Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry.

OBJECTIVE: Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression. METHODS: The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes. RESULTS: Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αßγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αß1ß2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1ß3γ2s, α2ß3γ2s, α3ß3γ2s and α5ß3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3' untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes. CONCLUSIONS: Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

Enabling Routine MHC-II-Associated Peptide Proteomics for Risk Assessment of Drug-Induced Immunogenicity.

Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides that can potentially activate T cells and contribute to the immunogenicity of a drug. Acceptance of the MAPPs technology as an appropriate preclinical (and potentially clinical) immunogenicity risk assessment tool depends not only on its technical stability and robustness but also on the ability to compare results across experiments and donors. To this end, we developed a specialized MAPPs data processing pipeline, dataMAPPs, which presents complex mass spectrometric data sets in the form of heat maps (heatMAPPs), enabling rapid and convenient comparison between conditions and donors. A customized normalization procedure based on identified endogenous peptides standardizes signal intensities within and between donors and enables cross-experimental comparison. We evaluated the technical reproducibility of the MAPPs platform using tool compounds with respect to the most prominent experimental factors and found that the systematic biological differences across donors by far outweighed any technical source of variation. We illustrate the capability of the MAPPs platform to generate data that may be used for preclinical risk assessment of drug-induced immunogenicity and discuss its applicability in the clinics.

Long-Term Electrical and Mechanical Function Monitoring of a Human-on-a-Chip System.

The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.

Safe Use of Contrast Media: What the Radiologist Needs to Know.

Iodinated and gadolinium-based contrast media are used on a daily basis in most radiology practices. These agents often are essential to providing accurate diagnoses, and are nearly always safe and effective when administered correctly. However, reactions to contrast media do occur and can be life threatening. Therefore, it is critical for faculty and staff to know how reactions to contrast agents manifest and how to treat them promptly. The decline in renal function seen occasionally after intravenous administration of iodinated contrast agents is poorly understood and likely multifactorial, and its association with the contrast medium may be overemphasized. However, it is important that radiologists be aware of current understanding and strategies to decrease the incidence of renal dysfunction. Nephrogenic systemic fibrosis, a skin disease, is an adverse reaction related to use of some gadolinium-based contrast agents in patients with chronic renal failure. The types of gadolinium most often associated with this condition and the indications for withholding gadolinium are important and are discussed in this article. The use of enteric contrast agents and contrast agents during pregnancy and nursing are reviewed briefly. Current knowledge for safe use of contrast media and key concepts that all radiologists should know are summarized in this review.

Poroelastic behavior and water permeability of human skin at the nanoscale.

Topical skin care products and hydrating compositions (moisturizers or injectable fillers) have been used for years to improve the appearance of, for example facial wrinkles, or to increase "plumpness". Most of the studies have addressed these changes based on the overall mechanical changes associated with an increase in hydration state. However, little is known about the water mobility contribution to these changes as well as the consequences to the specific skin layers. This is important as the biophysical properties and the biochemical composition of normal stratum corneum, epithelium, and dermis vary tremendously from one another. Our current studies and results reported here have focused on a novel approach (dynamic atomic force microscopy-based nanoindentation) to quantify biophysical characteristics of individual layers of ex vivo human skin. We have discovered that our new methods are highly sensitive to the mechanical properties of individual skin layers, as well as their hydration properties. Furthermore, our methods can assess the ability of these individual layers to respond to both compressive and shear deformations. In addition, since human skin is mechanically loaded over a wide range of deformation rates (frequencies), we studied the biophysical properties of skin over a wide frequency range. The poroelasticity model used helps to quantify the hydraulic permeability of the skin layers, providing an innovative method to evaluate and interpret the impact of hydrating compositions on water mobility of these different skin layers.

Development of automated detection of radiology reports citing adrenal findings.

