Electrical signature of heterogeneous human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune-evasive, and multipotent differentiation potential. The inherent heterogeneity of hMSCs poses a challenge for therapeutic treatments and necessitates the identification of robust biomarkers to ensure reproducibility in both in vivo and in vitro experiments. In this study, we utilized dielectrophoresis (DEP), a label-free electrokinetic phenomenon, to investigate the heterogeneity of hMSCs derived from bone marrow (BM) and adipose tissue (AD). The electrical properties of BM-hMSCs were compared to homogeneous mouse fibroblasts (NIH-3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK-293). The DEP profile of BM-hMSCs differed most from HEK-293 cells. We compared the DEP profiles of BM-hMSCs and AD-hMSCs and found that they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteoblast cells revealed that they behave differently in response to differentiation-inducing cytokines. Histology and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses of the differentiation-related genes revealed differences in heterogeneity between BM-hMSCs and AD-hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate differences in the heterogeneity of BM-hMSCs and AD-hMSCs. Our results demonstrate that using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.

Epidemiology of interstitial lung disease in patients with metastatic breast cancer at baseline and after treatment with HER2-directed therapy: a real-world data analysis.

PURPOSE: Using real-world data, interstitial lung disease (ILD) prevalence before and after HER2-directed therapy was estimated. Potential ILD risk factors in patients receiving HER2-directed therapy for metastatic breast cancer (mBC) were evaluated. METHODS: Adults with HER2-directed therapy for mBC initiated between September 25, 1998, and February 22, 2020 were, included. ILD was defined broadly as one or more of 64 lung conditions. Patients were followed until incident ILD, death, last contact, or study end. RESULTS: In total, 533 patients were identified with median age at mBC of 57, 51% had de novo mBC, 43% were ever smokers, 30% had lung metastases, 9% had thoracic radiation, 6% had chronic obstructive pulmonary disease, and 16% had prevalent ILD. ILD cumulative incidence at one year was 9% (95% CI 6%, 12%), with a median follow-up of 23 months. Smoking (HR 2.2, 95% CI 1.1, 4.8) and Black/African-American race (HR 3.4, 95% CI 1.6, 7.5) were significantly associated with ILD; HRs for preexisting lung conditions (HR 1.8, 95% CI 0.9, 3.8) and thoracic radiation (HR 2.3, 95% CI 0.8, 7.1) were not statistically significant. Prevalent ILD was associated with 13-fold greater occurrence of incident ILD. 85% of patients with prevalent or incident ILD were symptomatic. CONCLUSIONS: This real-world population of patients with mBC had a high prevalence of ILD prior to HER2-directed therapy, reflecting the multifactorial causation of interstitial lung changes. The cumulative incidence of ILD in patients receiving HER2-directed therapy for mBC augments prior reports. Symptomatic presentation suggests an opportunity for early intervention.

Effectiveness of Liposomal Bupivacaine Compared With Standard-of-Care Measures in Pediatric Cardiothoracic Surgery: A Retrospective Cohort Study.

OBJECTIVE: Effective postsurgical pain management is important for pediatric patients to improve outcomes while reducing resource use and waste. The authors examined opioid consumption and economic outcomes associated with liposomal bupivacaine (LB) or non-LB analgesia use in pediatric patients undergoing cardiothoracic surgery. DESIGN: The authors retrospectively analyzed Premier Healthcare Database records. SETTING: The data extracted from the database included patient records from hospitals across the United States in both rural and urban locations. PARTICIPANTS: The records included data from patients aged 12-to-<18 years. INTERVENTIONS: The records belonged to patients undergoing video-assisted thoracoscopic procedures (VATS) who received LB or non-LB analgesia after surgery. MEASUREMENTS AND MAIN RESULTS: Outcomes included in-hospital postsurgical opioid consumption in morphine milligram equivalents (MMEs), hospital length of stay (LOS), and total hospital costs; the LB and non-LB cohorts were compared using a generalized linear model with inverse probability of treatment weighting to balance the cohorts. For VATS procedures, pediatric patients receiving LB had significant reductions in in-hospital opioid consumption (632 v 991 MMEs; p < 0.0001), shorter LOS (5.1 v 5.6 days; p = 0.0023), and lower total hospital costs ($18,084 v $21,962; p < 0.0001) compared with those receiving non-LB analgesia. CONCLUSIONS: These results support use of LB in multimodal analgesia regimens for managing pain in pediatric patients after cardiothoracic surgery.

