Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages.

The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry.

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.

Comparison between a high-resolution single-stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs.

The capabilities of a high-resolution (HR), accurate mass spectrometer (Exactive-MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50,000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive-MS was compared with that of a triple quadrupole mass spectrometer (TQ-MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole-N-oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR-MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR-MS offers several benefits over TQ-MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR-MS delivers comparable results to TQ-MS in routine quantitative drug analyses. Considering the advantages of HR-MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed.

Rationale and design of a randomized, double-blind, parallel-group study of terutroban 30 mg/day versus aspirin 100 mg/day in stroke patients: the prevention of cerebrovascular and cardiovascular events of ischemic origin with terutroban in patients with a history of ischemic stroke or transient ischemic attack (PERFORM) study.

BACKGROUND: Ischemic stroke is the leading cause of mortality worldwide and a major contributor to neurological disability and dementia. Terutroban is a specific TP receptor antagonist with antithrombotic, antivasoconstrictive, and antiatherosclerotic properties, which may be of interest for the secondary prevention of ischemic stroke. This article describes the rationale and design of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic Attack (PERFORM) Study, which aims to demonstrate the superiority of the efficacy of terutroban versus aspirin in secondary prevention of cerebrovascular and cardiovascular events. METHODS AND RESULTS: The PERFORM Study is a multicenter, randomized, double-blind, parallel-group study being carried out in 802 centers in 46 countries. The study population includes patients aged > or =55 years, having suffered an ischemic stroke (< or =3 months) or a transient ischemic attack (< or =8 days). Participants are randomly allocated to terutroban (30 mg/day) or aspirin (100 mg/day). The primary efficacy endpoint is a composite of ischemic stroke (fatal or nonfatal), myocardial infarction (fatal or nonfatal), or other vascular death (excluding hemorrhagic death of any origin). Safety is being evaluated by assessing hemorrhagic events. Follow-up is expected to last for 2-4 years. Assuming a relative risk reduction of 13%, the expected number of primary events is 2,340. To obtain statistical power of 90%, this requires inclusion of at least 18,000 patients in this event-driven trial. The first patient was randomized in February 2006. CONCLUSIONS: The PERFORM Study will explore the benefits and safety of terutroban in secondary cardiovascular prevention after a cerebral ischemic event.

Improved SNR efficiency in gradient echo coronary MRA with high temporal resolution using parallel imaging.

Contemporary coronary magnetic resonance angiography techniques suffer from signal-to-noise ratio (SNR) constraints. We propose a method to enhance SNR in gradient echo coronary magnetic resonance angiography by using sensitivity encoding (SENSE). While the use of sensitivity encoding to improve SNR seems counterintuitive, it can be exploited by reducing the number of radiofrequency excitations during the acquisition window while lowering the signal readout bandwidth, therefore improving the radiofrequency receive to radiofrequency transmit duty cycle. Under certain conditions, this leads to improved SNR. The use of sensitivity encoding for improved SNR in three-dimensional coronary magnetic resonance angiography is investigated using numerical simulations and an in vitro and an in vivo study. A maximum 55% SNR enhancement for coronary magnetic resonance angiography was found both in vitro and in vivo, which is well consistent with the numerical simulations. This method is most suitable for spoiled gradient echo coronary magnetic resonance angiography in which a high temporal and spatial resolution is required.

Diagnostic accuracy of body mass index, body fat and waist in defining low aerobic fitness in adolescents

Objective: to assess the diagnostic accuracy of different anthropometric markers in defining low aerobic fitness among adolescents. Methods: cross-sectional study on 2,331 boys and 2,366 girls aged 10 - 18 years. Body mass index (BMI) was measured using standardized methods; body fat (BF) was assessed by bioelectrical impedance. Low aerobic fitness was assessed by the 20-meter shuttle run using the FITNESSGRAMR criteria. Waist was measured in a subsample of 1,933 boys and 1,897 girls. Overweight, obesity and excess fat were defined according to the International Obesity Task Force (IOTF) or FITNESSGRAMR criteria. Results: 38.5% of boys and 46.5% of girls were considered as unfit according to the FITNESSGRAMR criteria. In boys, the area under the ROC curve (AUC) and 95% confidence interval were 66.7 (64.1 - 69.3), 67.1 (64.5 - 69.6) and 64.6 (61.9 - 67.2) for BMI, BF and waist, respectively (P<0.02). In girls, the values were 68.3 (65.9 - 70.8), 63.8 (61.3 - 66.3) and 65.9 (63.4 - 68.4), respectively (P<0.001). In boys, the sensitivity and specificity to diagnose low fitness were 13% and 99% for obesity (IOTF); 38% and 86% for overweight + obesity (IOTF); 28% and 94% for obesity (FITNESSGRAMR) and 42% and 81% for excess fat (FITNESSGRAMR). For girls, the values were 9% and 99% for obesity (IOTF); 33% and 82% for overweight + obesity (IOTF); 22% and 94% for obesity (FITNESSGRAMR) and 26% and 90% for excess fat (FITNESSGRAMR). Conclusions: BMI, not body fat or waist, should be used to define low aerobic fitness. The IOTF BMI cut-points to define obesity have a very low screening capacity and should not be used.