The aim of this study was to determine the feasibility of automated detection of adrenal nodules, a common finding on CT, using a newly developed search engine that mines dictated radiology reports. To ensure Health Insurance Portability and Accountability Act compliance, we utilized a preexisting de-identified database of 32,974 CT reports from February 1, 2009 to February 28, 2010. Common adrenal descriptors from 29 staff radiologists were used to develop an automated rule-based algorithm targeting adrenal findings. Each sentence within the free text of reports was searched with an adapted NegEx negation algorithm. The algorithm was refined using a 2-week test period of reports and subsequently validated using a 6-week period. Manual review of the 3,693 CT reports in the validation period identified 222 positive reports while the algorithm detected 238 positive reports. The algorithm identified one true positive report missed on manual review for a total of 223 true positive reports. This resulted in a precision of 91% (217 of 238) and a recall of 97% (217 of 223). The sensitivity of the query was 97.3% (95% confidence interval (CI), 93.9-98.9%), and the specificity was 99.3% (95% CI, 99.1-99.6%). The positive predictive value of the algorithm was 91.0% (95% CI, 86.6-94.3%), and the negative predictive value was 99.8% (95% CI, 99.6-99.9%). The prevalence of true positive adrenal findings identified by the query (7.1%) was nearly identical to the true prevalence (7.2%). Automated detection of language describing common findings in imaging reports, such as adrenal nodules on CT, is feasible.

Quantitative trait analysis reveals transforming growth factor-beta2 as a positive regulator of early hematopoietic progenitor and stem cell function.

Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.

Microphysiological heart-liver body-on-a-chip system with a skin mimic for evaluating topical drug delivery.

Body-on-a-chip in vitro systems are a promising technology that aims to increase the predictive power of drug efficacy and toxicity in humans when compared to traditional animal models. Here, we developed a new heart-liver body-on-a-chip system with a skin surrogate to assess the toxicity of drugs that are topically administered. In order to test the utility of the system, diclofenac, ketoconazole, hydrocortisone and acetaminophen were applied topically through a synthetic skin surrogate (Strat-M membrane) and the toxicity results were compared to those of acute drug exposure from systemically applying the compounds. The heart-liver system was successful in predicting the effects for both cardiac and liver functions changes due to the compounds. The difference in the concentrations of drugs applied topically compared to systemically indicates that the barrier properties of the skin surrogate were efficient. One important advantage of this heart-liver system was the capability of showing differential effects of acute and chronic drug exposure which is necessary as part of the International Conference in Harmonisation (ICH) tri-partate guidelines. In conclusion, this work indicates a promising heart-liver body-on-a-chip system that can be used for the assessment of potential drug toxicity from dermal absorption as well as evaluate transport dynamics through the skin in the same system.

Psychosocial status in adolescents undergoing bariatric surgery.

BACKGROUND: Little is known about the psychosocial status of adolescents who undergo bariatric surgery. Our objective was to describe the psychological and behavioral characteristics of patients in this age group who underwent bariatric surgery at our institution. METHODS: A review of clinical charts of patients aged 14-21 years who had bariatric surgery at our institution between 2000 and 2005 was conducted. Abstracted data included clinical information and the results of a psychosocial evaluation consisting of a clinical interview with a psychologist and self-reported data from the Weight and Lifestyle Inventory and the Beck Depression Inventory-II. RESULTS: Twenty-five patient records were reviewed. Nineteen patients (76%) were female. The mean (+/-SD) age was 18.7 +/- 1.6 years, and mean body mass index was 50.6 +/- 7.9 kg/m(2). Depression was the most common psychiatric comorbidity (68%). Abnormal eating behaviors were frequent and included binge eating (48%), rapid eating (44%), having guilt associated with eating (36%), eating until uncomfortably full (36%), loss of control (24%), eating without hunger (24%), and eating alone (20%). Sixteen patients were judged to be appropriate for surgery by the bariatric surgery team; surgery was delayed for nine patients primarily because of concerns about ability to adhere to the postoperative diet. These patients were recommended for additional dietary counseling and/or psychotherapeutic treatment prior to surgery. CONCLUSIONS: Among adolescent bariatric surgery candidates, depression and aberrant eating behaviors were very common. Early identification and management of these conditions may enable most of these patients to undergo bariatric surgery and optimize the likelihood for a successful outcome.

Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system.

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well.

Age-related accumulation of a novel CD44 + CD25lowgammadelta T-cell population in hematopoietic organs of the mouse.

We discovered a novel population of gammadelta T cells in the mouse that accumulates with age in hematopoietic organs, but not in epithelia. These cells are CD25low (an unusual phenotype for gammadelta T cells in the mouse); express higher levels of TCRgammadelta and CD44 than do CD25- gammadelta T cells; mainly express Vgamma2, Vgamma3, and Vgamma4 chains; and are largely quiescent. A very similar cell population appears in the late stages of fetal thymus organ cultures, suggesting that the accumulation of CD44 + CD25lowTCRgammadelta + cells is a response to stress induced by aging in vivo or by culture in vitro. The precursors of CD44 + CD25lowTCRgammadelta + cells are generated during fetal or very young adult life, as this population was undetectable in aged recipients of bone marrow from old or young donors. CD44 + CD25lowTCRgammadelta + cells may be a biomarker of aging, but could also play a role in the inflammatory changes that accompany aging.

Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs.

We report on a functional human model to evaluate multi-organ toxicity in a 4-organ system under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 14 days. Computer simulations of the platform established flow rates and resultant shear stress within accepted ranges. Viability of the system was demonstrated for 14 days as well as functional activity of cardiac, muscle, neuronal and liver modules. The pharmacological relevance of the integrated modules were evaluated for their response at 7 days to 5 drugs with known side effects after a 48 hour drug treatment regime. The results of all drug treatments were in general agreement with published toxicity results from human and animal data. The presented phenotypic culture model exhibits a multi-organ toxicity response, representing the next generation of in vitro systems, and constitutes a step towards an in vitro "human-on-a-chip" assay for systemic toxicity screening.

Reducing Unnecessary Portable Pelvic Radiographs in Trauma Patients: A Resident-Driven Quality Improvement Initiative.

Quality improvement is increasingly important in the changing health care climate. We aim to establish a methodology and identify critical factors leading to successful implementation of a resident-led radiology quality improvement intervention at the institutional level. Under guidance of faculty mentors, the first-year radiology residents developed a quality improvement initiative to decrease unnecessary STAT pelvic radiographs (PXRs) in hemodynamically stable trauma patients who would additionally receive STAT pelvic CT scans. Development and implementation of this initiative required multiple steps, including: establishing resident and faculty leadership, gathering evidence from published literature, cultivating multidisciplinary support, and developing and implementing an institution-wide ordering algorithm. A visual aid and brief questionnaire were distributed to clinicians for use during treatment of trauma cases to ensure sustainability of the initiative. At multiple time points, pre- and post-intervention, residents performed a retrospective chart review to evaluate changes in imaging-ordering trends for trauma patients. Chart review showed a decline in the number of PXRs for hemodynamically stable trauma patients, as recommended in the ordering algorithm: 78% of trauma patients received both a PXR and a pelvic CT scan in the first 24 hours of the initiative, compared with 26% at 1 month; 24% at 6 months; and 18% at 10 to 12 months postintervention. The resident-led radiology quality improvement initiative created a shift in ordering culture at an institutional level. Development and implementation of this algorithm exemplified the impact of a multidisciplinary collaborative effort involving multiple departments and multiple levels of the medical hierarchy.

High-throughput flow cytometry cell-based assay to detect antibodies to N-methyl-D-aspartate receptor or dopamine-2 receptor in human serum.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Staphylococcus-infected tunneled dialysis catheters: is over-the-wire exchange an appropriate management option?

