Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH (-/-)) or activating Fc receptors (FcRγ(-/-)) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

OBJECTIVE To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). DESIGN Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). PROCEDURES Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. RESULTS Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. CONCLUSIONS AND CLINICAL RELEVANCE Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

Clinical, genetic, and functional characterization of adrenocorticotropin receptor mutations using a novel receptor assay.

The ACTH receptor (MC2R) is expressed predominantly in the adrenal cortex, but is one of five G protein-coupled, seven-transmembrane melanocortin receptors (MCRs), all of which bind ACTH to some degree. Testing of MC2R activity is difficult because most cells express endogenous MCRs; hence, ACTH will elicit background activation of assayable reporter systems. Inactivating mutations of MC2R lead to hereditary unresponsiveness to ACTH, also known as familial glucocorticoid deficiency (FGD). These patients are usually seen in early childhood with very low cortisol concentrations, normal mineralocorticoids, hyperpigmentation, and increased bodily growth. Several MC2R mutations have been reported in FGD, but assays of the activities of these mutants are cumbersome. We saw two patients with typical clinical findings of FGD. Genetic analysis showed that patient 1 was homozygous for the mutation R137W, and patient 2 was a compound heterozygote for S74I and Y254C. We tested the activity of these mutations in OS-3 cells, which are unresponsive to ACTH but have intact downstream cAMP signal transduction. OS-3 cells transfected with a cAMP-responsive luciferase reporter plasmid (pCREluc) were unresponsive to ACTH, but cotransfection with a vector expressing human MC2R increased luciferase activity more than 40-fold. Addition of ACTH to cells cotransfected with the pCREluc reporter and wild-type MC2R activated luciferase expression with a 50% effective concentration of 5.5 x 10(-9) M ACTH, which is similar to previously reported values. By contrast, the MC2R mutant R137W had low activity, and the S74I or Y254C mutants elicited no measurable response. This assay provides excellent sensitivity in an easily assayed transient transfection system, providing a more rapid and efficient measurement of ACTH receptor activity.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

Determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis: evaluation of assay performance and quality assurance over a 10-year period in the routine arena.

The performance of high-resolution CZE for determination of carbohydrate-deficient transferrin (CDT) in human serum based on internal and external quality data gathered over a 10-year period is reported. The assay comprises mixing of serum with a Fe(III) ion-containing solution prior to analysis of the iron saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. CDT values obtained with a human serum of a healthy individual and commercial quality control sera are shown to vary less than 10%. Values of a control from a specific lot were found to slowly decrease as function of time (less than 10% per year). Furthermore, due to unknown reasons, gradual changes in the monitored pattern around pentasialo-transferrin were detected, which limit the use of commercial control sera of the same lot to less than 2 years. Analysis of external quality control sera revealed correct classification of the samples over the entire 10-year period. Data obtained compare well with those of HPLC and CZE assays of other laboratories. The data gathered over a 10-year period demonstrate the robustness of the high-resolution CZE assay. This is the first account of a CZE-based CDT assay with complete internal and external quality assessment over an extended time period.

Groundwater dating with Atom Trap Trace Analysis of 39Ar

We report on the realization of Atom Trap Trace Analysis for39Ar and its first application to dating of groundwater samples. The presented system achieves an atmospheric39Ar count rate as high as 3.58 ± 0.10 atoms/h allowing for the determination of the39Ar concentration in less than a day. We demonstrate that the measured count rates are proportional to the39Ar concentration by intercomparison with Low-Level Counting results and by measurements on prepared argon samples with defined concentration. For a geophysical application, we degas three different groundwater samples and gas chromatographically extract the argon. The39Ar ages inferred from the count rates extend over the accessible dating range and are in agreement with the Low-Level Counting results as well as with complementary isotope data.