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PubMed2000

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers.

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.

Base Sequence↗

PubMed2002

Simultaneous separation and detection of actinides in acidic solutions using an extractive scintillating resin.

An extractive scintillating resin was evaluated for the simultaneous separation and detection of actinides in acidic solutions. The transuranic extractive scintillating (TRU-ES) resin is composed of an inert macroporous polystyrene core impregnated with organic fluors (diphenyloxazole and 1,4-bis-(4-methyl-5-phenyl-2-oxazolyl)benzene) and an extractant (octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide in tributyl phosphate). The TRU-ES resin was packed into FEP Teflon tubing to produce a flow cell (0.2-mL free column volume), which is placed into a scintillation detection system to obtain pulse height spectra and time series data during loading and elution of actinides onto/from the resin. The alpha-particle absolute detection efficiencies ranged from 77% to 96.5%, depending on the alpha energy and quench. In addition to the on-line analyses, off-line analyses of the effluent can be conducted using conventional detection methods. The TRU-ES resin was applied to the quantification of a mixed radionuclide solution and two actual waste samples. The on-line characterization of the mixed radionuclide solution was within 10% of the reported activities whereas the agreement with the waste samples was not as good due to sorption onto the sample container walls and the oxidation state of plutonium. Agreement between the on-line and off-line analyses was within 35% of one another for both waste samples.

Acids↗

PubMed2006

Tumor infiltrating T lymphocytes in colorectal cancer: Tumor-selective activation and cytotoxic activity in situ.

OBJECTIVE: To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. SUMMARY BACKGROUND DATA: Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8 TIL in situ in colorectal cancer patients have not yet been examined. METHODS: Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II-IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8 T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-gamma Elispot analysis. RESULTS: While absolute numbers of CD8 T cells were similar, CD4 T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8 TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. CONCLUSIONS: Tumor-selective activation and cytotoxic activity of CD8 TIL and tumor-selective migration of CD4 T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.

Adenocarcinoma↗

PubMed1995

Quantification of alpha 1-adrenoceptor subtypes in human tissues by competitive RT-PCR analysis.

Alpha 1-Adrenoceptors are a heterogeneous subfamily of receptors comprising at least three pharmacologically- and structurally- distinct subtypes named alpha 1a-, alpha 1b- and alpha 1d-adrenoceptors. A competitive reverse transcription-polymerase chain reaction methodology using subtype selective competitor RNAs as reference internal standards was adopted to determine the absolute amounts of each of the three cloned to date alpha 1-adrenoceptor subtype encoding mRNAs in selected human tissues. Our data demonstrated that each of these alpha 1-adrenoceptor subtypes exhibits a distinctive tissular expression pattern.

Base Sequence↗

PubMed1997

[Air pollution in Switzerland--quantification of health effects using epidemiologic data].

Public health costs ascribable to air pollution are socialized in our society. To quantify the damage to public health, epidemiologic studies are needed. We present the methods and epidemiologic background data which form the basis for estimating the public health effect ascribable to air pollution. The figures are presented per 1 million "average" Swiss population and per 10 micrograms/m3 increase in long-term annual mean particulate pollution (PM10). Quantification was restricted to the health effects given below (due to lack of complete data for other effects or to avoid duplicating health effects which may be described by overlapping measurements). In parenthesis we present (1) the mean effect estimates (+/-1 SE) (% increase per 10 micrograms/m3 increment in PM10) derived from national and international epidemiologic studies, and (2) the expected absolute additional health effects (+/-1 SE) per 1 million Swiss population and per 10 micrograms/m3 increment in PM10, based on Swiss population statistics: total mortality (long-term estimates from the 2 US cohort studies) (+4.4% [+/-1.1]/349 [+/-91] premature deaths per year); prevalence of chronic bronchitis in adults (+25% [+8.4]/increase in long-term prevalence by 3,513 [+/-1.475]); bronchitis among children (35% [+/-13]/5,180 [+/-2,590] additional children sufferers in a year); repeated cough among children (+54% [+/-8.8]/23,490 [+/-5,873] additional children per year); cough/phlegm in adults (+12.8% [+/-7.8]/1.56 [+/-1.08] million person-days per year); hospital admissions for respiratory diseases (+1.47% [+/-0.5]/71 [+/-24] additional admissions or 1,104 (+/-390) hospital days); admissions for cardiovascular diseases (+0.9% [+/-0.25]/70 [+19] admissions; 970 (+/-270) hospital days); restricted activity days (+10.5% [+/-0.77]/0.42 [+/-0.03] million person-days); days with asthma attacks among the 6.7% asthmatics (Swiss prevalence) (+5.3% [+/-2.1]/ 240,000 [+/-102,000] additional person-days). Conservative assumptions were used throughout the study and thus the true effects are likely to be larger. Estimates of Swiss population exposure distribution would be needed to apply these results to the entire population. This step, and monetary quantification of these total effects, were the second and third elements (not shown in this report) of this Swiss government funded project. Although individual relative risks of air pollution are rather slight, the public health burden of even moderate pollution may be substantial.

