Analyses and Utilization of Selectively Tuned Human Adipose-Derived Stromal/Stem Cell Exosomes.

We have developed a hollow fiber bioreactor-based production system for manufacturing large quantities of extracellular vesicles (EVs) containing exosomes from adult human adipose-derived stromal/stem cells (ASCs). By manipulating the cellular bioreactor environment, we have found that we can alter ASC EV production, secretion, and surface protein composition. The aims of this chapter are to describe the methodology for culturing and tuning of adipose ASCs in a bioreactor, along with the collection and isolation of the EVs containing exosomes demonstrating increased HSP70 content.

A 78-Year-Old Woman with Sudden Onset of Left-Sided Hemiballismus.

BACKGROUND Hemiballismus is the most severe form of chorea and is a hyperkinetic disorder characterized by involuntary, high-amplitude movements of the ipsilateral arm and leg, due to lesions of the contralateral side of the central nervous system. Ischemic or hemorrhagic strokes and nonketotic hyperglycemia are predominant etiologies of hemiballismus. Case reports highlighting hemiballismus associated with temporal and parietal lobe infarcts have been published, although research of frontal lobe involvement is limited. CASE REPORT A 78-year-old woman presented to the Emergency Department with sudden-onset left-sided hemiballismus. On examination, she was alert, oriented to self and time, and able to follow commands. Her neurologic examination was notable for left-sided hemiballismus, described by the provider as periodic, uncontrolled, and involving a "flinging" motion of the left upper and lower extremities, sparing the face. She was treated with benzodiazepines in the Emergency Department and administered intravenous levetiracetam. Computed tomography of the head without contrast revealed an old left basal ganglia lacunar infarct. The patient was then admitted to the inpatient service, where magnetic resonance imaging of the brain revealed an acute punctate left superior frontal gyrus cortical infarct. Outpatient electroencephalogram revealed right anterior hemisphere dysfunction. CONCLUSIONS We describe a patient with left-sided sudden onset hemiballismus with an acute infarct of the ipsilateral superior frontal gyrus. This case highlights that brain lesions separate from the basal ganglia can induce hemiballismus, particularly within the frontal lobe, which warrants further research into precentral sulcus functioning and its role in modulating motor activity.

Radiation doses in extraoral bitewing radiography compared with intraoral bitewing and panoramic radiography.

OBJECTIVES: We compared the effective dose (E) and thyroid equivalent dose of 2 extraoral bitewing (EOBW) units and compared E with their respective panoramic (PAN) modes and with intraoral bitewing radiography (IOBW). STUDY DESIGN: Child and adult anthropomorphic phantoms with dosimeters were used to evaluate Orthophos SL, Rayscan α+, and 1 intraoral unit using rectangular and circular collimation. Extraoral bitewing thyroid equivalent dose was assessed without and with thyroid shielding. RESULTS: Child and adult E values of EOBW were lower with Orthophos (3.6 and 8.6 µSv) than with Rayscan (28.1 and 30.2 µSv). For IOBW, E was lower with rectangular vs circular collimation for child (7.0 vs 11.8 µSv) and adult (4.6 vs 14.2 µSv). E values of EOBW were lower than PAN for Orthophos. The IOBW E was lower than Rayscan EOBW for child (≤11.8 vs 28.1 µSv) and adult (≤14.2 vs 30.2 µSv). Adult E for rectangular IOBW (4.6 µSv) was lower than EOBW with Orthophos (8.6 µSv) and Rayscan (30.2 µSv). Thyroid shielding reduced EOBW thyroid equivalent dose with Rayscan in the adult from 190.7 to 89.0 µSv. CONCLUSION: Orthophos provides significantly lower EOBW E than Rayscan, thus EOBW recommendations must be unit specific. For children, Orthophos EOBW could be an alternative to IOBW, for which rectangular collimation is recommended. Thyroid shielding reduced adult Rayscan equivalent dose but added imaging artifacts.

Bitewing radiography dosimetry of the PORTRAY stationary-intraoral tomosynthesis imaging system.

