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Assessing expertise using psychometric models usually yields a measure of ability that is difficult to generalize to the complexity of diagnoses in clinical practice. However, using an item response modeling framework, it is possible to create a decision-aligned response model that captures a clinician's decision-making behavior on a continuous scale that fully represents competing diagnostic possibilities. In this proof-of-concept study, the authors demonstrate the necessary statistical conceptualization of this model using a specific electrocardiogram (ECG) example.The authors collected a range of ECGs with elevated ST segments due to either ST-elevation myocardial infarction (STEMI) or pericarditis. Based on pilot data, 20 ECGs were chosen to represent a continuum from "definitely STEMI" to "definitely pericarditis," including intermediate cases in which the diagnosis was intentionally unclear. Emergency medicine and cardiology physicians rated these ECGs on a 5-point scale ("definitely STEMI" to "definitely pericarditis"). The authors analyzed these ratings using a graded response model showing the degree to which each participant could separate the ECGs along the diagnostic continuum. The authors compared these metrics with the discharge diagnoses noted on chart review.Thirty-seven participants rated the ECGs. As desired, the ECGs represented a range of phenotypes, including cases where participants were uncertain in their diagnosis. The response model showed that participants varied both in their propensity to diagnose one condition over another and in where they placed the thresholds between the 5 diagnostic categories. The most capable participants were able to meaningfully use all categories, with precise thresholds between categories.The authors present a decision-aligned response model that demonstrates the confusability of a particular ECG and the skill with which a clinician can distinguish 2 diagnoses along a continuum of confusability. These results have broad implications for testing and for learning to manage uncertainty in diagnosis.

Liver microphysiological systems (MPSs) are promising models for predicting hepatic drug effects. Yet, after a decade since their introduction, MPSs are not routinely used in drug development due to lack of criteria for ensuring reproducibility of results. We characterized the feasibility of a liver MPS to yield reproducible outcomes of experiments assaying drug toxicity, metabolism, and intracellular accumulation. The ability of the liver MPS to reproduce hepatotoxic effects was assessed using trovafloxacin, which increased lactate dehydrogenase (LDH) release and reduced cytochrome P450 3A4 (CYP3A4) activity. These observations were made in two test sites and with different batches of Kupffer cells. Upon culturing equivalent hepatocytes in the MPS, spheroids, and sandwich cultures, differences between culture formats were detected in CYP3A4 activity and albumin production. Cells in all culture formats exhibited different sensitivities to hepatotoxicant exposure. Hepatocytes in the MPS were more functionally stable than those of other culture platforms, as CYP3A4 activity and albumin secretion remained prominent for greater than 18 days in culture, whereas functional decline occurred earlier in spheroids (12 days) and sandwich cultures (7 days). The MPS was also demonstrated to be suitable for metabolism studies, where CYP3A4 activity, troglitazone metabolites, diclofenac clearance, and intracellular accumulation of chloroquine were quantified. To ensure reproducibility between studies with the MPS, the combined use of LDH and CYP3A4 assays were implemented as quality control metrics. Overall results indicated that the liver MPS can be used reproducibly in general drug evaluation applications. Study outcomes led to general considerations and recommendations for using liver MPSs. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Microphysiological systems (MPSs) have been designed to recreate organ‐ or tissue‐specific characteristics of extracellular microenvironments that enhance the physiological relevance of cells in culture. Liver MPSs enable long‐lasting and stable culture of hepatic cells by culturing them in three‐dimensions and exposing them to fluid flow. WHAT QUESTION DID THIS STUDY ADDRESS? What is the functional performance relative to other cell culture platforms and the reproducibility of a liver MPS for assessing drug development and evaluation questions, such as toxicity, metabolism, and pharmacokinetics? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The liver MPS systematically detected the toxicity of trovafloxacin. When compared with spheroids and sandwich cultures, this system had a more stable function and different sensitivity to troglitazone, tamoxifen, and digoxin. Quantifying phase II metabolism of troglitazone and intracellular accumulation of chloroquine demonstrated the potential use of the liver MPS for studying drug metabolism and pharmacokinetics. Quality control criteria for assessing chip function were key for reliably using the liver MPS. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Due to its functional robustness and physiological relevance (3D culture, cells expose to fluid flow and co‐culture of different cell types), the liver MPS can, in a reproducible manner: (i) detect inflammatory‐induced drug toxicity, as demonstrated with trovafloxacin, (ii) detect the toxicity of other drugs, such as troglitazone, tamoxifen, and digoxin, with different effects than those detected in spheroids and sandwich cultures, (iii) enable studies of hepatic function that rely on prolonged cellular activity, and (iv) detect phase II metabolites and drug accumulation to potentially support the interpretation of clinical data. The integration of MPSs in drug development will be facilitated by careful evaluation of performance and reproducibility as performed in this study.

Introduction Electrocardiogram interpretation is an essential skill for emergency and critical care nurses and physicians. There remains a gap in standardized curricula and evaluation strategies used to achieve and assess competence in electrocardiogram interpretation. The purpose of this study was to develop an importance ranking of the 120 American Heart Association electrocardiogram diagnostic labels with interdisciplinary perspectives to inform curriculum development. Methods Data for this mixed methods study were collected through focus groups and individual semi-structured interviews. A card sort was used to assign relative importance scores to all 120 American Heart Association electrocardiogram diagnostic labels. Thematic analysis was used for qualitative data on participants' rationale for the rankings. Results The 18 participants included 6 emergency and critical care registered nurses, 5 cardiologists, and 7 emergency medicine physicians. The 5 diagnoses chosen as the most important by all disciplines were ventricular tachycardia, ventricular fibrillation, atrial fibrillation, complete heart block, and normal electrocardiogram. The “top 20” diagnoses by each discipline were also reported. Qualitative thematic content analysis revealed that participants from all 3 disciplines identified skill in electrocardiogram interpretation as clinically imperative and acknowledged the importance of recognizing normal, life threatening, and time-sensitive electrocardiogram rhythms. Additional qualitative themes, identified by individual disciplines, were reported. Discussion This mixed-methods approach provided valuable interdisciplinary perspectives concerning electrocardiogram curriculum case selection and prioritization. Study findings can provide a foundation for emergency and critical care educators to create local ECG educational programs. Further work is recommended to validate the list amongst a larger population of emergency and critical care frontline nurses and physicians.