Adult↗

PubMed2005

Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides.

Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.

Animals↗

PubMed2001

Detection and quantification of HIV type 1 RNA in nasopharyngeal washes from HIV-infected subjects.

Human immunodeficiency virus type 1 (HIV) RNA load was measured in paired samples of peripheral blood plasma and nasopharyngeal (NP) washes from 97 Thai subjects infected with subtype E or B. HIV RNA was quantifiable in 93% of peripheral blood plasma samples tested and was inversely correlated (rho =-0.524; p < 0.001) with CD4 absolute count. HIV RNA was quantifiable in 29% of NP samples tested, and the median value was less than that of plasma viral load. HIV RNA load in NP samples was correlated (rho = 0.388; p < 0.001) with viral load in peripheral blood. HIV RNA was not detected in NP washes from subjects with undetectable plasma viral load. Virus isolation attempts on two NP samples were negative. The results do not support local HIV production in the nasopharynx, but extend current knowledge of HIV shedding to include the NP compartment.

CD4 Lymphocyte Count↗

PubMed2006

A novel 3-d model of chick chorioallantoic membrane for ameliorated studies in angiogenesis.

Decisive indulgence of angiogenesis requires a more holistic assessment and several in vivo assays have been developed that permit a more realistic appraisal of the angiogenic response. One of the most popular assays to study angiogenic activity is the chick chorioallantoic membrane (CAM). Although CAM assay is a vital technique used to study normal and putative angiogenesis, a serious drawback in its utilization is the lack of quantitative assessment of vascularization. In this study, we proposed a new 3-D model of the developing CAM for precise quantification of normal vasculature of CAM from Day 4 to Day 13 of incubation. Image probing technique was used to quantify different 3-D parameters of vascular microarchitecture. A significant increase (P < 0.05) in surface roughness (Sa) was observed at Day 5 of incubation, while highly significant increase (P < 0.01) in Sa values was observed at Day 6 of incubation. Maximum increase (P < 0.001) in Sa values was observed from Day 7 to Day 9 of incubation and slight decrease in Sa values was observed in successive days. Similar results were observed for root mean square values (Sq), absolute heights of the surface (Sz), and amount of lowest valleys (Sy) on CAM. A significant increase (P < 0.05) in developed surface area (Sdr) from Day 5 of incubation, reaching to its maximum (P < 0.01) at Day 9 also was noted. Similarly, significant increase (P < 0.05) in fluid core retention (Sci) illustrates presence of copious fluid in blood vessels. Image probing technique offers a useful modality for visualizing 3-D microvascular architecture of CAM to exaggerate the fine details and reveal the hidden information that can be helpful for precise quantification of angiogenesis. This approach can be used to evaluate the angiogenic and antiangiogenic potential of different biological substances and also can be a valuable independent prognostic indicator in a wide variety of human cancers.

Animals↗

PubMed2003

Flow cytometric quantification of apoptosis and proliferation in mixed lymphocyte culture.

BACKGROUND: The one-way mixed lymphocyte culture (MLC) is the classic culture used for studying the allogenic immunoresponse in vitro, but stimulator and responder cell identifications and quantification of apoptotic or proliferative responder cells are unreliable. METHODS: Peripheral blood mononuclear cells were labeled with 5- (and 6-) carboxy fluorescein diacetate succinimidyl ester (CFSE) and stimulated with allogenic unlabeled irradiated cells in unidirectional cultures. Apoptosis was determined by the 7-aminoactinomycin D technique, and the absolute number of each cell population was calculated by adding a fixed number of cells stained with propidium iodide as the reference standard for each test. RESULTS: CFSE labeling of cells under different cultures did not affect the results of proliferation or apoptosis. Data of apoptosis obtained with this method were comparable to those of the monoclonal antibody technique, and the proliferation level determined by [(3)H]-thymidine incorporation or counting the number of proliferative living cells, as proposed in this method, showed a good correlation. CONCLUSIONS: The method presented in this report allows the simultaneous determination of apoptosis and proliferation in MLCs and the analysis of cell phenotype, thereby avoiding the use of radioactivity. This assay opens new perspectives for a better understanding of the mechanisms implied in the establishment or break of tolerance to the graft in solid organ transplants.

Apoptosis↗

PubMed1986

A new approach to the measurement of phospholipase A2 in tissue homogenates and its application to human skin.