PURPOSE: Over-the-wire exchange of tunneled dialysis catheters is the standard of care per K/DOQI guidelines for treating catheter-related bacteremia. However, Gram-positive bacteremia, specifically with staphylococcus species, may compromise over-the-wire exchange due to certain biological properties. This study addressed the effectiveness of over-the-wire exchange of staphylococcus-infected tunneled dialysis catheters compared with non-staphylococcus-infected tunneled dialysis catheters. METHODS: Patients who received over-the-wire exchange of their tunneled dialysis catheter due to documented or suspected bacteremia were identified from a QA database. Study patients (n = 61) had positive cultures for Staphylococcus aureus, Staphylococcus epidermidis, or coagulase-negative staphylococcus not otherwise specified. Control patients (n = 35) received over-the-wire exchange of their tunneled dialysis catheter due to infection with any organism besides staphylococcus. Overall catheter survival and catheter survival among staphylococcal species were assessed. RESULTS: There was no difference in tunneled dialysis catheter survival between study and control groups (P = 0.46). Median survival time was 96 days for study catheters and 51 days for controls; survival curves were closely superimposed. There also was no difference among the three staphylococcal groups in terms of catheter survival (P = 0.31). The median time until catheter removal was 143 days for SE, 67 days for CNS, and 88 days for SA-infected catheters. CONCLUSIONS: There is no significant difference in tunneled dialysis catheter survival between over-the-wire exchange of staphylococcus-infected tunneled dialysis catheters and those infected with other organisms.

Transforming growth factor-beta2 is involved in quantitative genetic variation in thymic involution.

The mechanisms regulating thymic involution are unclear. In inbred mouse strains the rate of thymic involution and the function of the hematopoietic stem cell (HSC) compartment are subject to quantitative genetic variation. We have shown previously that transforming growth factor-beta2 (TGF-beta2) is a genetically determined positive regulator of HSCs. Here, we demonstrate that genetic variation in the rate of thymic involution correlates with genetic variation in the responsiveness of hematopoietic stem and progenitor cells to TGF-beta2. Corroborating these correlations, thymic cellularity and peripheral naive T-cell frequency were higher in old Tgfb2+/- mice than in wild-type littermates. The frequency of early T-cell precursors was increased in Tgfb2+/- mice, suggesting that TGF-beta2 affects the earliest stages of T-cell development in old mice. Reciprocal transplantation experiments indicated that TGF-beta2 expressed both in the (micro)environment and in the hematopoietic system can accelerate thymic involution; however, the age of the stem cells appeared irrelevant. Thus, although thymic involution is largely determined by the aged environment, TGF-beta2 plays a major modulatory role that is subject to genetic variation and is possibly mediated through its regulatory effects on early hematopoiesis.

Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice.

The number of bone marrow hematopoietic stem and progenitor cells as defined by the lineage(-), Sca1(++), c-kit(+) (LSK) phenotype and their proliferative capacity in vitro are subject to quantitative genetic variation, and several quantitative trait loci (QTL) have been identified in young mice. Because some traits affecting hematopoiesis also change with age in a mouse strain-dependent fashion, we performed quantitative trait analysis in aged BXD recombinant inbred (RI) mice for the number and frequency of LSK cells, and for their proliferative capacity in vitro. Several novel QTL were identified. The number and frequency of LSK cells in old mice correlated inversely with lifespan. Furthermore, 4 of 7 lifespan QTL overlap with QTL contributing to the number, frequency, or proliferative capacity of LSK cells in young or old mice. Taken together, these data establish a close genetic, and perhaps functional, link between genetic variation in lifespan and characteristics of stem and progenitor cells.

The positive regulatory effect of TGF-beta2 on primitive murine hemopoietic stem and progenitor cells is dependent on age, genetic background, and serum factors.

TGF-beta is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-beta isoform, TGF-beta2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-beta2, but not TGF-beta1 and TGF-beta3, had a biphasic dose response on the proliferation of purified lin-Sca1(++)kit(+) (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-beta2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-beta2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-beta2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-beta2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.