OBJECTIVE: This study measured effective (E) and equivalent doses from adult and child 3-dimensional (3D) and 2D posterior bitewing (PBW) examinations using the PORTRAY stationary-intraoral tomosynthesis radiography system. STUDY DESIGN: Adult and child phantoms and optically stimulated luminescent dosimeters were used to measure doses for adult-4 and child-2 projection PBW examinations acquired without (W/O) and with (W) a direct digital sensor in the beam path. Child doses without and with thyroid shielding were measured. RESULTS: Three-dimensional examination E values (µSv) W/O and W were 16.7 and 7.3 for adult, 9.2 and 3.5 for child, and 8.7 and 3.0 with thyroid shielding, respectively. Two-dimensional examination E values W/O and W were 4.3 and 1.5 for adult, 2.1 and 0.6 for child, and 2.0 and 0.5 with shielding, respectively. Sensor presence reduced E for all adult and child examinations (P = .0001). Child E was reduced compared with adult E for both sensor conditions in 3D (P < .0001) and 2D (P ≤ .0043) imaging. Adult and child 3D W/O and W equivalent thyroid doses did not differ (P ≥ .9996). However, child 2D W/O and W doses were lower (P ≤ .0002). Shielding produced no reduction (P ≥ .1128) for either 3D condition or 2D with the sensor (P = .6615) but reduced child 2D dose without the sensor. CONCLUSIONS: Inclusion of a sensor yielded significant reductions in adult and child E. Sensor presence impacted thyroid dose reduction more than shielding.

Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo.

Background: MicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum, and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis. Methods: Exosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models. Results: U-IPFexo or MF-IPFexo expressed miR-let-7d, miR-29a-5p, miR-181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 min after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models. Conclusions: Our results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models. Funding: This work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).

Renal Autologous Cell Therapy to Stabilize Function in Diabetes-Related Chronic Kidney Disease: Corroboration of Mechanistic Action With Cell Marker Analysis.

Introduction: Chronic kidney disease (CKD) is a worldwide disease without cure. Selected renal cells (SRCs) can augment kidney function in animal models. This study correlates the phenotypical characteristics of autologous homologous SRCs (formulated product called Renal Autologous Cell Therapy [REACT]) injected into patients' kidneys with advanced type 2 diabetes-related CKD (D-CKD) to clinical and laboratory findings. Methods: A total of 22 adults with type 2 D-CKD underwent a kidney biopsy followed by 2 subcortical injections of SRCs, 7 ± 3 months apart. There were 2 patients who had only 1 injection. We compared annualized estimated glomerular filtration rate (eGFR) slopes pre- and post-REACT injection using the 2009 CKD-EPI formula for serum creatinine (sCr) and the 2012 CKD-EPI Creatinine-Cystatin C equation and report clinical/laboratory changes. Fluorescent Activated Cell Sorting (FACS) Analysis for renal progenitor lineages in REACT and donor vascular endothelial growth factor A (VEGF-A) analysis were performed. Longitudinal parameter changes were analyzed with longitudinal linear mixed effects model. Results: At baseline, the mean diabetes duration was 18.4 ± 8.80 years, glycated hemoglobin (Hgb) was 7.0 ± 1.05, and eGFR was 40.3 ± 9.35 ml/min per 1.73 m2 using the 2012 CKD-EPI cystatin C and sCr formulas. The annualized eGFR slope (2012 CKD-EPI) was -4.63 ml/min per 1.73 m2 per year pre-injection and improved to -1.69 ml/min per 1.73 m2 per year post-injection (P = 0.015). There were 7 patients who had an eGFR slope of >0 ml/min per 1.73 m2 postinjection. SRCs were found to have cell markers of ureteric bud, mesenchyme cap, and podocyte sources and positive VEGF. There were 2 patients who had remote fatal adverse events determined as unrelated with the biopsies/injections or the REACT product. Conclusion: Our cell marker analysis suggests that SRCs may enable REACT to stabilize and improve kidney function, possibly halting type 2 D-CKD progression.

Opioid-Free Discharge is Not Associated With Increased Unplanned Healthcare Encounters After Ureteroscopy: Results From a Statewide Quality Improvement Collaborative.