We describe a new approach to the quantification of tissue phospholipase A2 based on the observation that the Vmax is increased by several orders of magnitude in the presence of high concentrations of dimethyl sulphoxide. The enzyme from human skin showed optimal activity at 30% dimethyl sulphoxide and pH 6-7.5, and had an absolute dependence on Ca2+ (apparent Km = 1.4 mmol/l). The relationship between reaction velocity and substrate concentration did not follow Michaelis-Menten kinetics. The enzyme was inhibited by p-bromophenacyl bromide, half-maximal inhibition occurring at 2.4 mumol/l. Surprisingly, assay of specimens of various human tissues indicated that, with the exception of pancreas, skin contained higher levels of phospholipase A2 activity than any other tissue investigated. Equally surprising was that primate skin (in particular human) seems considerably more active than that of other mammals. Speculatively, these findings may be related to the high levels of free fatty acids occurring on the skin surface.

Animals↗

PubMed2006

Simultaneous quantification of alpha-/beta-diastereomers of arteether, sulphadoxine and pyrimethamine: a promising anti-relapse antimalarial therapeutic combination, by liquid chromatography tandem mass spectrometry.

A rapid, sensitive, selective and specific HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of alpha-/beta-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) in rat blood plasma using propyl ether analogue of beta-arteether as internal standard. The method involved a single-step, liquid-liquid extraction with ethyl acetate and the analytes were chromatographed on a C18 chromatographic column by isocratic elution with methanol:ammonium acetate buffer (10 mM, pH 4) (90:10%, v/v) and analyzed by tandem mass spectrometry. The run time was 4.5 min and the weighted (1/x2) calibration curves were linear over a range of 0.78-400 ng ml-1. The method was validated fully and the lower limit of quantification (LLOQ) in plasma was 0.78 ng ml-1 for all the analytes. The intra- and inter-day precision and accuracy were found to be well within the acceptable limits (<15%) and the analytes were stable after three freeze-thaw (f-t) cycles. The absolute recoveries were consistent and reproducible. The assay method was applied to pre-clinical pharmacokinetic interaction studies of alpha-/beta-AE, SDX and PYR in rats.

Animals↗

PubMed1999

Simultaneous determination of tramadol and its major active metabolite O-demethyltramadol by high-performance liquid chromatography with electrochemical detection.

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O-demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10-1000 ng/ml for ODMT and 10-10000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 microl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.

Analgesics, Opioid↗

PubMed1998

White cell subsets in apheresis and filtered platelet concentrates.

BACKGROUND: White cell (WBC)-reduced platelet concentrates (PCs) are defined by their absolute WBC count, a criterion which provides no information regarding the various WBC subsets contained in the PC. These heterogeneous cells are known to mediate different physiologic and pathophysiologic functions and account for distinct adverse transfusion responses. This study describes a method which allows the detection and quantification of these subsets and characterizes their presence in a variety of platelet components. STUDY DESIGN AND METHODS: Random-donor pooled PCs (RD PCs) and single-donor apheresis PCs (SD PCs) were studied. RD PCs consisting of 6 units of 2- to 3-day old PCs were randomly assigned to be filtered with one of four WBC-reduction filters from three different manufacturers (n=34). The residual WBCs were pelleted by centrifugation and isolated on a density gradient. The various WBC subsets were quantified by flow cytometry in unfiltered and filtered PCs using fluorescence and two-angle light scatter. SD PCs obtained with two manufacturer's systems and three processing protocols (n=30) were studied in like manner. RESULTS: WBC counts for non-WBC-reduced PCs averaged 3 x 10(8) in RD PCs and ranged from 8.6 to 9.6 x 10(6) per SD PC. Residual WBC counts in filtered PCs ranged from 2.3 x 10(4) to 2.2 x 10(5) and those in WBC-reduced SD PCs averaged 2.2 x 10(5) per unit. The data demonstrate significant phenotypic differences among PCs produced with various procedures. All SD PCs and two of four filtered RD PCs contained five WBC populations including granulocytes and monocytes, while RD PCs filtered with the remaining manufacturer's devices contained only lymphocytes. CONCLUSION: The data confirm that distinct phenotypic differences exist among PCs prepared with different devices and/or procedures. It is suggested that as for non-generic pharmaceuticals, the clinical benefits of these various PCs should be individually proved.

Filtration↗

PubMed1999

Clinical application of breath biomarkers of oxidative stress status.

Isolation and quantification of volatile breath biomarkers indicative of relevant alterations in clinical status has required development of new techniques and applications of existing analytical chemical methods. The most significant obstacles to successful application of this type of sample have been reduction in required sample volume permitting replicate analysis (an absolute requirement for all clinical studies), separation of the analyte(s) of interest from background molecules, water vapor and other molecules with similar physical properties, introduction of automation in analysis and the use of selective detection systems (electron impact mass spectrometry, flame photometric, thermionic detectors), and automated sample collection from the human subject. Advances in adsorption technology and trace gas analysis have permitted rapid progress in this area of clinical chemistry.