Maryland's All-Payer Health Care System: A Light at the End of a Tunnel.

The state of Maryland, in collaboration with the Centers for Medicare & Medicaid Services, developed the first all-payer system model in the Unites States in 1971 and 35 years later in response to financial pressures undertook to modernize this program. The focus of the modernized program was to improve overall per-capita expenditure, quality of care, and the outcome of Marylanders' health. The financial status of Maryland hospitals was declining because of the rate setting of the Health Services Cost Review Commission while hospital admission rates and spending were increasing. This study showed positive change in moving Maryland health care delivery model in hospitals from volume-driven care to value-driven coordinated care. Maryland hospitals have changed their mind-sets to achieve the Triple Aim of cost reduction, health improvement, and quality-of-care improvement. The modernized model does require hospitals and business individuals to change their approach to be accountable in providing health care to all citizens, as well as trying to solve chronic social problems such as poverty and unequal access to health care.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras.

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Psoriasis disease severity affects patient satisfaction with treatment.

BACKGROUND: Patients with psoriasis are often dissatisfied with available treatments, but contributing factors are not well defined. OBJECTIVE: Examine relationships between psoriasis severity, patient characteristics, and treatment satisfaction. METHODS: Patients with psoriasis were classified into mild and moderate-to-severe groups based on self-reported data. Demographics, comorbidities, symptoms, and multiple treatment satisfaction outcomes were compared between groups. Predictors of patient satisfaction with treatment were examined using linear regression models. RESULTS: The analyses included 773 patients (407 mild; 366 moderate-to-severe). The percentage of patients reporting satisfaction with treatment was low overall, ranging from 8.6% to 61.7% for the mild and 13.9% to 49.5% for the moderate-to-severe group. Satisfaction among biologics users was also low (≤53%; 50% of satisfaction rates <40%). Regression results consistently showed greater dissatisfaction with current treatment among moderately to severely affected patients. CONCLUSION: Many psoriasis patients were dissatisfied with their treatment; moderate-to-severe patients expressed significantly less satisfaction than mild patients.

Electrical Impedance Spectroscopy as a Tool to Detect the Epithelial to Mesenchymal Transition in Prostate Cancer Cells.

Prostate cancer (PCa) remains a significant health threat, with chemoresistance and recurrence posing major challenges despite advances in treatment. The epithelial to mesenchymal transition (EMT), a biochemical process where cells lose epithelial features and gain mesenchymal traits, is linked to chemoresistance and metastasis. Electrical impedance spectroscopy (EIS), a novel label-free electrokinetic technique, offers promise in detecting cell phenotype changes. In this study, we employed EIS to detect EMT in prostate cancer cells (PCCs). PC3, DU145, and LNCaP cells were treated with EMT induction media for five days. EIS characterization revealed unique impedance spectra correlating with metastatic potential, distinguishing DU145 EMT+ and EMT- cells, and LNCaP EMT+ and EMT- cells (in combination with dielectrophoresis), with comparisons made to epithelial and mesenchymal controls. These changes were supported by shifts in electrical signatures, morphologies, and protein expression, including the downregulation of E-cadherin and upregulation of vimentin. No phenotype change was observed in PC3 cells, which maintained a mesenchymal phenotype. EMT+ cells were also distinguishable from mixtures of EMT+ and EMT- cells. This study demonstrates key advancements: the application of EIS and dielectrophoresis for label-free EMT detection in PCCs, characterization of cell electrical signatures after EMT, and EIS sensitivity to EMT transitions. Detecting EMT in PCa is important to the development of more effective treatments and overcoming the challenges of chemoresistance.

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies.

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3(fl) mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3(fl) mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3(fl) deletion does not cooperate with oncogenic Kras(G12D) expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

A locus coeruleus-dorsal CA1 dopaminergic circuit modulates memory linking.

