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Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy. The understanding of the mechanisms underlying the ability of disseminated tumor cells (DTCs) to form metastasis is incomplete. Here, by using high-resolution intravital imaging of the murine lung to track the fate of breast-derived DTCs, the authors show that macrophages within the primary tumor induce a pro-dissemination and pro-dormancy phenotype in tumor cells, favouring their extravasation in the lung.

We present a rare case of acute exercise-induced bilateral upper-arm compartment syndrome in a patient who, after a year-long hiatus from exercise, subjected his upper-extremities to the stress of over 100 pushups. The patient presented with severe pain of the bilateral biceps and triceps and complaints of dark urine. Decompressive fasciotomy was performed followed by an intensive care unit (ICU) stay for associated myoglobinuria secondary to rhabdomyolysis. The patient suffered no long-term sequelae as a result of his conditions and recovered full function of the bilateral upper-extremities. Albeit rare, acute exercise-induced compartment syndrome should be considered as a diagnosis following unaccustomed bouts of exercise.

Category: Ankle Introduction/Purpose: Ankle arthrodesis (AA) is frequently employed in the treatment of end-stage ankle arthritis, which is common following trauma and athletic injuries. While AA remains a popular therapeutic option, little data exists about activity and sporting capacity following AA. The objective of this research was to determine functional outcomes and sporting activity levels in patients following AA. Methods: Validated questionnaires were emailed to 35 patients with a history of AA at an average follow-up of 52 months. Functional outcomes were assessed using the Foot and Ankle Disability Index (FADI), as well as the associated FADI-Sport. Responses were compared to those from a control population of 24 patients scheduled for AA, at an average preoperative visit of 3 months. Activity levels were assessed prior to injury as well as pre- and post-operatively using the Tegner activity level scale. Results: Average Tegner scores of the 35 surgical patients decreased from 3.82 (SE±0.38) before their injury, to 1.15 (SE±0.19) immediately pre-op, with recovery to 2.67 (SE±0.26) following fusion. Average post arthrodesis FADI and FADI-sport scores in our patients were 76.5% (SE±3.19%) and 33.8% (SE±23.06%). For the preoperative control population, corresponding scores were 47.41% (SE=2.61%) and 22.24% (SE=1.03%). Conclusion: Following AA, we found that patients improved upon their pre-op Tegner score, although they did not return to their before injury level of sporting participation. Additionally, patients reported that they had no current dysfunction with their lower leg. Thus, patients undergoing AA should expect improved lower leg function, though will not likely return to their pre-injury level of activity.

Category: Basic Sciences/Biologics Introduction/Purpose: Vancomycin is often delivered locally for surgical site infection prophylaxis. Recent reports of possible osteotoxicity have led to uncertainty concerning vancomycin’s safety in the setting of arthrodesis and bone healing. Bone formation during arthrodesis takes place as recruited human mesenchymal stromal cells (hMSCs) proliferate and differentiate into mature osteoblasts. The purpose of this research was to determine the impact of vancomycin treatment on hMSCs during osteogenic differentiation. Methods: Human MSCs were cultured in MSC growth media to an appropriate confluence. Cells were cultured for 24 hours to facilitate adherence, after which the media was aspirated and replaced with osteogenic differentiation media (Lonza, Switzerland). Osteogenic differentiation media was supplemented with vancomycin powder to yield solutions with concentrations of 0, 50, 500 & 5000 µg/mL. Fresh vancomycin powder was added with every media change. MSCs viability and proliferation were assessed via live/dead staining with 1 µM calcein-AM and 0.5 µM ethidium homodimer-1 (EthD-1) after 1, 3, and 7 days of differentiation and vancomycin treatment. Mineralization of differentiated cells was assessed via staining with 40 mM alizarin red (ARS; pH 4.1) after 21 days. Semi-quantification of the degree of mineralization was performed by measuring absorbance values at 405 nm using a microplate reader. Microscopy was used for qualitative evaluation. Results: Cell viability decreased with increasing vancomycin concentrations. Impairment of hMSC proliferation was also observed with increasing concentrations of vancomycin. MSCs treated with 5000 µg/mL vancomycin demonstrated significantly less cell growth compared to all other treatment groups (P=0.0001). Absorbance measurements from each well stained with alizarin red was used for semi-quantification of the degree of mineralization. As vancomycin concentrations were increased, absorbance levels decreased (Figure). This reduction in mineralization was also demonstrated qualitatively; with alizarin red less apparent in the wells with increasing vancomycin concentrations (Figure). Conclusion: Local vancomycin is utilized for prevention of infection, often in procedures that necessitate the formation of new bone. Bone healing requires migration, proliferation and differentiation of hMSCs. This work demonstrates impaired viability and function of hMSCs following vancomycin as well as decreased osteoblastic mineralization. Future work will require in vivo studies aimed at determining relative nonunion rates in the setting of vancomycin prophylaxis. Still, the results of this study suggest that vancomycin may be toxic to hMSCs and caution should be exercised by providers when considering vancomycin in foot and ankle patients requiring bony healing following fracture or arthrodesis.