Animals↗

PubMed2006

Development of a nebulizer for a sheathless interfacing of NanoHPLC and ICPMS.

A novel nebulizer (nDS-200) working at sample uptake rates of less than 500 nL min(-1) was developed for a sheathless interfacing of nanoHPLC (75-microm column i.d.) with ICPMS. It was based on a hollow fused-silica needle of which the tip (i.d. 10 microm, o.d. 20 microm) centered in a 254-microm-i.d. sapphire orifice. The nebulizer, equipped with a 3-cm(3) drain-free vaporization chamber, enabled a stable introduction into an ICP of aqueous mobile phases containing up to 95% acetonitrile at eluent flow rates between 50 and 450 nL min(-1). The low dead volume of the interface resulted in a peak width of 1.3 s (at half-height) and the entirely preserved chromatographic resolution. An example application of the coupling to the analysis of a tryptic digest of a SIP18 protein containing two to nine selenomethionine residues was described. The absolute detection limit was 25 fg (80Se), which allowed the detection of low-abundant selenopeptides at the femtomole level. In contrast to electrospray MS, the ICPMS detection in nanoHPLC is unaffected by the coeluting matrix and concomitant compounds and offers an elegant method for the detection and quantification of minor heteroelement-containing species prior to or in parallel with ESI MS analysis.

Chromatography, High Pressure Liquid↗

PubMed2004

Isolation of genes associated with developmentally competent bovine oocytes and quantitation of their levels during development.

We have performed suppressive subtraction hybridization (SSH) of populations of developmentally competent and incompetent bovine oocytes from large (> or =5-mm) and small (< or =2-mm) follicles to isolate messenger RNA associated with the attainment of developmental competency. RNA was amplified in a linear fashion and then subjected to the SSH procedure to produce a library enriched for genes associated with competency. One thousand clones of this library were subjected to a differential screening approach to identify 31 potentially upregulated isolates. Sequencing revealed these to represent 21 genes. To rigorously identify the degree of upregulation and reproducibility thereof, we examined the expression of these genes in three separate pools of developmentally competent and incompetent oocytes by quantitative real-time PCR. Results indicated that upregulation varied from zero to threefold, showing that accurate quantification is essential for the interpretation of such differential screening experiments. Furthermore, it appears that the molecular causes for poor developmental capacity may be highly complex and be reliant on many small changes. We further characterized a selection of these novel and known maternally expressed genes for their absolute expression levels during maturation in the presence or absence of an inhibitor of transcription and during preattachment development. Last, the effect of nuclear transfer on the levels of these genes was assayed. Nuclear transfer was found to differentially affect transcript levels of genes expressed after embryonic genome activation but did not prevent the degradation of maternal transcripts or result in activation of maternal genes that are silent at blastocyst stages.

Animals↗

PubMed1991

Nondestructive detection of glutamate by 1H nuclear magnetic resonance spectroscopy in cortical brain slices from the guinea pig: evidence for changes in detectability during severe anoxic insults.

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.

Animals↗

PubMed1993

High proportion of gamma-delta T cell receptor positive T cells in bronchoalveolar lavage and peripheral blood of HIV-infected patients with Pneumocystis carinii pneumonias.

To investigate the local immunological situation in the lung of HIV-infected patients with Pneumocystis carinii pneumonias (HIV-PCP), we analyzed the proportion and the distribution of lymphocyte subpopulations and their state of activation in bronchoalveolar lavage (BAL) and peripheral blood of 21 HIV-PCP patients (CDC classification group IV) compared to 24 HIV-negative patients with interstitial lung diseases (ILD). Peripheral blood lymphocytes (PBL) and BAL cells were stained with monoclonal antibodies. Two-color cytofluorometric analysis (flow cytometry) was performed with a cytofluorograph (Epics Profile, Coulter Corp., Hialeah, Fla., USA). In BAL from HIV-PCP patients the number of CD3-positive lymphocytes was significantly increased, yet there was no difference in the number of macrophages and neutrophils when compared to patients with ILD. Quantification of lymphocyte subpopulations showed that the increased number of BAL CD3-positive lymphocytes in HIV-PCP patients was mainly due to a significantly increased number of CD8-positive T cells, while the pulmonary CD4-positive T cells were decreased both in relative and absolute numbers. As a consequence, an inverted pulmonary CD4/CD8 ratio resulted for HIV-infected patients with PCP. Analysis of in-vivo-activated T cells in BAL and peripheral blood when measured by the expression of IL-2R, HLA-DR and VLA-1 revealed increased numbers of IL-2R and HLA-DR bearing CD8-positive T cells but significantly decreased numbers of IL-2R and HLA-DR bearing CD4-positive T cells as well as a higher number of CD8/CD57 double positive T cells in HIV-infected individuals when compared to patients with ILD.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Related Opportunistic Infections↗