Comparing spatial distributions of ALA-PpIX and indocyanine green in a whole pig brain glioma model using 3D fluorescence cryotomography.

Significance: ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain. Aim: We aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography. Approach: A glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24 h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics. Results: Significant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively). Conclusions: This study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.

Centering the role of community health workers in social risk screening, referral, and follow-up within the primary care setting.

BACKGROUND: Community health workers (CHWs) remain an underutilized resource in social risk diagnostics in the primary care setting. This process evaluation study seeks to assess the role of CHWs in social risk screening, referral, and follow-up through process mapping to identify barriers to the process for future quality improvement efforts. METHODS: Researchers at the Arizona Prevention Research Center (AzPRC) engaged with two Federally Qualified Health Centers (FQHCs) in two of Arizona's major urban areas to evaluate their internal processes for social risk screening and intervention. The Consolidated Framework for Implementation Research (CFIR) was used to direct a process mapping exercise to visually describe the workflow, gaps, and barriers to identifying and addressing social risk. RESULTS: The process unveiled key areas for health system improvements in the community setting, the organizational setting, and in the implementation of social risk screening, referral, and follow-up. Further, process maps highlight the potential resources needed for effective CHW integration to address social risk in the primary care setting. CONCLUSIONS: Our findings demonstrate the importance of organizational tools, such as process mapping, to assist primary care settings in evaluating internal processes for quality improvement in addressing social risk and in effectively integrating the CHW workforce. Subsequent research will evaluate rates of social risk screening, referral, and follow-up within all of Arizona's FQHCs and propose models for CHW integration to address social risk in primary care and strengthen social risk screening reach and effectiveness.

Disparities in pediatric parotid cancer treatment and presentation: A National study.

OBJECTIVES: Although parotid gland malignancies are uncommon, they nevertheless represent a cause of morbidity and mortality in the pediatric population. Few studies have sought to identify disparities related to their presentation, treatment, and survival. There is a need to understand these variations to improve care for historically underrepresented groups. STUDY DESIGN: Retrospective Cohort Study. SETTING: Surveillance, Epidemiology, and End Results (SEER) Program Database. METHODS: Analysis of pediatric patients with parotid gland malignancies between 2000 and 2019. Race and ethnicity were classified as Non-Hispanic White, Non-Hispanic Black, Asian, and Hispanic for multivariable analysis. Outcomes included tumor size and stage at diagnosis, survival, and need for facial nerve sacrifice. Kaplan-Meier analysis was used to analyze survival. Multivariable logistic regression was conducted to identify predictors of outcomes. RESULTS: 149 patients met the criteria for inclusion. Stratified by race/ethnicity, Non-Hispanic Black (Median 23 mm, IQR 15-33), Asian (30 mm, 14-32), and Hispanic (23 mm, 20-28) patients had larger tumors at presentation than Non-Hispanic White patients (18 mm, 12-25, p = 0.017). Disease-specific survival differed by time-to-treatment (log-rank, p = 0.01) and overall survival differed by income (p < 0.001). On multivariable analysis, Hispanic patients were more likely to experience facial nerve sacrifice (OR 3.71, 95%CI 1.25-11.6, p = 0.020), and Non-Hispanic Black (OR 3.37, 0.95-11.6, = 0.053) and Asian (OR 5.67, 1.46-22.2, p = 0.011) patients presented with larger tumors compared to Non-Hispanic White patients. CONCLUSIONS: Variations in presentation and treatment exist across race and ethnicity in pediatric parotid cancer. Identifying these disparities may help improve access and outcomes for underserved patient populations. LEVEL OF EVIDENCE: III.

Testosterone Recovery Following Androgen Suppression and Prostate Radiotherapy (TRANSPORT): A Pooled Analysis of Five Randomized Trials from the Meta-Analysis of Randomized Trials in Cancer of the Prostate (MARCAP) Consortium.