Proteomics has the potential to identify pharmacodynamic (PD) biomarkers for similarity assessment of proposed biosimilars without relying on clinical efficacy end points. In this study, with 36 healthy participants randomized to therapeutic doses of interferon-beta 1a products (IFNβ-1a) or pegylated-IFNβ-1a (pegIFNβ-1a) approved to treat multiple sclerosis or placebo, we evaluated the utility of a proteomic assay that profiles 7,000 plasma proteins. IFNβ-1a and pegIFNβ-1a resulted in 248 and 528 differentially expressed protein analytes, respectively, between treatment and placebo groups over the time course. Thirty-one proteins were prioritized based on a maximal fold change ≥ 2 from baseline, baseline adjusted area under the effect curve (AUEC) and overlap between the 2 products. Of these, the majority had a significant AUEC compared with placebo in response to either product; 8 proteins showed 4-fold maximal change from baseline. We identified previously reported candidates, beta-2microglobulin and interferon-induced GTP-binding protein (Mx1) with ~ 50% coefficient of variation (CV) for AUEC, and many new candidates (including I-TAC, C1QC, and IP-10) with CVs ranging from 26%-129%. Upstream regulator analysis of differentially expressed proteins predicted activation of IFNβ1 signaling as well as other cytokine, enzyme, and transcription signaling networks by both products. Although independent replication is required to confirm present results, our study demonstrates the utility of proteomics for the identification of individual and composite candidate PD biomarkers that may be leveraged to support clinical pharmacology studies for biosimilar approvals, especially when biologics have complex mechanisms of action or do not have previously characterized PD biomarkers.

Unexpected mortality occurred in a group of 12 NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) and 12 NOD.Cg-Rag1(tm1Mom) Il2rg(tm1Wjl)/SzJ (NRG) immunodeficient mice. At 10 d after routine bone marrow–liver–thymus humanization surgery, 9 mice were found dead without observation of initiating clinical signs; 1 d later (day 11), 3 additional mice showed signs of morbidity, including severe hunching, lateral recumbency, slow movement, shallow respiration, and decreased response to external stimulus. All remaining mice rapidly decompensated and were found dead or were euthanized within 4 d after the first death. Histopathology revealed severe ascending pyelonephritis with numerous yeast. Cultures in some mice were positive for Enterococcus faecalis or Staphylococcus xylosus, 2 bacteria considered commensals in rodents. In addition, Candida albicans was cultured from some animals. Further investigation revealed that a restraining device used for tail vein injections was the likely fomite harboring Candida organisms. These findings indicate that ascending pyelonephritis, with Candida as the etiologic agent, can cause significant mortality in NSG and NRG immunodeficient mice.

Protamine sulfate (PS) is an FDA approved drug used to reverse heparin-induced anticoagulation in patients. Protamine sulfate is a mixture of primarily four ∼4 kDa arginine-rich cationic polypeptide chains derived from chum (Oncorhynchus keta) salmon sperm. Because the presence of residual host cell salmon DNA (resDNA) in PS drug product can pose safety concerns, processing steps during PS manufacturing are designed to target the reduction of these impurities. However, given protamine’s positively charged structure, isolating and measuring negatively charged residual DNA is challenging. Here, the development of a sensitive detection method using real-time quantitative polymerase chain reaction (qPCR) assay for a multicopy gene (5S ribosomal DNA) using custom-designed primers and TaqMan probes is described. The PS qPCR standard curve was accurate over a linear range of 0.0025–156.25 pg/μL using protease-digested research grade salmon sperm DNA (neat) as the reference standard. DNA present in PS drug products was extracted using an optimized two-hour procedure achieving ∼85% recovery values from 1 to 125 pg reference DNA spiked into PS (1 mg) samples. The procedure lower limit of quantitation (LLOQ) of 5 pg of DNA per mg of PS or 250 pg of DNA per 50 mg dose of PS was determined from DNA spike recovery curves using the acceptance criteria of 70–130% recovery with % CV ≤ 25%. Seven pharmaceutical-grade lots of PS were evaluated and the detectable amount of resDNA was below the LLOQ. This qPCR method demonstrated sensitivity 40-fold above the current guidelines for resDNA (10 ng DNA per dose). Overall, the approach offers a promising tool for monitoring resDNA in PS and potentially other challenging complex drug products with cationic character.