Individual memories are often linked so that the recall of one triggers the recall of another. For example, contextual memories acquired close in time can be linked, and this is known to depend on a temporary increase in excitability that drives the overlap between dorsal CA1 (dCA1) hippocampal ensembles that encode the linked memories. Here, we show that locus coeruleus (LC) cells projecting to dCA1 have a key permissive role in contextual memory linking, without affecting contextual memory formation, and that this effect is mediated by dopamine. Additionally, we found that LC-to-dCA1-projecting neurons modulate the excitability of dCA1 neurons and the extent of overlap between dCA1 memory ensembles as well as the stability of coactivity patterns within these ensembles. This discovery of a neuromodulatory system that specifically affects memory linking without affecting memory formation reveals a fundamental separation between the brain mechanisms modulating these two distinct processes.

Mosquito cryptochromes expressed in Drosophila confer species-specific behavioral light responses.

Cryptochrome (CRY) is a short-wavelength light-sensitive photoreceptor expressed in a subset of circadian neurons and eyes in Drosophila that regulates light-evoked circadian clock resetting. Acutely, light evokes rapid electrical excitation of the ventral lateral subset of circadian neurons and confers circadian-modulated avoidance behavioral responses to short-wavelength light. Recent work shows dramatically different avoidance versus attraction behavioral responses to short-wavelength light in day-active versus night-active mosquitoes and that these behavioral responses are attenuated by CRY protein degradation by constant light exposure in mosquitoes. To determine whether CRY1s mediate species-specific coding for behavioral and electrophysiological light responses, we used an "empty neuron" approach and transgenically expressed diurnal Aedes aegypti (AeCRY1) versus nocturnal Anopheles gambiae (AgCRY1) in a cry-null Drosophila background. AeCRY1 is much less light sensitive than either AgCRY1 or DmCRY as shown by partial behavioral rhythmicity following constant light exposure. Remarkably, expression of nocturnal AgCRY1 confers low survival to constant white light as does expression of AeCRY1 to a lesser extent. AgCRY1 mediates significantly stronger electrophysiological cell-autonomous responses to 365 nm ultraviolet (UV) light relative to AeCRY1. AgCRY1 expression mediates electrophysiological sensitivity to 635 nm red light, whereas AeCRY1 does not, consistent with species-specific mosquito red light responses. AgCRY1 and DmCRY mediate intensity-dependent avoidance behavior to UV light at different light intensity thresholds, whereas AeCRY1 does not, thus mimicking mosquito and fly behaviors. These findings highlight CRY as a key non-image-forming visual photoreceptor that mediates physiological and behavioral light responses in a species-specific fashion.

Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins.

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

MLL5 contributes to hematopoietic stem cell fitness and homeostasis.

MLL5 is a novel trithorax group gene and a candidate tumor suppressor gene located within a 2.5-Mb interval of chromosome band 7q22 that frequently is deleted in human myeloid malignancy. Here we show that inactivation of the Mll5 gene in mice results in a 30% reduction in the average representation of hematopoietic stem cells and in functional impairment of long-term hematopoietic repopulation potential under competitive conditions. Bone marrow cells from Mll5-deficient mice were defective in spleen colony-forming assays, and the mutant mice showed enhanced susceptibility to 5-fluorouracil-induced myelosuppression. Heterozygous and homozygous Mll5 mutant mice did not spontaneously develop hematologic cancers, and loss of Mll5 did not alter the phenotype of a fatal myeloproliferative disorder induced by oncogenic Kras in vivo. Collectively, the data reveal an important role for Mll5 in HSC homeostasis and provide a basis for further studies to explore its role in leukemogenesis.

Primary prevention of lead exposure: the Philadelphia lead safe homes study.

