharmaceutical but was more reproducible and robust.

A Human-derived Dual MRI/PET Reporter Gene System with High Translational Potential for Cell Tracking.

PURPOSE: Reporter gene imaging has been extensively used to longitudinally report on whole-body distribution and viability of transplanted engineered cells. Multi-modal cell tracking can provide complementary information on cell fate. Typical multi-modal reporter gene systems often combine clinical and preclinical modalities. A multi-modal reporter gene system for magnetic resonance imaging (MRI) and positron emission tomography (PET), two clinical modalities, would be advantageous by combining the sensitivity of PET with the high-resolution morphology and non-ionizing nature of MRI. PROCEDURES: We developed and evaluated a dual MRI/PET reporter gene system composed of two human-derived reporter genes that utilize clinical reporter probes for engineered cell detection. As a proof-of-concept, breast cancer cells were engineered to co-express the human organic anion transporter polypeptide 1B3 (OATP1B3) that uptakes the clinical MRI contrast agent gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA), and the human sodium iodide symporter (NIS) which uptakes the PET tracer, [18F] tetrafluoroborate ([18F] TFB). RESULTS: T1-weighted MRI results in mice exhibited significantly higher MRI signals in reporter-gene-engineered mammary fat pad tumors versus contralateral naïve tumors (p < 0.05). No differences in contrast enhancement were observed at 5 h after Gd-EOB-DTPA administration using either intravenous or intraperitoneal injection. We also found significantly higher standard uptake values (SUV) in engineered tumors in comparison to the naïve tumors in [18F]TFB PET images (p < 0.001). Intratumoral heterogeneity in signal enhancement was more conspicuous in relatively higher resolution MR images compared to PET images. CONCLUSIONS: Our study demonstrates the ability to noninvasively track cells engineered with our human-derived dual MRI/PET reporter system, enabling a more comprehensive evaluation of transplanted cells. Future work is focused on applying this tool to track therapeutic cells, which may one day enable the broader application of cell tracking within the healthcare system.

Concordance of regional hypoperfusion by pCASL MRI and 15O-water PET in frontotemporal dementia: Is pCASL an efficacious alternative?

BACKGROUND: Clinical diagnosis of frontotemporal dementia (FTD) remains a challenge due to the overlap of symptoms among FTD subtypes and with other psychiatric disorders. Perfusion imaging by arterial spin labeling (ASL) is a promising non-invasive alternative to established PET techniques; however, its sensitivity to imaging parameters can hinder its ability to detect perfusion abnormalities. PURPOSE: This study evaluated the similarity of regional hypoperfusion patterns detected by ASL relative to the gold standard for imaging perfusion, PET with radiolabeled water (15O-water). METHODS AND MATERIALS: Perfusion by single-delay pseudo continuous ASL (SD-pCASL), free-lunch Hadamard encoded pCASL (FL_TE-pCASL), and 15O-water data were acquired on a hybrid PET/MR scanner in 13 controls and 9 FTD patients. Cerebral blood flow (CBF) by 15O-water was quantified by a non-invasive approach (PMRFlow). Regional hypoperfusion was determined by comparing individual patients to the control group. This was performed using absolute (aCBF) and CBF normalized to whole-brain perfusion (rCBF). Agreement was assessed based on the fraction of overlapping voxels. Sensitivity and specificity of pCASL was estimated using hypoperfused regions of interest identified by 15O-water. RESULTS: Region of interest (ROI) based perfusion measured by 15O-water strongly correlated with SD-pCASL (R = 0.85 ± 0.1) and FL_TE-pCASL (R = 0.81 ± 0.14). Good agreement in terms of regional hypoperfusion patterns was found between 15O-water and SD-pCASL (sensitivity = 70%, specificity = 78%) and between 15O-water and FL_TE-pCASL (sensitivity = 71%, specificity = 73%). However, SD-pCASL showed greater overlap (43.4 ± 21.3%) with 15O-water than FL_TE-pCASL (29.9 ± 21.3%). Although aCBF and rCBF showed no significant differences regarding spatial overlap and metrics of agreement with 15O-water, rCBF showed considerable variability across subtypes, indicating that care must be taken when selecting a reference region. CONCLUSIONS: This study demonstrates the potential of pCASL for assessing regional hypoperfusion related to FTD and supports its use as a cost-effective alternative to PET.

Noninvasive Quantification of Cerebral Blood Flow Using Hybrid PET/MR Imaging to Extract the [15 O]H2 O Image-Derived Input Function Free of Partial Volume Errors.