Clinical spectrum of hypertrophic cardiomyopathy (HC) has been expanded to include patients with mild or no thickening of the left ventricle (LV), who nevertheless have outflow tract obstruction at rest or after exercise, due to systolic anterior motion (SAM) and ventricular septal contact, with mitral valve elongation and papillary muscles anomalies. Apical ballooning mimicking a takotsubo syndrome (TS) wall motion pattern can occur in HC with mild septal thickening when latent obstruction becomes unrelenting. To define the prevalence of anatomic abnormalities characteristic of HC in patients diagnosed with TS, we analyzed echocardiograms of 44 unselected TS patients, age 67±12 years, 95% women including studies performed before the event (n = 11, median 515 days) and after recovery of left ventricular function (n = 33, median 92 days, interquartile range = 29 to 327) and compared the findings to 60 age and sexed matched controls. Analysis of echocardiograms was blinded to event timing, and patient vs. control status. During the ballooning event, 13 patients (30%) had SAM including 9 with LV outflow obstruction, peak gradients 71±40 mmHg, as well as: ventricular septal thickening (16 ± 4 mm), elongated anterior leaflets (30 ± 3mm), and increased mitral coaptation to posterior wall distance (17 ± 5 mm), consistent with diagnosis of the HC phenotype. Compared to 31 TS patients without SAM, study patients with SAM had longer anterior leaflets (30 ± 3 vs 26 ± 4 mm, p = 0.006), thicker septum (16 ± 4 vs 12 ± 3 mm), increased coaptation to posterior wall distance (17 ± 5 vs 14 ± 4 mm, p0.04) and reduced distance from coaptation to septum (19 ± 5 vs 27 ± 5, p0.001). In the 13 patients with SAM, morphologic characteristics of HC persisted after normalization of LV function. In conclusion, a subset of patients experiencing TS events demonstrates a constellation of morphologic abnormalities characteristic of HC that persist after recovery of LV wall motion. These findings suggest that dynamic outflow obstruction may cause apical ballooning in susceptible patients.

Aortic root thrombus is an uncommon complication of continuous-flow left ventricular assist devices (LVAD). We present the case of a 71-year-old man with ischemic cardiomyopathy who underwent destination therapy HeartMate II LVAD placement. Eighteen months later, he presented with a cerebrovascular accident followed by myocardial infarction. Transesophageal echocardiography revealed an aortic root thrombus spanning the left and noncoronary cusps and obliterating the left main coronary artery. We discuss the incidence, risk factors, and management of aortic root thrombus in LVAD patients. To our knowledge, this is the first report of three-dimensional echocardiography used to characterize an LVAD-associated aortic root thrombus.

Pancreas-enriched microRNAs have been experimentally investigated in rodents as candidate serum biomarkers of pancreatic injury with several different acute pancreatic injury models. In the present study, temporal and magnitude responses of exocrine pancreas-enriched miR-216a, miR-216b, and miR-217 and endocrine-enriched miR-375 and miR-148a were measured by droplet digital PCR in serum in a caerulein model of pancreatic injury in the dog. All 5 microRNAs followed a similar time course that mirrored the responses of the conventional serum pancreatic injury biomarkers, amylase and lipase. Detection was improved through the use of assays designed against microRNA isomers (isomirs) identified by sequencing. Serum biomarker increases were concordant with histopathology defined acinar cell injury. Minimal islet cell changes were noted. The pancreas-enriched microRNAs demonstrated similar or greater sensitivity, a larger range of response, and a higher correlation to acinar cell injury compared to amylase and lipase. Our results further support the translational potential of pancreas-enriched microRNAs as sensitive biomarkers of acinar cell injury with evidence from an additional non-clinical model system.

BACKGROUND Acute left ventricular (LV) apical ballooning with normal coronary angiography occurs rarely in obstructive hypertrophic cardiomyopathy (OHCM); it may be associated with severe hemodynamic instability. METHODS, RESULTS We searched for acute LV ballooning with apical hypokinesia/akinesia in databases of two HCM treatment programs. Diagnosis of OHCM was made by conventional criteria of LV hypertrophy in the absence of a clinical cause for hypertrophy and mitral-septal contact. Among 1519 patients, we observed acute LV ballooning in 13 (0.9%), associated with dynamic left ventricular outflow tract (LVOT) obstruction and high gradients, 92 ± 37 mm Hg, 10 female (77%), age 64 ± 7 years, LVEF 31.6 ± 10%. Septal hypertrophy was mild compared to that of the rest of our HCM cohort, 15 vs 20 mm (P

Protamine sulfate (PS) is an approximately 4 kDa cationic polypeptide derived from chum salmon used to reverse heparin-induced anticoagulation in patients. Because the presence of residual host cell salmon DNA (resDNA) in PS drug product can pose safety concerns, processing steps during PS manufacturing are designed to target the reduction of these impurities. However, given protamine׳s positively charged structure, isolating and measuring negatively charged residual DNA is challenging. Suitable resDNA methods for PS require the generation of host DNA reference materials, efficient DNA extraction procedures and assay sensitivity and accuracy as high as possible. Here, optimization data are shown for the extraction of DNA present in PS drug products and for the generation of reference standard from protease-digested research grade chum salmon DNA. The lower limit of quantitation (LLOQ) for the reference standard determined from protease-digested DNA (0.0025–156.25 pg/μL) was 0.0025 pg/μL. The extraction procedure LLOQ, determined from DNA (0.01–1.25 pg/μL) spiked into PS samples, was 5 pg DNA per mg PS. The data supporting the LLOQs were evaluated using acceptance criteria of 70–130% recovery with % correlation coefficient (CV) ≤ 25% for DNA concentrations and curve metrics (slopes, R2 and y-intercepts) within 2SD of the mean. The data presented here complement a broader study (Sommers et al., 2018) [1] and are particularly useful for the development of resDNA methods for challenging drug products. Keywords: Protamine sulfate, Real-time polymerase chain reaction, Residual host cell DNA, TaqMan, DNA reference standard

