Native mitral valve staphylococcus endocarditis with a very unusual complication: Ruptured posterior mitral valve leaflet aneurysm.

Infective endocarditis (IE) is a life-threatening disease associated with in-hospital mortality of nearly one in five cases. IE can destroy valvular tissue, which may rarely progress to aneurysm formation, most commonly at the anterior leaflet in instances of mitral valve involvement. We present a remarkable case of a patient with IE and a rare complication of a ruptured aneurysm of the posterior leaflet of the mitral valve. Two- and Three-dimensional transesophageal echocardiography, intra-operative videography, and histopathologic analysis revealed disruption at this unusual location-at the junction of the P2 and P3 scallops, surrounded by an annular abscess.

Importance Ranking of Electrocardiogram Rhythms: A Primer for Curriculum Development.

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.

Hypertrophic cardiomyopathy with dynamic obstruction and high left ventricular outflow gradients associated with paradoxical apical ballooning.

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 < 0.00001). An elongated anterior mitral leaflet or anteriorly displaced papillary muscles occurred in 77%. Course was complicated by cardiogenic shock and heart failure in 5, and refractory heart failure in 1. High-dose beta-blockade was the mainstay of therapy. Three patients required urgent surgical relief of LVOT obstruction, 2 for refractory cardiogenic shock, and one for refractory heart failure. In the three patients, surgery immediately normalized refractory severe LV dysfunction, and immediately reversed cardiogenic shock and heart failure. All have normal LV systolic function at 45-month follow-up, and all have survived. CONCLUSIONS: Acute LV apical ballooning, associated with high dynamic LVOT gradients, may punctuate the course of obstructive HCM. The syndrome is important to recognize on echocardiography because it may be associated with profound reversible LV decompensation.

Summarizing performance for genome scale measurement of miRNA: reference samples and metrics.

BACKGROUND: The 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. RESULTS: Three 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. CONCLUSIONS: The 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.

Aortic root thrombus complicated by left main coronary artery occlusion visualized by 3D echocardiography in a patient with continuous-flow left ventricular assist device.

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.

Evaluation of miR-216a and miR-217 as potential biomarkers of acute pancreatic injury in rats and mice.

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.

Modeling Diagnostic Expertise in Cases of Irreducible Uncertainty: The Decision-Aligned Response Model.

PURPOSE: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluating the Utility of Proteomics for the Identification of Circulating Pharmacodynamic Biomarkers of IFNß-1a Biologics.

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 (IFNß-1a) or pegylated-IFNß-1a (pegIFNß-1a) approved to treat multiple sclerosis or placebo, we evaluated the utility of a proteomic assay that profiles > 7,000 plasma proteins. IFNß-1a and pegIFNß-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 IFNß1 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.

Comparison of the diagnostic accuracy of di-22:6-bis(monoacylglycerol)phosphate and other urinary phospholipids for drug-induced phospholipidosis or tissue injury in the rat.

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.

Comparative profile of commercially available urinary biomarkers in preclinical drug-induced kidney injury and recovery in rats.

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.

An adaptable method using human mixed tissue ratiometric controls for benchmarking performance on gene expression microarrays in clinical laboratories.

BACKGROUND: Molecular biomarkers that are based on mRNA transcripts are being developed for the diagnosis and treatment of a number of diseases. DNA microarrays are one of the primary technologies being used to develop classifiers from gene expression data for clinically relevant outcomes. Microarray assays are highly multiplexed measures of comparative gene expression but have a limited dynamic range of measurement and show compression in fold change detection. To increase the clinical utility of microarrays, assay controls are needed that benchmark performance using metrics that are relevant to the analysis of genomic data generated with biological samples. RESULTS: Ratiometric controls were prepared from commercial sources of high quality RNA from human tissues with distinctly different expression profiles and mixed in defined ratios. The samples were processed using six different target labeling protocols and replicate datasets were generated on high density gene expression microarrays. The area under the curve from receiver operating characteristic plots was calculated to measure diagnostic performance. The reliable region of the dynamic range was derived from log(2) ratio deviation plots made for each dataset. Small but statistically significant differences in diagnostic performance were observed between standardized assays available from the array manufacturer and alternative methods for target generation. Assay performance using the reliable range of comparative measurement as a metric was improved by adjusting sample hybridization conditions for one commercial kit. CONCLUSIONS: Process improvement in microarray assay performance was demonstrated using samples prepared from commercially available materials and two metrics - diagnostic performance and the reliable range of measurement. These methods have advantages over approaches that use a limited set of external controls or correlations to reference sets, because they provide benchmark values that can be used by clinical laboratories to help optimize protocol conditions and laboratory proficiency with microarray assays.