OBJECTIVES: To evaluate patient factors associated with post-ureteroscopy opioid prescriptions, provider-level variation in opioid prescribing, and the relationship between opioid-free discharges and ED visits. METHODS: This is a retrospective analysis of a prospective cohort study of adults age 18 years and older who underwent primary ureteroscopy for urinary stones from June 2016 to September 2019 within the Michigan Urological Surgery Improvement Collaborative (MUSIC) Reducing Operative Complications from Kidney Stones (ROCKS) quality improvement initiative. Postoperative opioid prescription trends and variation among practices and surgeons were examined. Multivariable logistic regression models defined risk factors for receipt of opioid prescriptions. The association among opioid prescriptions and postoperative ED visits within 30 days of surgery was assessed among complete case and propensity matched cohorts, matched on all measured characteristics other than opioid receipt. RESULTS: 13,143 patients underwent ureteroscopy with 157 urologists across 28 practices. Post-ureteroscopy opioid prescriptions and ED visits declined (86% to 39%, P<.001; 10% to 6%, P<.001, respectively). Practice and surgeon-level opioid prescribing varied from 8% to 98%, and 0% to 98%, respectively. Patient-related factors associated with opioid receipt included male, younger age, and history of chronic pain. Procedure-related factors associated with opioid receipt included pre- and post-ureteroscopy ureteral stenting and access sheath use. An opioid-free discharge was not associated with increased odds of an ED visit (OR 0.77, 95% CI 0.62-0.95, P=.014). CONCLUSIONS: There was no increase in ED utilization among those not prescribed an opioid after ureteroscopy, suggesting their routine use may not be necessary in this setting.

Comparative dosimetry of radiography device, MSCT device and two CBCT devices in the elbow region.

The aim of the study was to estimate and to compare effective doses in the elbow region resulting from four different x-ray imaging modalities. Absorbed organ doses were measured using 11 metal oxide field effect transistor (MOSFET) dosimeters that were placed in a custom-made anthropomorphic elbow RANDO phantom. Examinations were performed using Shimadzu FH-21 HR radiography device, Siemens Sensation Open 24-slice MSCT-device, NewTom 5G CBCT device, and Planmed Verity CBCT device, and the effective doses were calculated according to ICRP 103 recommendations. The effective dose for the conventional radiographic device was 1.5 µSv. The effective dose for the NewTom 5G CBCT ranged between 2.0 and 6.7 µSv, for the Planmed Verity CBCT device 2.6 µSv and for the Siemens Sensation MSCT device 37.4 µSv. Compared with conventional 2D radiography, this study demonstrated a 1.4-4.6 fold increase in effective dose for CBCT and 25-fold dose for standard MSCT protocols. When compared with 3D CBCT protocols, the study showed a 6-19 fold increase in effective dose using a standard MSCT protocol. CBCT devices offer a feasible low-dose alternative for elbow 3D imaging when compared to MSCT.

Ureteral Stent Placement following Ureteroscopy Increases Emergency Department Visits in a Statewide Surgical Collaborative.

PURPOSE: Ureteral stents are commonly placed after ureteroscopy. Although studies indicate that stents are associated with patient discomfort, their impact on downstream health services use is unclear. We examined patterns of stent utilization in Michigan and their association with unplanned health care encounters. MATERIALS AND METHODS: We used the Michigan Urological Surgery Improvement Collaborative's Reducing Operative Complications from Kidney Stones (MUSIC ROCKS) clinical registry to identify ureteroscopy cases between 2016 and 2019. Factors associated with stent placement were examined using bivariate and multivariable statistics. Using multivariable logistic regression, we evaluated whether stent placement was associated with emergency department visits and hospitalizations within 30 days. RESULTS: We identified 9,662 ureteroscopies and a stent was placed in 7,025 (73%) of these. Frequency of stent use across the 137 urologists varied (11%-100%, p <0.001) and was not associated with total case volume. Factors associated with stent use included age and stone size. Pre-stented cases and renal stones had a decreased odds of stent placement. On multivariable analysis after adjusting for risk factors, stent placement was associated with a 1.25 higher odds of emergency department visit (OR 1.25, 95% CI 1.01-1.54, p=0.043) but not hospitalization (OR 1.28, 95% CI 0.94-1.76, p=0.12). In a single high volume practice, 0.5% of cases that omitted a stent required urgent stenting postoperatively. CONCLUSIONS: There is substantial variation in the use of stents in Michigan, irrespective of case volume. Stent placement significantly increased the odds of an emergency department visit after surgery. Importantly, stent omission rarely required subsequent urgent stent placement.