BackgroundThe potential utility of microRNA as biomarkers for early detection of cancer and other diseases is being investigated with genome-scale profiling of differentially expressed microRNA. Processes for measurement assurance are critical components of genome-scale measurements. Here, we evaluated the utility of a set of total RNA samples, designed with between-sample differences in the relative abundance of miRNAs, as process controls.ResultsThree pure total human RNA samples (brain, liver, and placenta) and two different mixtures of these components were evaluated as measurement assurance control samples on multiple measurement systems at multiple sites and over multiple rounds. In silico modeling of mixtures provided benchmark values for comparison with physical mixtures. Biomarker development laboratories using next-generation sequencing (NGS) or genome-scale hybridization assays participated in the study and returned data from the samples using their routine workflows. Multiplexed and single assay reverse-transcription PCR (RT-PCR) was used to confirm in silico predicted sample differences. Data visualizations and summary metrics for genome-scale miRNA profiling assessment were developed using this dataset, and a range of performance was observed. These metrics have been incorporated into an online data analysis pipeline and provide a convenient dashboard view of results from experiments following the described design. The website also serves as a repository for the accumulation of performance values providing new participants in the project an opportunity to learn what may be achievable with similar measurement processes.ConclusionsThe set of reference samples used in this study provides benchmark values suitable for assessing genome-scale miRNA profiling processes. Incorporation of these metrics into an online resource allows laboratories to periodically evaluate their performance and assess any changes introduced into their measurement process.

Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection. Circulating proinflammatory cytokines and/or miRNAs that track with the onset and progression of lethal A/Puerto Rico/8/34 (PR8) influenza A virus infection and their response to oseltamivir treatment were investigated up to 10 days after infection. Changes in plasma cytokines (IL-1β, IL-10, IL-12p70, IL-6, KC, TNF-α, and IFN-γ) and several candidate miRNAs were profiled. Among the cytokines analyzed, IL-6 and KC/GRO cytokines appeared to correlate with peak viral titer. Over the selected 48 miRNAs profiled, certain miRNAs were up- or downregulated in a manner that was dependent on the oseltamivir treatment and disease severity. Our findings suggest that IL-6 and KC/GRO cytokines can be a potential disease severity biomarker and/or marker for the progression/remission of infection. Further studies to explore other cytokines, miRNAs, and lung injury proteins in serum with different subtypes of influenza A viruses with varying disease severity may provide new insight into other unique biomarkers.

Background The American College of Cardiology (ACC), in collaboration with the National Board of Medical Examiners (NBME), developed the first standardized in-training examination (ITE) for cardiovascular disease fellows-in-training (FITs). In addition to testing knowledge, this examination uses the newly developed ACC Curricular Milestones to provide specific, competency-based feedback to program directors and FITs. The ACC ITE has been administered more than 5,000 times since 2011. Objectives This analysis sought to report the initial experience with the ITE, including feasibility and reliability of test development and implementation, as well as the ability of this process to provide useful feedback in key content areas. Methods The annual ACC ITE has been available to cardiovascular disease fellowship programs in the United States since 2011. Questions for this Web-based, secure, multiple-choice examination were developed by a group of cardiovascular disease specialists and each question was analyzed by the NBME to ensure quality. Scores were equated and standardized to allow for comparability. Trainees and program directors were provided detailed feedback, including a list of the curricular competencies tested by those questions answered incorrectly. Results The ITE was administered 5,118 times. In 2013, the examination was taken by 1,969 fellows, representing 194 training programs. Among the 3 training years, there was consistency in the examination scores. Total test scores and scores within each of the content areas increased with each FIT year (there was a statistically significant difference in each cohort’s average scale score across administration years). There was also significant improvement in examination scores across the fellowship years. Conclusions The ACC ITE is a powerful tool available to all training programs to assess medical knowledge. This examination also delivers robust and timely feedback addressing individual knowledge gaps, and thus, may serve as a basis for improving training curricula.

Primary tumors of the aorta are rare entities. We report the unusual manifestation of an aortic intimal sarcoma that presented as a brain metastasis in a 56-year-old, otherwise healthy woman. After the brain mass had been resected, multiple imaging methods revealed pseudocoarctation and the primary tumor in the aortic arch. To our knowledge, this is the first report of the diagnosis of an aortic intimal sarcoma with use of real-time, 3-dimensional transesophageal echocardiography.

Pericarditis and ST-segment elevation myocardial infarction (STEMI) are often confused on electrocardiogram (EKG) interpretation and have markedly different implications. We sought to create an accurate statistical model to represent skill in distinguishing these 2 diagnoses. From a pilot study, 20

Understanding what qualities of an electrocardiogram (EKG) make it difficult for learners to interpret is crucial to developing teaching models for EKG learning. We hypothesized that EKGs could be ranked on a scale from simple pattern recognition to those requiring complex deductive analysis, and

Aortocava fistula is a rare condition ranging from 0.22% to 6% of all ruptured aortic aneurysms. Recognition and diagnosis of this entity can often be difficult and requires heightened clinical suspicion to ensure that prompt surgical management leads to a favorable outcome. We herein describe the diagnosis and the technical points of successful endovascular management of aortocaval fistula in the setting of a ruptured abdominal aortic aneurysm.