OBJECTIVE: Lead exposure in children can lead to neuropsychological impairment. This study tested whether primary prevention interventions in the newborn period prevent elevated blood lead levels (BLLs). METHODS: The Philadelphia Lead Safe Homes (LSH) Study offered parental education, home evaluation, and lead remediation to the families of urban newborns. Households were randomized to a standard lead education group or maintenance education group. We conducted home visits at baseline, six months, and 12 months. To compare BLLs, we identified a matched comparison group. RESULTS: We enrolled and randomized 314 newborns in the intervention component; 110 completed the study. There were few significant differences between the randomized groups. In the combined intervention groups, positive results on visual inspection declined from baseline to 12 months (97.0% to 90.6%, p = 0.007). At baseline, 36.9% of homes were above the U.S. Environmental Protection Agency's lead dust standard, compared with 26.9% at 12 months (p = 0.032), mainly due to a drop in windowsill dust levels. Both groups showed a significant increase in parental scores on a lead education test. Children in the intervention and matched control groups had similar geometric mean initial BLLs (2.6 vs. 2.7, p = 0.477), but a significantly higher percentage of children in the intervention group had an initial blood lead screening compared with those in the matched group (88.9% vs. 84.4%, p = 0.032). CONCLUSIONS: A study of primary prevention of lead exposure showed a higher blood lead screening rate for the combined intervention groups and mean BLLs at one year of age not statistically different from the comparison group. Most homes had lead hazards. Lead education significantly increased knowledge.

Validation of a Mortality Composite Score in the Real-World Setting: Overcoming Source-Specific Disparities and Biases.

PURPOSE: This study tested whether a composite mortality score could overcome gaps and potential biases in individual real-world mortality data sources. Complete and accurate mortality data are necessary to calculate important outcomes in oncology, including overall survival. However, in the United States, there is not a single complete and broadly applicable mortality data source. It is further likely that available data sources are biased in their coverage of sex, race, age, and socioeconomic status (SES). METHODS: Six individual real-world data sources were combined to develop a high-quality composite mortality score. The composite score was benchmarked against the gold standard for mortality data, the National Death Index. Subgroup analyses were then conducted to evaluate the completeness and accuracy by sex, race, age, and SES. RESULTS: The composite mortality score achieved a sensitivity of 94.9% and specificity of 92.8% compared with the National Death Index, with concordance within 1 day of 98.6%. Although some individual data sources show significant coverage gaps related to sex, race, age, and SES, the composite score maintains high sensitivity (84.6%-96.1%) and specificity (77.9%-99.2%) across subgroups. CONCLUSION: A composite score leveraging multiple scalable sources for mortality in the real-world setting maintained strong sensitivity, specificity, and concordance, including across sex, race, age, and SES subgroups.

Customizable Natural Language Processing Biomarker Extraction Tool.

PURPOSE: Natural language processing (NLP) in pathology reports to extract biomarker information is an ongoing area of research. MetaMap is a natural language processing tool developed and funded by the National Library of Medicine to map biomedical text to the Unified Medical Language System Metathesaurus by applying specific tags to clinically relevant terms. Although results are useful without additional postprocessing, these tags lack important contextual information. METHODS: Our novel method takes terminology-driven semantic tags and incorporates those into a semantic frame that is task-specific to add necessary context to MetaMap. We use important contextual information to capture biomarker results to support Community Health System's use of Precision Medicine treatments for patients with cancer. For each biomarker, the name, type, numeric quantifiers, non-numeric qualifiers, and the time frame are extracted. These fields then associate biomarkers with their context in the pathology report such as test type, probe intensity, copy-number changes, and even failed results. A selection of 6,713 relevant reports contained the following standard-of-care biomarkers for metastatic breast cancer: breast cancer gene 1 and 2, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and programmed death-ligand 1. RESULTS: The method was tested on pathology reports from the internal pathology laboratory at Henry Ford Health System. A certified tumor registrar reviewed 400 tests, which showed > 95% accuracy for all extracted biomarker types. CONCLUSION: Using this new method, it is possible to extract high-quality, contextual biomarker information, and this represents a significant advance in biomarker extraction.

ADHD among a cohort of ethnic minority women.

Attention deficit-hyperactivity disorder has focused primarily on Caucasian boys and men and resulted in limited insight about the experiences of attention deficit-hyperactivity disorder among women from an ethnically diverse background. This descriptive qualitative study explored the experience of 16 women diagnosed with attention deficit-hyperactivity disorder who were also engaged in their academic pursuits. Three themes emerged: (i) internalized chaos, (ii) cultivation of self-understanding, and (iii) commitment to building capacity. Comorbid psychological disorders (e.g., major depression and anxiety) among women with attention deficit-hyperactivity disorder were associated with increased impairment. Broader criteria that demonstrate equitable assessment and recognition of attention deficit-hyperactivity disorder symptoms for both genders are requisite to promote effective interventions and recovery.