Intraoral radiographs: A comparison of dose and risk reduction with collimation and thyroid shielding.

BACKGROUND: The National Commission on Radiation Protection and Measurements has reinforced its recommendation for the use of rectangular collimation for intraoral radiography in its Report No. 177 published in 2019. This study compared effective dose (E) using circular and rectangular collimator (RC) modalities. METHODS: The authors exposed 18 projections for adult and 12 projections for child full-mouth series using an original equipment 6 centimeter diameter circular collimator (circular), original equipment rectangular positioning indicator device (Focus-RC), and 5 universal RC modalities (JadRad-RC, Rinn-RC, Durr-RC, DEXshield-RC, and TruAlign-RC) for adult and child phantoms. The authors acquired dosimetry using optically stimulated luminescence dosimeters. Exposures were made with a Focus (Instrumentarium) intraoral source using 70 peak kilovoltage and total milliamperes of 5.34 (adult) and 2.7 (child). RESULTS: Adult E was lowest for Focus-RC (54 microsieverts), which also produced the greatest exposure area reduction (51%) compared with circular, followed by JadRad-RC (55 µSv), Durr-RC (58 µSv), Rinn-RC (62 µSv), DEXshield-RC (70 µSv), TruAlign-RC (85 µSv), and circular (86 µSv). Child E followed a similar trend: Focus-RC (44 µSv), JadRad-RC (44 µSv), Durr-RC (45 µSv), Rinn-RC (48 µSv), DEXshield-RC (53 µSv), TruAlign-RC (85 µSv), and circular (89 µSv). When used with thyroid shielding, circular collimation thyroid dose was reduced by as much as 59%. CONCLUSIONS: Focus-RC techniques yielded the greatest dose reduction compared with alternative RC and circular. In addition to shape, collimator dimensions should be considered as significant factors affecting patient E. RC alone yielded a greater reduction in thyroid dose than did circular with thyroid shielding. PRACTICAL IMPLICATIONS: This study's findings underscore the updated recommendations of the National Commission on Radiation Protection and Measurements Report No.177, which emphasized the benefits and important practical considerations of RC with intraoral imaging.

Bitewing radiography dosimetry of a stationary intraoral tomosynthesis imaging system.

OBJECTIVES: This study assessed effective doses (E) from conventional and stationary intraoral tomosynthesis (s-IOT) radiography for posterior bitewing (PBW) examinations and evaluated the effect of sensor attenuation. STUDY DESIGN: An adult human tissue-equivalent phantom and optically stimulated luminescent dosimeters were used. Series of 4 PBW radiographs were acquired with circular and rectangular collimation. s-IOT PBW radiographs were acquired with built-in rectangular collimation. Radiographs were acquired without and with a sensor in the beam path. RESULTS: E (in µSv) was 15.7 and 8.2 for conventional-circular, 4.6 and 1.1 for conventional-rectangular, and 11.9 and 5.9 for s-IOT in sensor-absent and sensor-present scenarios, respectively. For sensor-absent exposures, E for conventional-rectangular was 29.3% and E for s-IOT was 75.8% of the conventional-circular dose. With the sensor present, these values were 13.4% and 72.0%, respectively (P < .001). Sensor-present E was lower than sensor-absent E for all modalities (P < .001). Reductions in equivalent doses were similar to effective dose reductions. CONCLUSIONS: For PBW examinations, E for s-IOT was smaller than for conventional radiography with circular collimation, but larger than for conventional radiography with rectangular collimation. The presence of a sensor maintained these differences but reduced E for all modalities.

Estimation of effective doses from MDCT and CBCT imaging of extremities.

Little research has been published on the radiation burden of cone beam computed tomography (CBCT) in comparison with multi-row detector computed tomography (MDCT) for extremity imaging. This manuscript reports effective doses produced by standard and reduced dose imaging protocols for a 128-slice MDCT unit and a dedicated extremity imaging device using CBCT technology. Anthropomorphic phantoms with x-ray attenuation characteristics simulating adult hand-wrist, foot-ankle, and knee areas were modified to receive optically stimulated luminescent dosimeters (OSL). Standard and lower kVp reduced dose (Lite) exposures were compared. Effective doses for four age groups (five years old to adult) were calculated following 2007 recommendations of the ICRP. Standard CBCT imaging protocols produced effective doses (1.3-21.1 µSv) that were on the order of daily US ubiquitous background radiation doses. CBCT doses were on average 90% less than comparable MDCT views of the same anatomy (9.1-204 µSv). Child doses were significantly greater than adult doses (p = 0.0001). CBCT doses could be further reduced by 36%-51% with Lite exposure protocols. These protocols can be recommended for children and in cases where increased image noise will not interfere with the diagnostic task.