BACKGROUND: Quantification of cerebral blood flow (CBF) with [15 O]H2 O-positron emission tomography (PET) requires arterial sampling to measure the input function. This invasive procedure can be avoided by extracting an image-derived input function (IDIF); however, IDIFs are sensitive to partial volume errors due to the limited spatial resolution of PET. PURPOSE: To present an alternative hybrid PET/MR imaging of CBF (PMRFlowIDIF ) that uses phase-contrast (PC) MRI measurements of whole-brain (WB) CBF to calibrate an IDIF extracted from a WB [15 O]H2 O time-activity curve. STUDY TYPE: Technical development and validation. ANIMAL MODEL: Twelve juvenile Duroc pigs (83% female). POPULATION: Thirteen healthy individuals (38% female). FIELD STRENGTH/SEQUENCES: 3 T; gradient-echo PC-MRI. ASSESSMENT: PMRFlowIDIF was validated against PET-only in a porcine model that included arterial sampling. CBF maps were generated by applying PMRFlowIDIF and two previous PMRFlow methods (PC-PET and double integration method [DIM]) to [15 O]H2 O-PET data acquired from healthy individuals. STATISTICAL TESTS: PMRFlow and PET CBF measurements were compared with regression and correlation analyses. Paired t-tests were performed to evaluate differences. Potential biases were assessed using one-sample t-tests. Reliability was assessed by intraclass correlation coefficients. Statistical significance: α  = 0.05. RESULTS: In the animal study, strong agreement was observed between PMRFlowIDIF (average voxel-wise CBF, 58.0 ± 16.9 mL/100 g/min) and PET (63.0 ± 18.9 mL/100 g/min). In the human study, PMRFlowDIM (y = 1.11x - 5.16, R2  = 0.99 ± 0.01) and PMRFlowPC-PET (y = 0.87x + 3.82, R2  = 0.97 ± 0.02) performed similarly to PMRFlowIDIF, and CBF was within the expected range (eg, 49.7 ± 7.2 mL/100 g/min for gray matter). DATA CONCLUSION: Accuracy of PMRFlowIDIF was confirmed in the animal study with the primary source of error attributed to differences in WB CBF measured by PC MRI and PET. In the human study, differences in CBF from PMRFlowIDIF , PMRFlowDIM , and PMRFlowPC-PET were due to the latter two not accounting for blood-borne activity. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 1.

A Noninvasive Method for Quantifying Cerebral Metabolic Rate of Oxygen by Hybrid PET/MRI: Validation in a Porcine Model.

The gold standard for imaging the cerebral metabolic rate of oxygen (CMRO2) is positron emission tomography (PET); however, it is an invasive and complex procedure that also requires correction for recirculating 15O-H2O and the blood-borne activity. We propose a noninvasive reference-based hybrid PET/magnetic resonance imaging (MRI) method that uses functional MRI techniques to calibrate 15O-O2-PET data. Here, PET/MR imaging of oxidative metabolism (PMROx) was validated in an animal model by comparison to PET-alone measurements. Additionally, we investigated if the MRI-perfusion technique arterial spin labelling (ASL) could be used to further simplify PMROx by replacing 15O-H2O-PET, and if the PMROx was sensitive to anesthetics-induced changes in metabolism. Methods: 15O-H2O and 15O-O2 PET data were acquired in a hybrid PET/MR scanner (3 T Siemens Biograph mMR), together with simultaneous functional MRI (OxFlow and ASL), from juvenile pigs (n = 9). Animals were anesthetized with 3% isoflurane and 6 mL/kg/h propofol for the validation experiments and arterial sampling was performed for PET-alone measurements. PMROx estimates were obtained using whole-brain (WB) CMRO2 from OxFlow and local cerebral blood flow (CBF) from either noninvasive 15O-H2O-PET or ASL (PMROxASL). Changes in metabolism were investigated by increasing the propofol infusion to 20 mL/kg/h. Results: Good agreement and correlation were observed between regional CMRO2 measurements from PMROx and PET-alone. No significant differences were found between OxFlow and PET-only measurements of WB oxygen extraction fraction (0.30 ± 0.09 and 0.31 ± 0.09) and CBF (54.1 ± 16.7 and 56.6 ± 21.0 mL/100 g/min), or between PMROx and PET-only CMRO2 estimates (1.89 ± 0.16 and 1.81 ± 0.10 mLO2/100 g/min). Moreover, PMROx and PMROxASL were sensitive to propofol-induced reduction in CMRO2 Conclusion: This study provides initial validation of a noninvasive PET/MRI technique that circumvents many of the complexities of PET CMRO2 imaging. PMROx does not require arterial sampling and has the potential to reduce PET imaging to 15O-O2 only; however, future validation involving human participants are required.