BACKGROUND AND OBJECTIVE: Time to testosterone recovery (TR) following androgen deprivation therapy (ADT) with gonadotropin-releasing hormone agonists varies widely. We evaluate TR kinetics and the oncological impact of an effective castration period in patients receiving definitive radiotherapy and ADT for prostate cancer. METHODS: We obtained individual patient data from randomized controlled trials of radiotherapy with ADT and prospectively collected serial testosterone data from the MARCAP Consortium. We estimated the times to noncastrate TR (>1.7 nmol/l) and nonhypogonadal TR (>8.0 nmol/l) were estimated for each prescribed ADT duration, and developed corresponding nomograms. The association between effective castration period and metastasis-free survival (MFS) for any given ADT duration was evaluated via multivariable Cox regression. We conducted cubic spline analyses to assess nonlinear associations. KEY FINDINGS AND LIMITATIONS: We included 1444 men from five trials in the analysis, of whom 115 received 4 mo, 880 received 6 mo, 353 received 18 mo, 36 received 28 mo, and 60 received 36 mo of ADT. Times to noncastrate TR and to nonhypogonadal TR varied considerably by ADT duration. Higher baseline testosterone and lower age were associated with a higher likelihood of TR (p < 0.001 for both). Effective castration period was not linearly associated with MFS for any ADT duration on Cox regression. Cubic spline analysis revealed that the optimal effective castration period for an MFS benefit was 10.6 mo for men who received 6 mo of ADT and 18 mo for men who received 18 mo of ADT. CONCLUSIONS AND CLINICAL IMPLICATIONS: Time to TR varies according to the ADT duration, baseline testosterone, and age. The relationship between effective castration period and MFS may be nonlinear, with a longer effective castration period being helpful for men receiving 6 mo of ADT.

Effect of concomitant medications on treatment response and survival in non-metastatic castrate resistant prostate cancer: Exploratory analysis of the SPARTAN trial.

BACKGROUND: We performed an exploratory analysis of the SPARTAN trial to determine whether concomitant exposure to several classes of commonly prescribed medications influenced the effect of apalutamide on overall survival (OS) and metastasis-free survival (MFS) in patients with non-metastatic castration-resistant prostate cancer (nmCRPC). PATIENTS AND METHODS: SPARTAN was a phase III randomized controlled trial in which nmCRPC patients were randomly assigned in a 2:1 ratio to receive androgen deprivation therapy with or without apalutamide. We focused on 5 commonly prescribed classes of medications: metformin, statins, angiotensin converting enzyme inhibitors (ACEI), acetylsalicylic acid (ASA), and proton pump inhibitors (PPI) based on a plausible biological and clinical rationale. To determine the potential effect modification, we applied multivariable Cox regression models for OS and MFS separately with additional interaction terms. To determine the independent association of concomitant medications with OS and MFS, we used IPTW-based log-rank test. A 2-sided p < 0.01 was considered statistically significant. RESULTS: We did not find statistically significant differences in effect from apalutamide on OS across subgroups stratified by concomitant exposure to any of the medication classes. While there was some difference in the treatment effect from apalutamide on MFS between patients with concomitant statins (adjusted hazard ratio [aHR]: 0.20; 95 % CI: 0.15-0.28) versus without concomitant statins (aHR: 0.31 [0.24-0.39]), this did not reach the pre-specified threshold of statistical significance (p = 0.011). On IPTW-based analysis, patients treated concomitantly with metformin (median: not reached versus 31 months; p = 0.002), or ACEI (median: 37 versus 29 months, p = 0.006) had significantly improved MFS. CONCLUSIONS: In this post-hoc exploratory analysis of SPARTAN, effects of apalutamide on MFS and OS were consistent across subgroups stratified by exposure to concomitant medications. Exposure to concomitant metformin and ACEI was independently associated with a significant improvement in MFS.

Dynamic oxygen assessment techniques enable determination of anesthesia's impact on tissue.