Hand-wrist, knee, and foot-ankle dosimetry and image quality measurements of a novel extremity imaging unit providing CBCT and 2D imaging options.

PURPOSE: Radiation dose is a general concern in diagnostic imaging and a special concern for children who are at greater risk from radiation effects. This study evaluates effective doses (E) produced during 2D and volume imaging with a novel cone beam computed tomography (CBCT) based extremity imaging device. The device's compact size and protocol options offer image choices that enhance the potential for reduced dose and improved diagnostics when evaluating sports injuries. METHODS: Hand-wrist, foot-ankle, and knee phantoms were developed for use with optically stimulated luminescent dosimeters (OSL). Dosimetry of transmission radiographs (2D) and CBCT volumes (3D) was assessed for Standard and lowered dose (Lite) exposure protocols. Effective dose was calculated for child and adult age groups. Image quality was assessed with contrast-to-noise ratio (CNR) and modulation transfer function (MTF). A figure of merit was calculated as the square of CNR divided by E. RESULTS: Standard 2D doses ranged from 0.001-0.06 µSv for adults, 0.001-0.05 µSv for 15-yr olds, 0.003-0.13 µSv for 10-yr olds, and 0.005-0.20 for 5-yr olds. Dose reductions with Lite protocols ranged from 26 to 51%. Standard CBCT doses ranged from 0.3-4.6 µSv for adults, 0.3-4.4 µSv for 15-yr olds, 0.6-9.8 µSv for 10-yr olds, and 1-22.6 µSv for 5-yr olds. For both 2D and 3D a trend of increasing imaging dose with reduced age was present and was statistically significant for children below the age of 10 (P = 0.0009). Lite dose reductions averaged 47%. CNR was statistically reduced in Lite dose scans (P = 0.0384) but was not statistically different using FOM analysis (P = 0.3089). MTF was not significantly affected by the two dose protocols (P = 0.8104). CONCLUSION: CBCT effective doses calculated from anthropomorphic phantom exposures following manufacturer suggested protocols appear to be substantially less than previously reported doses for similar MDCT extremity examinations. In this study, effective dose from 2D radiographic imaging was approximately two orders of magnitude less than CBCT imaging. Doses were on the order of a few minutes to hours of ubiquitous per-capita background dose for 2D imaging and a few hours to days for 3D imaging. Dose significantly increased for children younger than age 10. Lite protocols resulted in substantial dose reductions and can be recommended for children and those examinations where reduced CNR will not affect diagnosis. Flexibility of 2D and 3D imaging options with low-dose protocols make this technology a promising option for radiographic evaluation of the extremities. Efficacy studies are needed to determine when MDCT, CBCT or Digital Radiography are best used for particular diagnostic tasks.

Initial Assessment of Variability of Responses to Toxicants in Donor-Specific Endothelial Colony Forming Cells.