Do Self-Reported Drug Allergies Influence Clinically Significant Outcome Improvement Following Osteochondral Allograft Transplantation? A Nested Cohort Study.

OBJECTIVE: To compare clinical outcomes for patients who underwent osteochondral allograft transplantation (OCA) based on the presence or absence of one or more self-reported drug allergies. DESIGN: Prospective data were collected from 245 consecutive patients after OCA of the knee from one large academic institution. Patient-reported allergies were obtained via chart review. Patient-reported outcome measures, including activities of daily living of the Knee Outcome Survey (KOS-ADL), Marx Activity Scale, International Knee Documentation Committee (IKDC), and visual analogue scale (VAS) pain were all collected. The minimal clinically important difference (MCID) for each outcome was quantified using a distribution-based method. Independent t tests were used to compare patient-reported outcome measures between those with and without self-reported allergies, while chi-square analysis of association was used to compare rates of MCID achievement. RESULTS: Of 245 patients included, 83 (33.9%) reported having at least one drug allergy at the time of OCA. There were no statistically significant differences with regard to patient demographics, including age, body mass index, gender, or sports participation between those with and without a reported allergy. Similarly, there were no significant differences found between baseline preoperative patient-reported outcomes. Overall, both cohorts demonstrated a significant improvement from baseline scores at 2 years postoperatively. There were no differences found between any patient-reported outcome at 2 years postoperatively. The presence of at least one self-reported drug allergy was not a significant risk factor for failing to achieve the MCID in any specific outcome measure. CONCLUSIONS: The presence of one or more drug allergy was not associated with worse patient-reported outcomes or lower rates of clinically significant outcome improvement after OCA.

Plasma radio-metabolite analysis of PET tracers for dynamic PET imaging: TLC and autoradiography.

BACKGROUND: In molecular imaging with dynamic PET, the binding and dissociation of a targeted tracer is characterized by kinetics modeling which requires the arterial concentration of the tracer to be measured accurately. Once in the body the radiolabeled parent tracer may be subjected to hydrolysis, demethylation/dealkylation and other biochemical processes, resulting in the production and accumulation of different metabolites in blood which can be labeled with the same PET radionuclide as the parent. Since these radio-metabolites cannot be distinguished by PET scanning from the parent tracer, their contribution to the arterial concentration curve has to be removed for the accurate estimation of kinetic parameters from kinetic analysis of dynamic PET. High-performance liquid chromatography has been used to separate and measure radio-metabolites in blood plasma; however, the method is labor intensive and remains a challenge to implement for each individual patient. The purpose of this study is to develop an alternate technique based on thin layer chromatography (TLC) and a sensitive commercial autoradiography system (Beaver, Ai4R, Nantes, France) to measure radio-metabolites in blood plasma of two targeted tracers-[18F]FAZA and [18F]FEPPA, for imaging hypoxia and inflammation, respectively. RESULTS: Radioactivity as low as 17 Bq in 2 µL of pig's plasma can be detected on the TLC plate using autoradiography. Peaks corresponding to the parent tracer and radio-metabolites could be distinguished in the line profile through each sample (n = 8) in the autoradiographic image. Significant intersubject and intra-subject variability in radio-metabolites production could be observed with both tracers. For [18F]FEPPA, 50% of plasma activity was from radio-metabolites as early as 5-min post injection, while for [18F]FAZA, significant metabolites did not appear until 50-min post. Simulation study investigating the effect of radio-metabolite in the estimation of kinetic parameters indicated that 32-400% parameter error can result without radio-metabolites correction. CONCLUSION: TLC coupled with autoradiography is a good alternative to high-performance liquid chromatography for radio-metabolite correction. The advantages of requiring only small blood samples (~ 100 µL) and of analyzing multiple samples simultaneously, make the method suitable for individual dynamic PET studies.

Validation protocol for current good manufacturing practices production of [15O]water for hybrid PET/MR studies.