Tissue oxygenation is well understood to impact radiosensitivity, with reports demonstrating a significant effect of breathing condition and anesthesia type on tissue oxygenation levels and radiobiological response. However, the temporal kinetics of intracellular and extracellular oxygenation have never been quantified, on the timescale that may affect radiotherapy studies. C57BL/6 mice were anesthetized using isoflurane at various percentages or ketamine/xylazine (ket/xyl: 100/10 mg/kg) (N = 48). Skin pO2 was measured using Oxyphor PdG4 and tracked after anesthetization began. Oxyphor data was validated with relative measurements of intracellular oxygen via protoporphyrin IX (PpIX) delayed fluorescence (DF) imaging. Ex vivo localization of both PdG4 Oxyphor and PpIX were quantified. Under all isoflurane anesthesia conditions, leg skin pO2 levels significantly increased from 12-15 mmHg at the start of anesthesia induction (4-6 minutes) to 24-27 mmHg after 10 minutes (p < 0.05). Ketamine/xylazine anesthesia led to skin pO2 maintained at 15-16 mmHg throughout the 10-minute study period (p < 0.01). An increase of pO2 in mice breathing isoflurane was demonstrated with Oxyphor and PpIX DF, indicating similar intracellular and extracellular oxygenation. These findings demonstrate the importance of routine anesthesia administration, where consistency in the timing between induction and irradiation may be crucial to minimizing variability in radiation response.

Defining the Landscape of Educational Experiences in Transplant Infectious Diseases: A National Survey of Infectious Diseases Fellows in the United States.

Background: Transplant infectious diseases (TID) is a growing area of expertise within infectious diseases (ID), but TID training is not standardized. Previous surveys of fellows identified opportunities to improve TID education resources but did not explore didactic, clinical, and nonclinical experiences comprehensively. Methods: The American Society of Transplantation ID Community of Practice surveyed adult and pediatric fellows in US-based general ID or dedicated TID training programs to explore their didactic exposure, clinical experiences, and non-direct patient care activities in TID. Results: A total of 234 fellows initiated the survey, and 195 (83%) (190 general ID and 19 TID fellows, including 125 adult, 76 pediatric, and 8 combined adult-pediatric fellows) completed the entire survey. More than half of the fellows described receiving no formal curricular content on most foundational topics in transplant medicine. Almost all respondents (>90%) had some inpatient TID experience, but for >60% of fellows this was <12 weeks annually. Clinical exposure varied by fellow and patient type-in an average month rotating on an inpatient TID service, more than half of adult fellows had evaluated ≥10 kidney, liver, or hematopoietic stem cell transplant recipients but <10 heart, lung, pancreas, or intestinal recipients; pediatric fellows saw <10 of all patient types. Nearly half (46%) of general ID fellows had not spent any time in the dedicated TID clinic at their program. Few fellows had participated in protocol development, organ selection meetings, or donor evaluations. Conclusions: This survey highlights important gaps in TID training. Given the increasing need for TID specialists, updated curricula and educational resources are needed.

Early therapeutic drug monitoring of methotrexate and its association with acute kidney injury: A retrospective cohort study.

INTRODUCTION: High-dose methotrexate (HDMTX) use can be limited by the development of acute kidney injury (AKI). Early AKI detection is paramount to prevent further renal injury and irreversible toxicities. This study sought to determine whether early elimination patterns of MTX would be useful as a biomarker of AKI in HDMTX treatment. METHODS: This retrospective cohort study included two sites that collected ≥2 MTX levels within 16 h from completion of MTX infusion. Early levels were tagged and MTX elimination half-life (t½) were calculated from combinations of two of three different early time periods. Receiver operating characteristic (ROC) curves were synthesized for each elimination t½ (biomarker) with respect to AKI and delayed methotrexate elimination (DME); the biomarker with the highest area under the ROC curve (AUC) was tested in a multiple variable logistic regression model. RESULTS: Data from 169 patients who received a total of 556 courses of HDMTX were analyzed. ROC analysis revealed MTX elimination t½ calculated from the second and third time periods had the highest AUC for AKI at 0.62 (interquartile range [IQR] 0.56-0.69) and DME at 0.86 (IQR 0.73-1.00). After adjusting for age, sex, dose (mg/m2), infusion duration, HDMTX course, and baseline estimated glomerular filtration rate, it remained significant for AKI with an OR of 1.29 and 95% confidence interval of 1.03-1.65. CONCLUSION: Early MTX elimination t½ measured within 16 h of infusion completion was significantly associated with the development of AKI and serves as an early clearance biomarker that may identify patients who benefit from increased hydration, augmented leucovorin rescue, and glucarpidase administration.