Explanatory models and help-seeking behavior for attention-deficit/hyperactivity disorder among a cohort of postsecondary students.

The authors present findings from a qualitative descriptive study that explored how a diverse ethnic group of postsecondary students diagnosed with attention-deficit/hyperactivity disorder (ADHD) conceptualized their condition and how this conceptualization shaped their efforts to seek help. Kleinman's explanatory model, the organizing framework, called for participants to describe the etiology, symptom onset, pathophysiology, course, and treatment of ADHD. Twenty-seven participants from four academic institutions took part in the study. A common explanatory model of ADHD was not shared; however, gender and age differences were apparent. These finding have implications for nurses when providing culturally appropriate care to individuals with ADHD in their practice settings.

Integrative Review of Lower Extremity Nerve Injury During Vaginal Birth.

OBJECTIVE: To describe the incidence, health effects, risk factors, and practice implications of lower extremity nerve injury (LENI) related to vaginal births. DATA SOURCES: We searched MEDLINE, CINAHL, and PubMed from 2000 to 2020 for peer-reviewed published case reports and research studies of LENI related to vaginal births. STUDY SELECTION: We identified 188 potential records, and 20 met inclusion criteria (six research studies and 14 case studies). DATA EXTRACTION: Three independent reviewers extracted details of injuries and births into an Excel spreadsheet and analyzed data using SPSS. DATA SYNTHESIS: Using birth data from each case study and from four of the six research articles, we found the incidence of LENI in vaginal births was 0.3% to 1.8%. The description of health effects includes affected nerves and the location, description, and duration of symptoms. Analyses of risk factors were limited by missing birth data (length of second stage, birth weight, etc). Vaginal births with LENI were 76% spontaneous, 77% with neuraxial anesthesia, and 64% first vaginal birth. Practice implications focused on prevention through specific positioning strategies. Despite nurses being the primary caregivers during labor, LENI was reported most often in anesthesia journals with virtually no reports in nursing journals. CONCLUSION: LENI is a potential complication of vaginal birth, and little published research is available on prevention and prognosis. While obstetric and anesthesia factors can cause or contribute to nerve injury, LENI is usually caused by positioning and is considered preventable. Care recommendations include the following: avoid prolonged hyperflexion of women's thighs and knees; minimize time in lithotomy, squatting, or kneeling positions; prevent hand or other deep pressure on lateral knee and posterior thigh areas; avoid motor-blocking neuraxial (epidural) anesthesia; and implement frequent repositioning. The paucity of literature contributes to the lack of awareness of LENI among clinicians.

Randomized clinical trial of a novel donor-derived cfDNA test to detect rejection in CPV-simulated renal transplant patients.

PURPOSE: Five-year kidney graft loss currently stands at about 30%. We evaluate the clinical utility of a blood test measuring donor-derived cell-free DNA that detects rejection earlier and, potentially, improves diagnostic and therapeutic accuracy. METHODS: In a randomized controlled experiment, we measured the clinical practice of 175 practicing nephrologists, both with and without the use of dd-cfDNA testing. Providers cared for six simulated post-renal transplant patient cases whose ages ranged from 30 to 75 years and were 3-24 months post-transplant with typical presentations. RESULTS: 154 nephrologists completed two rounds of simulated cases. At baseline, the study arms performed similarly, demonstrating no significant differences either in primary diagnosis (p = 0.853), decisions to biopsy or refer to transplant center (p = 1.000), or therapeutic management (p = 0.488). After introduction of the dd-cfDNA test, intervention nephrologists were more likely to arrive at the diagnosis of rejection (OR 4.00, 95% CI 1.93-8.30), make a correct decision on biopsy/transplant center referral (OR 11.07, 95% CI 4.87-25.16), and properly adjust therapeutic management (OR 2.37, 95% CI 1.07-5.24). CONCLUSION: A sample of nationally representative, practicing nephrologists given dd-cfDNA to evaluate post-transplant patients were more likely to correctly diagnose early and subclinical allograft rejection, to send for biopsy or refer to transplant center, and to appropriately change treatment than those nephrologists without dd-cfDNA access.