INTRODUCTION: Oxygen-15 (O; t½ = 122.4 s) has been used for nuclear imaging experiments since the beginning of the field. With the advent of simultaneous hybrid PET/MR technology, [O]water has seen a resurgence and remains the gold standard method for quantitative blood flow studies. The short half-life presents a nontrivial challenge to applying current good manufacturing practices production methods to maintain patient safety. METHODS: A two-vial production method was devised to ensure adequate mixing of [O]water vapour into buffered isotonic saline. For production validation, six batches of [O]water were prepared: sterility, quality control testing and four patient doses. The final dose also underwent quality tested. Routine quality control testing included the following: radiochemical identity and purity, radionuclidic identity and purity, appearance, pH, pyrogenicity, and filter integrity. Sterility was retrospectively confirmed. For validation, breakthrough Pt concentration was also measured. RESULTS: Consistent yields of 10-12 GBq (270-325 mCi) were obtained 3 min after bombardment. Overall, 26 [O]water batches underwent quality control testing under this protocol and all met or exceeded release specifications for clinical use. CONCLUSION: The multiple batch protocol proved to be a safe and effective means for producing [O]water. Furthermore, this protocol could be readily adapted by any facility attempting to produce [O]water for clinical studies. Compared with previous attempts at our site, the protocol outlined here was more consistent and reliable, improved production workflow and led to more available radioactivity for participant injection and QC testing.

Effects of tissue culture on the biomechanical properties of porcine meniscus explants.

BACKGROUND: The meniscus is critical for the normal functioning of the knee joint. The specific aim of this study was to validate an in vitro culture model of meniscus explants for testing the impact of culture conditions on meniscus biomechanical properties. We hypothesized that culturing menisci in the presence of intermediate and high concentration of serum would have a positive effect on the compressive stiffness of the meniscus. METHODS: Unconstrained microindentation testing was performed on porcine meniscus explants cultured with varying concentrations 1%, 5%, or 10% of fetal bovine serum media. Meniscus explants that were not cultured were used as a control. These tests quantified the Young's Modulus of the listed groups of cultured and uncultured explant tissues. FINDINGS: The Young's modulus for 10% cultured explants were significantly higher compared to the control, 1%, and 5% cultured meniscus explants. There was no statistical significance when the Young's modulus between control, 1%, and 5% cultured explants were compared. INTERPRETATION: These results suggest that low concentrations of serum do not impart an anabolic effect on meniscus tissue explant biomechanical properties.

Biologic Adjuvants for the Management of Osteochondral Lesions of the Talus.

Surgical techniques for the management of recalcitrant osteochondral lesions of the talus have improved; however, the poor healing potential of cartilage may impede long-term outcomes. Repair (microfracture) or replacement (osteochondral transplants) is the standard of care. Reparative strategies lead to production of fibrocartilage, which, compared with the native type II articular cartilage, has decreased mechanical and wear properties. The success of osteochondral transplants may be hindered by poor integration between grafts and host that results in peripheral cell death and cyst formation. These challenges have led to the investigation of biologic adjuvants to augment treatment. In vitro and in vivo models have demonstrated promise for cartilage regeneration by decreasing inflammatory damage and increasing the amount of type II articular cartilage. Further research is needed to investigate optimal formulations and time points of administration. In addition, clinical trials are needed to investigate the long-term effects of augmentation.

Customized platelet-rich plasma with transforming growth factor ß1 neutralization antibody to reduce fibrosis in skeletal muscle.

UNLABELLED: The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-ß1, that can cause fibrosis and inhibit optimal muscle healing. OBJECTIVE: To test if neutralizing TGF-ß1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. METHODS: PRP was isolated from in-bred Fisher rats. TGF-ß1 neutralization antibody (Ab) was used to block the TGF-ß1 within the PRP prior to injection. The effects of customized PRP (TGF-ß1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. RESULTS: A significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. CONCLUSION: Neutralizing TGF-ß1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.

Radiosynthesis and ex vivo evaluation of [(18)F]-(S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one for imaging MAO-B with PET.

Carbon-11 labeled SL25.1188 ((S)-5-(methoxymethyl)-3-(6-(4,4,4-trifluorobutoxy)benzo[d]isoxazol-3-yl)oxazolidin-2-one) is a reversible radiotracer for monoamine oxidase B that was recently evaluated in healthy volunteers by positron emission tomography (PET). Herein we report the preparation and ex vivo evaluation of a fluorinated SL25.1188 derivative as a candidate (18)F-labeled PET radiotracer. (S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxy methyl)oxazolidin-2-one (1) was labeled with fluorine-18 in 51% uncorrected radiochemical yield having high radiochemical purity (>98%) and specific activity (109±26GBq/µmol). Ex vivo biodistribution studies demonstrated low radioactivity retention, specific binding and metabolic stability within rat brains. High uptake of radioactivity in bone is consistent with metabolic defluorination. In vitro binding assays of longer chain fluoroalkoxy derivatives revealed that the length of the carbon chain is an integral feature in MAO-B inhibitor potency and selectivity within this scaffold.