LC/MS- and NMR-based global metabolomics analyses were utilized to study the changes in rat urine in response to gentamicin treatment. Sprague–Dawley rats were dosed with gentamicin sulfate at 0, 75, 150 or 300 mg/kg/day for one, two or three consecutive days. Four animals from each group were sacrificed to harvest kidney tissue and to collect urine on days 1, 2, 3, 7, 10, 15, 18, 22, 29, 36 and 44 for a total of 11 different time points. Both uni- and multivariate statistical analyses were employed to identify the significantly changed metabolites in urine at the different dose levels and time points. Increases and decreases in amino acids including tyrosine, valine and hydroxyproline reflected histopathology changes of kidney injury development and/or kidney injury recovery. Glucosuria was noted much earlier than changes in the classic kidney function biomarkers, blood urea nitrogen and serum creatinine. Dopamine-related compounds, homovanillic acid sulfate (HVA-SO4) and homoveratric acid sulfate (HVrA-SO4) were significantly increased at early time points and could be early indicators of a renal adaptive response to gentamicin-induced renal injury. Furthermore, the drug efficacy of gentamicin was evaluated through the detection of changes in gut microflora-related compounds (e.g. indole-containing metabolites). Metabolomics was successful in identifying valine, hydroxyproline, HVA-SO4 and HVrA-SO4 that might serve as potential early injury biomarkers or adaptive markers of gentamicin-induced renal injury, and in assessing gentamicin efficacy through changes in compounds reported to be related to gut microflora. However, caution should be taken in direct translation of the biomarkers reported in clinical settings because a much higher dose of gentamicin than the normal therapeutic dose (~1–2 mg/kg) was used to cause kidney damaged.

Cardiotoxicity was an unanticipated side effect elicited by the clinical use of imatinib (Imb). This toxicity has been examined in only a limited number of experimental studies. The present study sought, by a variety of approaches, to identify important characteristics of Imb-induced cardiac alterations. Male spontaneously hypertensive rats (SHRs) received oral doses of 10, 30, or 50 mg/kg Imb or water daily for 10 d. Cardiac lesions, detected at all doses, were characterized by cytoplasmic vacuolization and myofibrillar loss. In a second experiment, cardiac lesions were found in Sprague Dawley (SD) and SHR rats given 50 or 100 mg/kg Imb for 14 d. Mean cardiac lesion scores and serum levels of cardiac troponin I were higher in SHRs than in SD rats. Imb induced myocyte death by necrosis, autophagy, and apoptosis. Dose-related increases in cardiac expression were observed for several genes associated with endoplasmic reticulum stress response, protein folding, and vascular development and remodeling. Imb caused alterations in isolated myocytes (myofibrillar loss, highly disrupted and disorganized sarcomeric α-actinin, apoptosis, and increased lactate dehydrogenase release) at low concentrations (5 mM). The authors conclude that Imb exerts cardiotoxic effects that are manifest through a complex pattern of cellular alterations, the severity of which can be influenced by arterial blood pressure.

We designed a study to provide reversibility and comparative injury data for several candidate urinary biomarkers of kidney injury in the United States Food and Drug Administration biomarker qualification process. The nephrotoxin gentamicin was given to rats once on each of three days and the animals were killed during dosing or over the following 42 days. Between days one and three, all biomarkers except albumin were elevated, peaked at day 7, and returned to control levels by day 10 (μ- and α-glutathione S-transferases, and renal papillary antigen-1) or day 15 (kidney injury molecule-1, lipocalin-2, osteopontin, and clusterin). All biomarkers performed better during injury than during recovery except osteopontin, which performed equally well in both time periods. During the evolution of injury, kidney injury molecule-1, renal papillary antigen-1, and clusterin best mirrored the histopathologic lesions. During injury resolution, kidney injury molecule-1, osteopontin, and blood urea nitrogen best reflected recovery. Based on histopathology, necrosis, or apoptosis scoring, kidney injury molecule-1 was the best biomarker of overall renal injury. Evaluation by regeneration score showed that renal papillary antigen-1 best reflected tubular and/or collecting duct regeneration, especially during recovery. Thus, these biomarkers performed with different effectiveness when evaluated by individual pathological processes such as necrosis, apoptosis, and regeneration.

Data regarding effects of lower-dose GH on cardiopulmonary function in GH-deficient (GHD) adults are limited.The objective was to assess effects of lower-dose GH on exercise capacity and echocardiographic parameters in GHD adults.The study was a 6-month double-blind, placebo-controlled randomized trial.The study was conducted at the General Clinical Research Center.Thirty hypopituitary adults with GHD were studied.Subjects were randomized to recombinant human GH or placebo for 6 months, followed by open-label recombinant human GH for 12 months.Primary endpoints were exercise duration, maximal oxygen consumption, and left ventricular ejection fraction. Secondary endpoints were echocardiographic indices of systolic and diastolic function, left ventricular mass, lipids, and body composition.In the 6-month double-blind phase, mean GH dose was 0.64 mg/d. Mean IGF-I sd score increased from -4.5 to -1.0. Exercise duration, maximal oxygen consumption, left ventricular ejection fraction, and other echocardiographic parameters were normal at baseline and did not change. GH decreased total and low-density lipoprotein cholesterol by 7.5% (P = 0.016) and 14.7% (P = 0.002) (P = 0.04 vs. placebo). Mean lean body mass increased by 2.2 kg (P = 0.004), fat mass decreased by 1.7 kg (P = 0.21), and percent body fat decreased by 2.5% (P = 0.018), although between-group changes were not significant.Human GH did not improve exercise performance or echocardiographic parameters or decrease fat mass but significantly decreased total and low-density lipoprotein cholesterol, increased IGF-I, and increased lean body mass. These results indicate that responses to human GH are variable and should be assessed at baseline and during treatment.