There is increased interest in using high throughput in vitro assays to characterize human population variability in response to toxicants and drugs. Utilizing primary human endothelial colony-forming cells (ECFCs) isolated from blood would be highly useful for this purpose because these cells are involved in neonatal and adult vasculogenesis. We characterized the cytotoxicity of four known toxic chemicals (NaAsO2, CdCl2, tributyltin [TBT], and menadione) and their four relatively nontoxic counterparts (Na2HAsO4, ZnCl2, SnCl2, and phytonadione, respectively) in eight ECFC clones representing four neonatal donors (2 male and 2 female donors, 2 clones per donor). ECFCs were exposed to 9 concentrations of each chemical in duplicate; cell viability was evaluated 48 h later using the fluorescent vital dye fluorescent dye 5-Carboxyfluorescein Diacetate (CFDA), yielding concentration-effect curves from each experiment. Technical (day-to-day) variability of the assay, assessed from three independent experiments, was low: p-values for the differences of results were 0.74 and 0.64 for the comparison of day 2 vs. day 1 and day 3 vs. day 1, respectively. The statistical analysis used to compare the entire concentration-effect curves has revealed significant differences in levels of cytotoxicity induced by the toxic and relatively nontoxic chemical counterparts, demonstrating that donor-specific ECFCs can clearly differentiate between these two groups of chemicals. Partitioning of the total variance in the nested design assessed the contributions of between-clone and between-donor variability for different levels of cytotoxicity. Individual ECFC clones demonstrated highly reproducible responses to the chemicals. The most toxic chemical was TBT, followed by NaAsO2, CdCl2, and Menadione. Nontoxic counterparts exhibited low cytotoxicity at the higher end of concentration ranges tested. Low variability was observed between ECFC clones obtained from the same donor or different donors for CdCl2, NaAsO2, and TBT, but for menadione, the between-donor variability was much greater than the between-clone variability. The low between-clone variability indicates that an ECFC clone may represent an individual donor in cell-based assays, although this finding must be confirmed using a larger number of donors. Such confirmation would demonstrate that an in vitro ECFC-based testing platform can be used to characterize the inter-individual variability of neonatal ECFCs exposed to drugs and/or environmental toxicants.

Recognizing the Continuous Nature of Expression Heterogeneity and Clinical Outcomes in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) has been previously classified into putative discrete prognostic subtypes by gene expression profiling. To investigate the robustness of these proposed subtype classifications, we evaluated 12 public datasets, together with a new dataset of 265 ccRCC gene expression profiles. Consensus clustering showed unstable subtype and principal component analysis (PCA) showed a continuous spectrum both within and between datasets. Considering the lack of discrete delineation and continuous spectrum observed, we developed a continuous quantitative prognosis score (Continuous Linear Enhanced Assessment of RCC, or CLEAR score). Prognostic performance was evaluated in independent cohorts from The Cancer Genome Atlas (TCGA) (n = 414) and EMBL-EBI (n = 53), CLEAR score demonstrated both superior prognostic estimates and inverse correlation with anti-angiogenic tyrosine-kinase inhibition in comparison to previously proposed discrete subtyping classifications. Inverse correlation with high-dose interleukin-2 outcomes was also observed for the CLEAR score. Multiple somatic mutations (VHL, PBRM1, SETD2, KDM5C, TP53, BAP1, PTEN, MTOR) were associated with the CLEAR score. Application of the CLEAR score to independent expression profiling of intratumoral ccRCC regions demonstrated that average intertumoral heterogeneity exceeded intratumoral expression heterogeneity. Wider investigation of cancer biology using continuous approaches may yield insights into tumor heterogeneity; single cell analysis may provide a key foundation for this approach.

Pediatric Phantom Dosimetry of Kodak 9000 Cone-beam Computed Tomography.

PURPOSE: The purpose of the study was to evaluate the radiation dose of the Kodak 9000 cone-beam computed tomography (CBCT) device for different anatomical areas using a pediatric phantom. METHODS: Absorbed doses resulting from maxillary and mandibular region three by five cm CBCT volumes of an anthropomorphic 10-year-old child phantom were acquired using optical stimulated dosimetry. Equivalent doses were calculated for radiosensitive tissues in the head and neck area, and effective dose for maxillary and mandibular examinations were calculated following the 2007 recommendations of the International Commission on Radiological Protection (ICRP). RESULTS: Of the mandibular scans, the salivary glands had the highest equivalent dose (1,598 microsieverts [µSv]), followed by oral mucosa (1,263 µSv), extrathoracic airway (pharynx, larynx, and trachea; 859 µSv), and thyroid gland (578 µSv). For the maxilla, the salivary glands had the highest equivalent dose (1,847 µSv), followed closely by oral mucosa (1,673 µSv), followed by the extrathoracic airway (pharynx, larynx, and trachea; 1,011 µSv) and lens of the eye (202 µSv). CONCLUSION: Compared to previous research of the Kodak 9000, completed with the adult phantom, a child receives one to three times more radiation for mandibular scans and two to 10 times more radiation for maxillary scans.

Exosomes for repair, regeneration and rejuvenation.