Category: Ankle Introduction/Purpose: Ankle arthrodesis (AA) is frequently employed in the treatment of end-stage ankle arthritis, which is common following trauma and athletic injuries. While AA remains a popular therapeutic option, little data exists about activity and sporting capacity following AA. The objective of this research was to determine functional outcomes and sporting activity levels in patients following AA. Methods: Validated questionnaires were emailed to 35 patients with a history of AA at an average follow-up of 52 months. Functional outcomes were assessed using the Foot and Ankle Disability Index (FADI), as well as the associated FADI-Sport. Responses were compared to those from a control population of 24 patients scheduled for AA, at an average preoperative visit of 3 months. Activity levels were assessed prior to injury as well as pre- and post-operatively using the Tegner activity level scale. Results: Average Tegner scores of the 35 surgical patients decreased from 3.82 (SE±0.38) before their injury, to 1.15 (SE±0.19) immediately pre-op, with recovery to 2.67 (SE±0.26) following fusion. Average post arthrodesis FADI and FADI-sport scores in our patients were 76.5% (SE±3.19%) and 33.8% (SE±23.06%). For the preoperative control population, corresponding scores were 47.41% (SE=2.61%) and 22.24% (SE=1.03%). Conclusion: Following AA, we found that patients improved upon their pre-op Tegner score, although they did not return to their before injury level of sporting participation. Additionally, patients reported that they had no current dysfunction with their lower leg. Thus, patients undergoing AA should expect improved lower leg function, though will not likely return to their pre-injury level of activity.

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 (AT2) cells act as stem cells and can replace both themselves and alveolar type 1 cells (AT1); however, the complex orchestration of AT2 stem cell activity following lung injury is poorly understood owing to the inability of tracking individual stem cells and their dynamic behavior over time. Here, we apply live time lapse imaging to ex vivo mouse precision cut lung slice (PCLS) culture and in vivo mouse lung to track individual GFP-labeled AT2 cells for 72h following intra-tracheal administration of bleomycin. We observe highly dynamic movement of AT2 cells, including migration within and between alveoli, as well as the emergence of at least three distinct morphokinetic AT2 cell states. Small molecule-based inhibition of Rho-associated protein kinase (ROCK) pathway significantly reduced motility of AT2 stem cells following injury and reduced expression of Krt8, a known marker of intermediate progenitor cells. Together, our results uncover motility of alveolar stem cells as a new injury response mechanism in the lung and reveal properties of stem cell motility at high cellular resolution.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Introduction, results and discussion have been updated.

Sustained virologic response (SVR) is defined as aviremia 24 weeks after completion of antiviral therapy for chronic hepatitis C virus (HCV) infection. In analyses of SVR durability, the incidence of late relapse is extremely low (<1%). Histologic regression of both necroinflammation and fibrosis has been demonstrated in paired liver biopsy samples in SVR-achieving patients. More noteworthy is the sustained responder's favorable prognosis even with baseline cirrhosis; despite mostly retrospective analyses, relative to nonresponders or to those untreated, patients with SVR have significantly fewer liver-related complications, less hepatocellular carcinoma, and fewer liver-related deaths. Although HCV is associated with insulin resistance, successful eradication of HCV appears to reduce the risk of impaired fasting glucose and diabetes development. In summary, chronic HCV infection is curable with SVR attainment, and with cure comes improved liver histology and more favorable clinical outcomes, in comparison with patients who do not achieve the same therapeutic milestone.