Sensitive biomarkers are needed to detect kidney injury at the earliest stages. The objective of this study was to determine whether the appearance of kidney injury molecule-1 (Kim-1) protein ectodomain in urine and kidney injury molecule-1/hepatitis A viral cellular receptor-1 (Kim-1/Havcr1) gene expression in kidney tissue may be more predictive of renal injury after exposure to nephrotoxicants when compared to traditionally used biomarkers. Male Sprague-Dawley rats were injected with a range of doses of gentamicin, mercury (Hg; HgCl2), or chromium (Cr; K2Cr2O7). The results showed that increases in urinary Kim-1 and kidney Kim-1/Havcr1 gene expression paralleled the degree of severity of renal histopathology and were detected at lower doses of nephrotoxicants when compared to blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-beta-D-glucosaminidase (NAG). In a time course study, urinary Kim-1 was elevated within 24 h after exposure to gentamicin (100 mg/kg), Hg (0.25 mg/kg), or Cr (5 mg/kg) and remained elevated through 72 h. NAG responses were nephrotoxicant dependent with elevations occurring early (gentamicin), late (Cr), or no change (Hg). At 72 h, after treatment with any of the three nephrotoxicants, there was increased Kim-1 immunoreactivity and necrosis involving approximately 50% of the proximal tubules; however, only urinary Kim-1 was significantly increased, while BUN, serum creatinine, and NAG were not different from controls. In rats treated with the hepatotoxicant galactosamine (1.1 mg/kg), serum alanine aminotransferase was increased, but no increase in urinary Kim-1 was observed. Urinary Kim-1 and kidney Kim-1/Havcr1 expression appear to be sensitive and tissue-specific biomarkers that will improve detection of early acute kidney injury following exposure to nephrotoxic chemicals and drugs.

The spectrum and intensity of drug activity can be influenced by the level of organ development. In some instances responses are of sufficient magnitude to provoke an adverse drug reaction (ADR) (Makri et al., 2004; Stephenson, 2005). An ADR in pediatric patients may be different from that occurring in adults receiving the same drug. The incidence of ADRs in pediatric patients could be related to the nonlinear maturation of certain pathways responsible for drug absorption, distribution, metabolism, and excretion (ADME) (Faustman et al., 2000; Pirmohamed et al., 1998). The biological conditions present at a young age that could alter drug ADME include: differences in levels of drug metabolizing enzymes, stomach pH, gastrointestinal emptying time, levels of serum albumin, and in the nonadult body H2O:fat ratio (Cresteil et al., 1985; Heyman, 1998). In addition, drug activities can also be affected by developmental differences in the ratio of metabolites as well as lower levels of both biliary activity and renal excretion (Bates and Balistreri, 2004). One of the limiting factors in understanding ADRs in pediatric populations is the lack of appropriate animal models that can be used to predict the possible consequences of exposure to drugs during the early years of human development. Gentamicin (GM), an aminoglycoside antibiotic, prescribed for the treatment of life-threatening infections (gram-negative and -positive bacilli) is associated with a 10-15% incidence of nephrotoxicity (acute renal failure [ARF] and increases in serum creatinine [Cr]) (Ali, 1995; Mingeot-Leclercq and Tulkens, 1999). GM-induced nephrotoxicity is attributed to an accumulation of a small percentage (∼5%) of the administered dose in renal cortical tubular epithelial cells (Nagai and Takano, 2004; Wiland and Szechcinski, 2003). The exact pathgenic mechanism responsible for GM nephrotoxicity is not completely delineated. Several hypotheses have been suggested including the generation of reactive oxygen species, renal cortical phospholipidosis, and inhibition of Na+--K+- ATPase (Ali, 1995). There is a continuing need to develop specific and sensitive biomarkers for the early detection of renal toxicity. At present, the most common means of detecting renal injury is to monitor levels of blood urea nitrogen (BUN) and serum Cr. Detectable changes in the concentration of these two substances are observed only after a considerable amount of renal function is lost. This study was initiated to develop and characterize an animal model that could mimic the different stages of human development and to use this model to evaluate potential age-related differences in renal sensitivity to a nephrotoxic agent (GM).

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.

A significant limitation to the analytical accuracy and precision of dual-labeled spotted cDNA microarrays is the signal error due to dye bias. Transcript-dependent dye bias may be due to gene-specific differences of incorporation of two distinctly different chemical dyes and the resultant differential hybridization efficiencies of these two chemically different targets for the same probe. Several approaches were used to assess and minimize the effects of dye bias on fluorescent hybridization signals and maximize the experimental design efficiency of a cell culture experiment. Dye bias was measured at the individual transcript level within each batch of simultaneously processed arrays by replicate dual-labeled split-control sample hybridizations and accounted for a significant component of fluorescent signal differences. This transcript-dependent dye bias alone could introduce unacceptably high numbers of both false-positive and false-negative signals. We found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and therefore measurable and correctable. The additional microarrays and reagents required for paired technical replicate dye-swap corrections commonly performed to control for dye bias could be costly to end users. Incorporating split-control microarrays within a set of concurrently processed hybridizations to specifically measure dye bias can eliminate the need for technical dye swap replicates and reduce microarray and reagent costs while maintaining experimental accuracy and technical precision. These data support a practical and more efficient experimental design to measure and mathematically correct for dye bias.

Understanding the strengths and limitations of alternative models, such as the Tg.AC assay, for evaluation of the potential carcinogenicity of pharmaceuticals requires assessment of assay specificity through studies that specifically target biologically active compounds that are known to not be carcinogens in rodents. To identify drugs that might provoke a false positive response in the Tg.AC assay, we screened pharmaceuticals for in vitro induction of the gadd153 promoter and the zeta-globin promoter. We have previously found a high correlation between induction of the gadd153 promoter in HepG2 cells and activity in the Tg.AC assay. The three drugs selected through screening 99 noncarcinogenic pharmaceuticals were amiloride, dipyridamole, and pyrimethamine. A 26-week skin paint study was conducted in hemizygous Tg.AC mice with the three drugs at two doses selected by a 4-week dose range finding study. Evidence of systemic toxicity was observed in animals dosed chronically with pyrimethamine or amiloride, but no skin papillomas were observed in mice treated with amiloride, dipyridamole, or pyrimethamine for 26 weeks. All male mice and 80% of female mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in acetone developed a maximal tumor burden. However, mice treated with TPA in a vehicle containing 2.4% DMSO had greatly reduced incidences of papillomas. In summary, the correct negative response was shown in the Tg.AC assay for three noncarcinogenic pharmaceuticals, which adds further favorable evidence of appropriate specificity of this model system. However, vehicle composition must be carefully selected because the outcome of this assay can be confounded by certain commonly used solvents.