INTRODUCTION: Application of regenerative medicine strategies for repair of organs/tissue impacted by chronic disease is an active subject for product development. Such methodologies emphasize the role of stem cells as the active biological ingredient. However, recent developments in elucidating mechanisms of action of these therapies have focused on the role of paracrine, 'action-at-a-distance' modus operandi in mediating the ability to catalyze regenerative outcomes without significant site-specific engraftment. A salient component of this secreted regenerative milieu are exosomes: 40-100 nm intraluminal vesicles that mediate transfer of proteins and nucleic acids across cellular boundaries. AREAS COVERED: Here, we synthesize recent studies from PubMed and Google Scholar highlighting how cell-based therapeutics and cosmeceutics are transitioning towards the secretome generally and exosomes specifically as a principal modulator of regenerative outcomes. EXPERT OPINION: Exosomes contribute to organ development and mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. Encapsulation of the active biological ingredients of regeneration within non-living exosome carriers may offer process, manufacturing and regulatory advantages over stem cell-based therapies.

Technical Performance of Universal and Enhanced Intraoral Imaging Rectangular Collimators.

PURPOSE: The purpose of this study was to compare the number and type of technical errors between 2 rectangular collimators, time/motion effort and radiographer preference. METHODS: Subjects (n=17) were recruited to expose an 18 projection full mouth series (FMX) using Tru-Align™ (enhanced) and Rinn® (universal) collimator devices. Both FMXs were exposed using photostimulable phosphor (PSP) digital sensors on a DXTTR manikin with an intraoral x-ray unit. A 5-question survey evaluated ease of device use, time required and device preference. Data were analyzed using frequencies, paired t-test, ANOVA and least squares means using a general linear model. RESULTS: A lower mean number of technique errors per FMX occurred with the enhanced device (9.7) compared to the universal device (12.1). Collimator centering errors occurred 3-times more often with the universal device. The mean numbers of diagnostically unacceptable errors per FMX were similar (Universal=3.2 vs Enhanced=2.9). The least squares means adjusted model showed a statistically significant difference of errors between the 2 devices (p=0.0478) and errors by location when comparing posterior to anterior and posterior to bitewing (p<0.0001). Subjects (94%) preferred the enhanced device and found it easier to use compared to the universal device. Significantly less time was needed to expose an FMX (4 min) when using the enhanced device (p=0.0001). CONCLUSION: The enhanced device enabled subjects to expose diagnostically acceptable radiographs more efficiently with fewer collimator centering errors; however, it does so with a 35% greater exposure area and a concomitant increase in patient dose.

Isolation, culturing, and characterization of cardiac muscle cells from nonhuman primate heart tissue.

Cardiac safety pharmacology requires in vitro testing of all drug candidates before clinical trials in order to ensure they are screened for cardiotoxic effects which may result in severe arrhythmias and, ultimately, cardiomyopathy (Chi, Nat Rev Drug Discov 12:565-567, 2013). Given the physiological similarities between nonhuman primates and humans, isolated primate cardiac muscle cells are an ideal animal model for such in vitro testing. The aims of this chapter are to describe two methods for isolating and culturing primate cardiac muscle cells. One method uses mechanical dissociation of the tissue followed by placing the small pieces onto a Petri dish and culturing these tissue explants. The other method also uses mechanical dissociation but is then followed by enzymatic digestion and culturing of the cell suspension. Methods are also described for phenotypically characterizing cardiac muscle cells by flow cytometry. Based on the location within the heart tissue chosen for cell isolation, a dividing population of cardiac muscle cells expressing cardiomyocyte cell markers was obtained.

Cell-based therapeutic products: potency assay development and application.

Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Ideally, a potency assay will leverage the product's mechanism of action. Alternatively, the assay may focus on a therapeutically relevant biological activity. The absence of rigorous mechanistic data for the majority of cell-based therapeutics currently in the process research pipeline has impeded efforts to design and validate indices of product potency. Development of a systematic battery of parallel functional assays that, taken together, can address all potential mechanisms of action believed to be relevant for the product platform is recommended. Such an approach is especially important during preclinical development. Here, we summarize the principal and unique challenges facing the development of functionally relevant and rigorous potency assays for cell-based therapeutics. We present perspectives regarding potency assay development for these products as illustrated by our experiences in process R&D of cryopreserved hepatocytes (Incara Pharmaceuticals) and selected renal cells (Tengion).