A multifaceted evaluation of imatinib-induced cardiotoxicity in the rat.

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.

Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation.

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.

Normal intima-media thickness on carotid ultrasound reliably excludes an ischemic cause of cardiomyopathy.

BACKGROUND: Coronary artery disease (CAD) is the most common cause of left ventricular systolic dysfunction (LVSD). Patients with ischemia as the cause of LVSD may warrant revascularization. Angiography is the most accurate method of CAD diagnosis but is invasive, expensive, and associated with some risk. Noninvasive imaging for CAD often involves expensive equipment, radiation exposure, medication, and/or contrast administration. Carotid ultrasound with measurement of intima-media thickness (IMT) is safe and inexpensive. Carotid IMT is well correlated with the presence of CAD. We assessed the accuracy of carotid ultrasound for identification of CAD as a potential etiology of LVSD. METHODS: Patients with LVSD (ejection fraction < or =40%) of uncertain etiology referred for angiography underwent carotid ultrasound. Patients with history of myocardial infarction were excluded. Two experienced cardiologists blinded to CAD status determined common carotid artery (CCA) IMT and plaque. Significant CAD was defined as > or =50% stenosis of any major artery. Ischemic LVSD was defined as (1) left main and/or proximal left anterior descending coronary artery > or =75% or (2) > or =2 major arteries with > or =75% stenosis. RESULTS: Mean ejection fraction was 27% +/- 10% in 150 patients. Significant CAD was found in 64 (42.7%) and ischemic LVSD in 40 (26.7%). Carotid plaque was seen in 95 (63.3%). Mean CCA IMT was > or =0.9 mm in 69 (46.0%). The combination of mean CCA IMT <0.9 mm and no plaque had negative predictive value for ischemic LVSD of 98%. CONCLUSIONS: Carotid ultrasound with IMT measurement is a valuable screening tool for excluding an ischemic etiology of LVSD when CAD is suspected.

Phase II study of darinaparsin in patients with advanced hepatocellular carcinoma.

BACKGROUND: Darinaparsin is a novel organic arsenic that reaches higher intracellular concentration with decreased toxicity compared to inorganic arsenic. We conducted a multi-center phase II study with darinaparsin in patients with advanced HCC. METHODS: Eligibility criteria included unresectable or metastatic measurable HCC, up to two prior systemic treatments, ECOG performance status < or = 2, Child Pugh Class A or B and adequate organ functions. Darinaparsin was administered at 420 mg/m(2) intravenously, twice weekly at least 72 h apart for 3 weeks in a 4-week cycle. The primary end point was response rate. A Simon two-stage design was used. RESULTS: Among 15 patients in the first stage, no objective responses were observed. Two patients had stable disease. The median number of cycles on study per patient was 2 (1-6). The median progression free survival and overall survival were 55 days (95% confidence interval: 50-59) and 190 days (95% confidence interval: 93-227), respectively. No treatment related hospitalizations or deaths occurred. Treatment related grade 1-2 toxicities included nausea, vomiting (26.7% each), fatigue (20%), anorexia and diarrhea (13.3% each). Grade 3 anorexia, wheezing, agitation, abdominal pain and SGPT were observed in 1 patient each (6.7%). One patient experienced grade 4 hypoglycemia (6.7%). CONCLUSIONS: Darinaparsin could be safely administered with tolerable toxicity profiles, and no QTc prolongation in patients with advanced HCC. However, at this dose and schedule, it has shown no objective responses in HCC and this trial was terminated as planned after the first stage of efficacy analysis.

Distinctive Hypertrophic Cardiomyopathy Anatomy and Obstructive Physiology in Patients Admitted With Takotsubo Syndrome.

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, p < 0.04) and reduced distance from coaptation to septum (19 ± 5 vs 27 ± 5, p < 0.001). 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.

Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium.

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.

Outbreak of Opportunistic Ascending Pyelonephritis with Numerous Yeast after Experimental Humanization Surgery in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ and NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ Immunodeficient Mice.

Unexpected mortality occurred in a group of 12 NOD.Cg-NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and 12 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/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.

Development of methods for data quantitation of spiked salmon host cell DNA in protamine sulfate by qPCR.

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, R 2 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.

Quantitation of residual host cell DNA in protaminesulfate drug product by qPCR.

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.