Giant cell myocarditis is a rare and often fatal disease. The most obvious presentation often described in the literature is one of rapid hemodynamic deterioration due to cardiogenic shock necessitating urgent consideration of mechanical circulatory support and heart transplantation. We present the case of a 60-year-old man whose initial presentation was consistent with myopericarditis but who went on to develop a rapid decline in left ventricular systolic function without overt hemodynamic compromise or dramatic symptomatology. Giant cell myocarditis was confirmed via endomyocardial biopsy. Combined immunosuppression with corticosteroids and calcineurin inhibitor resulted in resolution of symptoms and sustained recovery of left ventricular function one year later. Our case highlights that giant cell myocarditis does not always present with cardiogenic shock and should be considered in the evaluation of new onset cardiomyopathy of uncertain etiology as a timely diagnosis has distinct clinical implications on management and prognosis.

Severe aortic plaques seen on transesophageal echocardiography (TEE) are a high-risk cause of stroke and peripheral embolization. Evidence to guide therapy is lacking. Retrospective information was obtained regarding the occurrence of embolic events (stroke, transient ischemic attacks, or peripheral emboli) in 519 patients with severe thoracic aortic plaque seen on TEE since 1988. Treatment with statins, warfarin, or antiplatelet medications was noted. Treatment was not randomized. In a matched-paired analysis, each patient taking each class of therapy was matched for age, gender, previous embolic event, hypertension, diabetes, congestive failure, and atrial fibrillation to someone not taking that medication. Multivariate analysis was also performed. An embolic event occurred in 111 patients (21%). Multivariate analysis showed that statin use was independently protective against recurrent events (p = 0.0001). Matched analysis also showed a protective effect of statins (p = 0.0004; absolute risk reduction 17%, relative risk reduction 59%, number needed to treat [n = 6]). No protective effect was found for warfarin or antiplatelet drugs. The odds ratio for embolic events was 0.3 (95% confidence interval [CI] 0.2 to 0.6) for statin therapy, 0.7 (95% CI 0.4 to 1.2) for warfarin, and 1.4 (95% CI 0.8 to 2.4) for antiplatelet agents. Thus, there is a protective effect of statin therapy, and no significant benefit of warfarin or antiplatelet drugs on the incidence of stroke and other embolic events in patients with severe thoracic aortic plaque on TEE.

Current therapy for chronic coronary artery disease (CAD) is extremely variable and under-utilizes preventive measures that have demonstrated benefit. Therefore, we sought to determine whether a novel hybrid prognostic and computer-based clinical decision support system (ohms|cad®; Interactive Heart Management Corporation, Laurence Harbor, New Jersey), that provides the treating physician with real-time, patient-specific recommendations, could improve outcomes, decrease utilization of services, and lower the costs of CAD. This was an observational study in a managed-care group practice with 39,565 members. A subpopulation of 1,628 patients participated in the study after being identified as being at increased risk for CAD. Over the 21 months after introduction of ohms|cad, utilization of cardiovascular services, coronary disease outcomes, and costs were compared with a baseline year by computing the relative risks of discrete CAD-related events. The use of ohms|cad significantly reduced adverse CAD outcom...

Mild injury of the exocrine pancreas is often asymptomatic and can be under- or mis-diagnosed. The pancreas-enriched microRNAs miR-216a and miR-217 were evaluated as potential serum biomarkers of exocrine pancreas injury in rodent models of acute pancreatitis induced by caerulein, l-arginine, and pancreatic duct ligation. Both microRNAs showed time- and dose- relevant responses to pancreatic injury and wider dynamic ranges of response than serum amylase or lipase. Pancreas-selective microRNAs were found to be relatively sensitive serum biomarkers of pancreatic injury in rodents with potentially greater specificity than the current standard assays.

The Tg.AC transgenic mouse skin paint assay is one of the short-term carcinogenesis models that has been proposed as a replacement for 1 species in the conventional 2-yr bioassay required for safety testing of pharmaceuticals. In our initial efforts to evaluate the sensitivity and specificity of this model for human pharmaceuticals, 61% of the hemizygous Tg.AC mice in the positive control groups were refractory to treatment with 12- O-tetradecanoylphorbol 13-acetate (TPA). Tg.AC mice are reported to carry ≤10 copies of a transgcne consisting of a ζ-globin promoter fused to the v-Ha-ras structural gene with a terminal simian virus 40 (SV40) polyaden-ylation signal. Southern blot hybridization of genomic DNA from 66 tail biopsies using a ζ-globin probe revealed that all of the hemizygous Tg.AC mice screened contained approximately 40 copies of the transgene and that mice unresponsive to TPA had lost a 2-kb BamHI fragment containing ζ-gIobin promoter sequence. By systematic screening of Tg.AC breeder mice for this diagnostic marker of phenotypic responsiveness, it should be possible to selectively enrich the Tg.AC mouse colony to consist exclusively of responders and to guard against further genetic instability.