Description and Characteristics of Ocular Tumor Lysis Syndrome.

Introduction: The aim of the study was to describe and evaluate characteristics of ocular tumor lysis syndrome (OTLS) in eyes with uveal melanoma. Methods: Retrospective chart review of all patients with OTLS at the University of Colorado from 2009 to 2021. Data collected included patient demographics, tumor characteristics, radiation dosimetry, gene expression profiling (GEP), OTLS characteristics, management, and outcomes. Results: Seven eyes of seven patients with uveal melanoma treated with I-125 brachytherapy developed OTLS. Average age was 59 years (range 32-83). Mean apical height was 8.6 mm (range 6-11); mean diameter was 12.7 mm (range 8.5-15.3). All tumors were treated with plaques ≥16 mm in diameter. On presentation, 5/7 tumors had subretinal fluid, and 6/7 had collar-button configuration. OTLS presented as extensive pigment dispersion in the vitreous in all eyes, subretinal pigment and/or retinal detachment in 4/7 eyes, vitreous hemorrhage in 2/7 eyes, and anterior chamber pigment in 3/7 eyes. Four tumors were GEP class 1, two were class 2, and one was unclassified. Biopsy route was trans-scleral in 4/7 eyes and trans-vitreal in 3/7 eyes. OTLS occurred 2-4 weeks after an intraocular procedure in 5/7 eyes. All underwent pars plana vitrectomy. Cytology of the vitreous, obtained in five cases, showed pigment laden macrophages and hemorrhage, but only 1/5 eyes had viable malignant cells. Four eyes were stable at the last follow-up, two were enucleated, and one had no light perception from pigmentary glaucoma. Poor vision (<20/200) occurred in 6/7 cases. Three patients died from metastasis (tumors were GEP class 2, GEP class without subclassification, and no GEP classification performed). Conclusions: OTLS is a rare but devastating complication of uveal melanoma. Common characteristics included large plaque diameter, presence of subretinal fluid, and collar-button shape. The extensively dispersed pigment is typically not malignant. Though poor vision is common, enucleation may be avoided in most eyes through vitreoretinal surgical repair.

Tumour lysis syndrome.

Tumour lysis syndrome (TLS) represents a critical oncological emergency characterized by extensive tumour cell breakdown, leading to the swift release of intracellular contents into the systemic circulation, outpacing homeostatic mechanisms. This process results in hyperuricaemia (a by-product of intracellular DNA release), hyperkalaemia, hyperphosphataemia, hypocalcaemia and the accumulation of xanthine. These electrolyte and metabolic imbalances pose a significant risk of acute kidney injury, cardiac arrhythmias, seizures, multiorgan failure and, rarely, death. While TLS can occur spontaneously, it usually arises shortly after the initiation of effective treatment, particularly in patients with a large cancer cell mass (defined as ≥500 g or ≥300 g/m2 of body surface area in children). To prevent TLS, close monitoring and hydration to improve renal perfusion and urine output and to minimize uric acid or calcium phosphate precipitation in renal tubules are essential. Intervention is based on the risk of a patient of having TLS and can include rasburicase and allopurinol. Xanthine, typically enzymatically converted to uric acid, can accumulate when xanthine oxidases, such as allopurinol, are administered during TLS management. Whether measurement of xanthine is clinically useful to optimize the use of allopurinol or rasburicase remains to be determined.

Cancer in Patients With Incidental Asymmetric Oropharynx Positron Emission Tomography Uptake.