Previous work has suggested that Emergency Department rotational patient assignment (a system in which patients are algorithmically assigned to physicians) is associated with immediate (first-year) improvements in operational metrics. We sought to determine if these improvements persisted over a longer follow-up period. Single-site, retrospective analysis focused on years 2–4 post-implementation (follow-up) of a rotational patient assignment system. We compared operational data for these years with previously published data from the last year of physician self-assignment and the first year of rotational patient assignment. We report data for patient characteristics, departmental characteristics and facility characteristics, as well as outcomes of length of stay (LOS), arrival to provider time (APT), and rate of patients who left before being seen (LBBS). There were 140,673 patient visits during the five year period; 138,501 (98.7%) were eligible for analysis. LOS, APT, and LBBS during follow-up remained improved vs. physician self-assignment, with improvements similar to those noted in the first year of implementation. Compared with the last year of physician self-assignment, approximate yearly average improvements during follow-up were a decrease in median LOS of 18min (8% improvement), a decrease in median APT of 21min (54% improvement), and a decrease in LBBS of 0.69% (72% improvement). In a single facility study, rotational patient assignment was associated with sustained operational improvements several years after implementation. These findings provide further evidence that rotational patient assignment is a viable strategy in front-end process redesign.

Background/Objectives: Road tests and driving simulators are most commonly used in research studies and clinical evaluations of older drivers. Our objective was to describe the process and associated challenges in adapting an existing, commercial, off-the-shelf (COTS), in-vehicle device for naturalistic, longitudinal research to better understand daily driving behavior in older drivers. Design: The Azuga G2 Tracking Device TM was installed in each participant's vehicle, and we collected data over 5 months (speed, latitude/longitude) every 30-seconds when the vehicle was driven.  Setting: The Knight Alzheimer's Disease Research Center at Washington University School of Medicine. Participants: Five individuals enrolled in a larger, longitudinal study assessing preclinical Alzheimer disease and driving performance.  Participants were aged 65+ years and had normal cognition. Measurements:  Spatial components included Primary Location(s), Driving Areas, Mean Centers and Unique Destinations.  Temporal components included number of trips taken during different times of the day.  Behavioral components included number of hard braking, speeding and sudden acceleration events. Methods:  Individual 30-second observations, each comprising one breadcrumb, and trip-level data were collected and analyzed in R and ArcGIS.  Results: Primary locations were confirmed to be 100% accurate when compared to known addresses.  Based on the locations of the breadcrumbs, we were able to successfully identify frequently visited locations and general travel patterns.  Based on the reported time from the breadcrumbs, we could assess number of trips driven in daylight vs. night.  Data on additional events while driving allowed us to compute the number of adverse driving alerts over the course of the 5-month period. Conclusions: Compared to cameras and highly instrumented vehicle in other naturalistic studies, the compact COTS device was quickly installed and transmitted high volumes of data. Driving Profiles for older adults can be created and compared month-to-month or year-to-year, allowing researchers to identify changes in driving patterns that are unavailable in controlled conditions.

BackgroundAssessment of lymphatic status via sentinel lymph node (SLN) biopsy is an integral and crucial part of melanoma surgical oncology. The most common technique for sentinel node mapping is preoperative planar scintigraphy of an injected gamma-emitting lymphatic tracer followed by intraoperative node localization using a non-imaging gamma probe with auditory feedback. In recent years, intraoperative visualization of SLNs in 3D has become possible by coupling the probe to an external system capable of tracking its location and orientation as it is read out, thereby enabling computation of the 3D distribution of the tracer (freehand SPECT). In this project, the non-imaging probe of the fhSPECT system was replaced by a unique handheld gamma camera containing an array of sodium iodide crystals optically coupled to an array of silicon photomultipliers (SiPMs). A feasibility study was performed in which preoperative SLN mapping was performed using camera fhSPECT and the number of detected nodes was compared to that visualized by lymphoscintigraphy, probe fhSPECT, and to the number ultimately excised under non-imaging probe guidance. ResultsAmong five subjects, SLNs were detected in nine lymphatic basins, with one to five SLNs detected per basin. A basin-by-basin comparison showed that the number of SLNs detected using camera fhSPECT exceeded that using lymphoscintigraphy and probe fhSPECT in seven of nine basins and five of five basins, respectively. (Probe fhSPECT scans were not performed for four basins.) It exceeded the number excised under non-imaging probe guidance for seven of nine basins and equaled the number excised for the other two basins.ConclusionsFreehand SPECT using a prototype SiPM-based gamma camera demonstrates high sensitivity for detection of SLNs in a preoperative setting. Camera fhSPECT is a potential means for efficiently obtaining real-time 3D activity distribution maps in applications such as image-guided percutaneous biopsy, and surgical SLN biopsy or radioguided tumor excision.