Plasma homocyst(e)ine [H(e)] levels correlate with the prevalence of arterial occlusive diseases. Recently, transesophageal echocardiography (TEE) has been used to evaluate patients with atherosclerotic plaques in the thoracic aorta. The purpose of this study was to determine whether H(e) levels correlate with the degree of atherosclerotic plaque in the thoracic aorta (ATH) as seen on TEE. Maximum plaque areas for three locations in the thoracic aorta (arch, proximal descending, and distal descending) were measured with TEE in 156 patients. Maximum plaque areas for these locations were added to yield an estimate of ATH. ATH and H(e) levels, and levels of folic acid, vitamin B 12 , and pyridoxal 5`-phosphate were measured in a double-blind manner. Univariate analysis demonstrated a significant correlation of H(e) with ATH ( r = 0.3, p < 0.001). On multivariate analysis, H(e) was independently predictive of ATH ( r for the model including H(e) was 0.63, p < 0.0001). Plasma H(e) levels are therefore significantly and independently correlated with the degree of atherosclerosis in the thoracic aorta. (Am Heart J 1997;133:534-40.)

We report a case of a large saphenous vein graft (SVG) aneurysm masquerading as a right atrial mass on transesophageal echocardiogram. Cardiac magnetic resonance angiography reliably made a diagnosis of SVG aneurysm extrinsically compressing right atrium. This case illustrates the importance of using combined imaging modalities for the diagnosis and management of cardiac masses.

Transesophageal echocardiography (TEE) is superior to other imaging techniques for the diagnosis of aortic trauma. It can accurately, rapidly, and safely diagnose life-threatening conditions such as aortic rupture, pseudoaneurysm, traumatic aortic dissection, and intramural hematoma. TEE is useful for the diagnosis and prevention of iatrogenic aortic trauma induced by cardiovascular surgery, intraaortic procedures, and cardiopulmonary resuscitation. (ECHOCARDIOGRAPHY, Volume 13, March 1996)

The diagnosis of paradoxical embolization typically requires evidence of acute systemic arterial occlusion, systemic venous thrombosis, and a pathway for the thromboembolus to bypass the pulmonary vasculature. However, even when all of these findings are present, they cumulatively represent only a “smoking gun” from which a diagnosis is inferred. It is rare that one captures the actual process of paradoxical embolization “red-handed.” These transthoracic and transesophageal echocardiographic images demonstrate a thrombus …

After graft repair of an ascending aortic aneurysm, a patient was seen by us with a chest x-ray film indicating a retained foreign body. Mediastinal exploration had been unrevealing. Transesophageal echocardiography demonstrated the nature and exact location of the foreign body and therefore was instrumental in directing its retrieval.

Pulmonary artery obstruction may be caused by tumor within or external to the arteries. Presented here is a patient with life-threatening compromise in pulmonary flow that was caused by a pulmonary neoplasm. The Doppler echocardiogram showed subtotal narrowing of the right pulmonary artery and total occlusion of the left pulmonary artery. In addition, the beneficial effects of chemotherapy were documented by Doppler.

Phospholipidosis (PLD), an abnormal accumulation of phospholipids within tissues, is observed during the preclinical testing of many pharmaceutical drugs. Diagnosis of PLD is currently based on morphologic criteria. An understanding of the clinical incidence of PLD and its possible relationship to adverse drug reactions has been hampered by the absence of noninvasive biomarkers for PLD. The uncommon phospholipid di-docosahexaenoyl bis(monoacylglycerol) phosphate (di-22:6-BMP) has been proposed as a potential urinary biomarker for PLD, but data on the utility of serum di-22:6-BMP measurements in diagnosing PLD is more limited. In this report, we compared the performance of serum and urinary di-22:6-BMP as biomarkers for PLD in rats treated with the PLD-inducing drugs amiodarone and 4,4'-diethylaminoethoxyhexestrol dihydrochloride or the hepatotoxicant acetaminophen (APAP). Serum levels of di-22:6-BMP showed a higher correlation to a generalized PLD incidence score than did levels in urine, but were unexpectedly elevated in rats with marked levels of APAP-induced liver necrosis. When samples were filtered based on serum ALT or liver histopathology thresholds, the diagnostic accuracy of serum di-22:6-BMP for PLD improved to the high level observed for urinary di-22:6-BMP without sample exclusion. These data help define the potential context-of-use of serum di-22:6-BMP as a non-clinical biomarker of PLD.

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

The case of an 82-year-old patient with signs and symptoms of superior vena cava syndrome is described. A multiplane transesophageal echocardiogram demonstrated anatomic and Doppler flow evidence of superior vena caval obstruction due to compression by a dissected, aneurysmal ascending aorta. Multiplane transesophageal echocardiography is particularly useful in the diagnosis of superior vena cava syndrome when it is a sequela of thoracic aortic disease.

The left atrial appendage of patients with mitral valve disease is commonly a source of thromboembolus and is often ligated during mitral valve surgery to diminish this risk. However, ligation is often incomplete. We describe a patient with a stroke whose only source of embolus was an incompletely ligated left atrial appendage. Attempts to exclude the left atrial appendage from the arterial circulation by suture ligation may not decrease the risk of thromboemboli and instead may increase such risk. (J Am Soc Echocardiogr 2001;14:396-8.)

Pulsed wave, continuous wave, and color flow Doppler imaging as well as intravenously administered agitated saline solution can detect intracardiac shunts during transthoracic echocardiography. Ultrasonographic contrast agents have greatly improved the visualization of left heart chambers and can enhance signals from blood flow within chambers and across valves, increasing the sensitivity of Doppler techniques. We describe a patient in whom the use of echo contrast media during transthoracic echocardiography allowed the detection of a previously unseen atrial septal defect. Combining such modalities may help to increase the sensitivity of transthoracic echocardiography and may eliminate the need for transesophageal echocardiography in selected patients. (J Am Soc Echocardiogr 2001;14:155-7.)