Sensitivity analysis, dominant factors, and robustness of the ECETOC TRA v3, Stoffenmanager 4.5, and ART 1.5 occupational exposure models

Occupational exposure modeling is widely used in the context of the E.U. regulation on the registration, evaluation, authorization, and restriction of chemicals (REACH). First tier tools, such as European Centre for Ecotoxicology and TOxicology of Chemicals (ECETOC) targeted risk assessment (TRA) or Stoffenmanager, are used to screen a wide range of substances. Those of concern are investigated further using second tier tools, e.g., Advanced REACH Tool (ART). Local sensitivity analysis (SA) methods are used here to determine dominant factors for three models commonly used within the REACH framework: ECETOC TRA v3, Stoffenmanager 4.5, and ART 1.5. Based on the results of the SA, the robustness of the models is assessed. For ECETOC, the process category (PROC) is the most important factor. A failure to identify the correct PROC has severe consequences for the exposure estimate. Stoffenmanager is the most balanced model and decision making uncertainties in one modifying factor are less severe in Stoffenmanager. ART requires a careful evaluation of the decisions in the source compartment since it constitutes ∼75% of the total exposure range, which corresponds to an exposure estimate of 20-22 orders of magnitude. Our results indicate that there is a trade off between accuracy and precision of the models. Previous studies suggested that ART may lead to more accurate results in well-documented exposure situations. However, the choice of the adequate model should ultimately be determined by the quality of the available exposure data: if the practitioner is uncertain concerning two or more decisions in the entry parameters, Stoffenmanager may be more robust than ART.

Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.

OBJECTIVES: Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics. MATERIALS AND METHODS: Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC. RESULTS: All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively. CONCLUSIONS: 5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.

Accuracy of MRI for the diagnosis of metastatic cervical lymphadenopathy in patients with thyroid cancer.

PURPOSE: The aim of this study was to systematically compare a comprehensive array of magnetic resonance (MR) imaging features in terms of their sensitivity and specificity to diagnose cervical lymph node metastases in patients with thyroid cancer. MATERIALS AND METHODS: The study included 41 patients with thyroid malignancy who underwent surgical excision of cervical lymph nodes and had preoperative MR imaging ≤4weeks prior to surgery. Three head and neck neuroradiologists independently evaluated all the MR images. Using the pathology results as reference, the sensitivity, specificity and interobserver agreement of each MR imaging characteristic were calculated. RESULTS: On multivariate analysis, no single imaging feature was significantly correlated with metastasis. In general, imaging features demonstrated high specificity, but poor sensitivity and moderate interobserver agreement at best. CONCLUSIONS: Commonly used MR imaging features have limited sensitivity at correctly identifying cervical lymph node metastases in patients with thyroid cancer. A negative neck MR scan should not dissuade a surgeon from performing a neck dissection in patients with thyroid carcinomas.

(MA)LDI MS Imaging at High Specificity and Sensitivity

(Matrix-assisted) laser desorption/ionization ((MA)LDI) mass spectrometry imaging (MSI) has been driven by remarkable technological developments in the last couple of years. Although molecular information of a wide range of molecules including peptides, lipids, metabolites, and xenobiotics can be mapped, (MA)LDI MSI only leads to the detection of the most abundant soluble molecules in the cells and, consequently, does not provide access to the least expressed species, which can be very informative in the scope of disease research. Within a short period of time, numerous protocols and concepts have been developed and introduced in order to increase MSI sensitivity, including in situ tissue chemistry and solvent-free matrix depositions. In this chapter, we will discuss some of the latest developments in the field of high-sensitivity MSI using solvent-free matrix depositions and will detail protocols of two methods with their capability of enriching molecular MSI signal as demonstrated within our laboratory.