Importance: Asymmetric oropharynx uptake on positron emission tomography (PET)/computed tomography (CT) is a common incidental finding and often prompts otolaryngology referral to rule out malignancy; however, the true risk of malignancy based on this finding is unknown. Objective: To identify the incidence of oropharynx cancer in patients with incidental asymmetric oropharynx PET uptake. Design, Setting, and Participants: In this retrospective cohort study, patients 18 years and older undergoing PET/CT scans at Mayo Clinic between January 2001 and December 2018 were included. Patients with a history or pretest suspicion of oropharynx cancer were excluded. Data were analyzed from March 2021 to December 2023. Exposure: Blinded radiologic review of imaging studies, including measurement of maximum standardized uptake values (SUVmax) of the ipsilateral side of concern and contralateral side. Retrospective medical record review for associated clinical data. Main Outcomes and Measures: The primary study outcome was the incidence of oropharynx cancer diagnosis in patients with asymmetric oropharynx PET uptake. The primary outcome was formulated before data collection. Results: Of the 1854 patients identified with asymmetric oropharynx PET uptake, 327 (17.6%) met inclusion criteria. Of these, 173 (52.9%) were male, and the median (range) age was 65.0 (24.8-90.7) years. The mean (SD) follow-up interval was 52.1 (43.4) months. A total of 18 of 327 patients (5.5%) were newly diagnosed with oropharynx cancer. The most common diagnosis was squamous cell carcinoma (n = 9), followed by lymphoma (n = 8), and sarcoma (n = 1). Patients with an incidental diagnosis of oropharynx cancer had higher mean (SD) ipsilateral SUVmax (8.7 [3.7] vs 5.3 [1.9]) and SUVmax ratio (3.0 [1.6] vs 1.6 [0.6]) compared with patients with normal examination findings. SUVmax ratio and difference were found to be good discriminators of oropharynx cancer, with areas under the receiver operating characteristic curve of 86.3% (95% CI, 76.4-94.6) and 85.8% (95% CI, 74.8-94.6), respectively. Patients with a new diagnosis of oropharynx cancer were more likely to have a corresponding CT abnormality than those with normal examination findings (6 of 18 [33%] vs 24 of 295 [8.1%]). Patients with concerning lesions on oropharynx palpation by an otolaryngology health care professional were significantly more likely to be diagnosed with oropharynx cancer compared with patients with normal examination findings (odds ratio, 28.4; 95% CI, 6.6-145.8). Conclusions and Relevance: In this cohort study, while incidental asymmetric oropharynx PET uptake was common, a new diagnosis of oropharynx cancer was not and potentially results in a large volume of unnecessary referrals and work-up. Using SUVmax ratio, SUVmax difference, and CT correlation may increase the benefit of referral. Patients with a palpable oropharynx lesion and asymmetric oropharynx PET uptake should undergo confirmatory biopsy.

Consensus recommendations for systemic therapies in the management of relapsed Ewing sarcoma: A report from the National Ewing Sarcoma Tumor Board.

Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in children, adolescents, and young adults. Debate and controversy remain in the management of relapsed/refractory ES (RR-ES). The authors leveraged the expertise assembled by the National Ewing Sarcoma Tumor Board, a multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES. In this review, they focus on select topics that apply to the management of patients with RR-ES. The specific topics covered include the initial approach of such patients and discussion of the goals of care, the role of molecular testing, chemotherapy regimens and novel agents to consider, the role of maintenance therapy, and the use of high-dose chemotherapy with autologous stem cell rescue. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, these guidelines are intended to support clinicians and provide some clarity and recommendations for the management of patients with RR-ES. PLAIN LANGUAGE SUMMARY: Ewing sarcoma (ES) is a bone and soft tissue cancer that most often occurs in teenagers and young adults. This article uses the experience of the National Ewing Sarcoma Tumor Board, a multi-institution, multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES and to address questions related to the treatment of patients with relapsed ES. Although not intended to replace the clinical judgement of treating physicians and limited by available data, these consensus recommendations will support clinicians who treat patients with this challenging malignancy, made even more difficult when it recurs.

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by FcγRIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

Enantioselective Nickel-Catalyzed α-Spirocyclization of Lactones.

Herein we report a strategy for the enantioselective synthesis of spirocycles containing all-carbon quaternary centers via nickel-catalyzed intramolecular addition of lactone enolates to aryl nitriles. The established lactone α-spirocyclization efficiently and enantioselectively forges 5-, 6-, and 7-membered rings, performing best in the synthesis of 7-membered rings (up to 90% ee). This discovery represents an expansion of the synthetic toolkit for enantioselective spirocyclization, providing access to chiral, pharmaceutically relevant spirocyclic products.

Acantholytic Dyskeratoses of the Vulva: Clinicopathologic Characterization of 16 Cases and Review of the Literature.

The vulva and perineum are rarely involved by acantholytic dyskeratoses, including Hailey-Hailey disease, Darier disease, papular acantholytic dyskeratosis of the genitocrural area, acantholytic dyskeratotic acanthoma, and warty dyskeratoma. These entities show broad histomorphologic overlap, generally requiring clinical correlation for definitive classification. This institutional series aims to better characterize vulvar acantholytic dyskeratoses and provide a practical literature review and diagnostic aid for gynecologic pathologists. Our institutional archives contained 16 vulvar acantholytic dyskeratoses diagnosed between 1990 and 2023. Affected patients were 36 to 79 (mean, 58) years old and presented with one or more asymptomatic (n = 9) or pruritic (n = 6) lesions involving the vulva (predominantly the labia majora), with additional perineal involvement in 2. Four patients have known Hailey-Hailey disease. Eleven cases comprised singular, raised, erythematous, or skin-colored papules, measuring 0.2 to 0.6 (mean, 0.3) cm. Two patients had oligofocal (both with known Hailey-Hailey disease) vulvar lesions, and 2 had multifocal vulvar lesions (one with known Hailey-Hailey disease). Histologically, all showed acantholysis and dyskeratoses (abundant in 8, focal in 8, with corps ronds generally more conspicuous than corps grains). Additional features included suprabasal clefting (n = 14), dermal papillomatosis (n = 12), and acanthosis (n = 8). Adnexal involvement was rare (n = 1). No histologic features reliably distinguished sporadic versus syndromic acantholytic dyskeratoses. Sporadic lesions were cured by local excision. Patients with Hailey-Hailey disease were variably responsive to corticosteroids. Neither our series nor the literature indicate a significant correlation between sporadic or syndromic acantholytic dyskeratosis and squamous cell carcinoma. Important differential diagnoses include pemphigus vulgaris and pemphigus vegetans, for which direct immunofluorescence may be performed, when indicated.

Severe trauma leads to sustained muscle loss, induced frailty, and distinct temporal changes in myokine and chemokine profiles of older patients.

INTRODUCTION: Sarcopenia is a known risk factor for adverse outcomes across multiple disease states, including severe trauma. Factors such as age, hyperinflammation, prolonged immobilization, and critical illness may not only exacerbate progression of this disease but may also contribute to the development of induced sarcopenia, or sarcopenia secondary to hospitalization. This study seeks to (1) determine the effects of severe traumatic injury on changes in skeletal muscle mass in older adults; (2) test whether changes in skeletal muscle mass are associated with clinical frailty, physical performance, and health-related quality of life; and (3) examine trauma-induced frailty and temporal changes in myokine and chemokine profiles. METHODS: A prospective, longitudinal cohort study of 47 critically ill, older (≥45 years) adults presenting after severe blunt trauma was conducted. Repeated measures of computed tomography-based skeletal muscle index, frailty, and quality of life were obtained in addition to selected plasma biomarkers over 6 months. RESULTS: Severe trauma was associated with significant losses in skeletal muscle mass and increased incidence of sarcopenia from 36% at baseline to 60% at 6 months. Severe trauma also was associated with a transient worsening of induced frailty and reduced quality of life irrespective of sarcopenia status, which returned to baseline by 6 months after injury. Admission biomarker levels were not associated with skeletal muscle index at the time points studied but demonstrated distinct temporal changes across our entire cohort. CONCLUSIONS: Severe blunt trauma in older adults is associated with increased incidence of induced sarcopenia and reversible induced frailty. Despite muscle wasting, functional decline is transient, with a return to baseline by 6 months, suggesting a need for holistic definitions of sarcopenia and further investigation into long-term functional outcomes in this population.

Development and optimization of a diluted whole blood ELISpot assay to test immune function.

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.