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PubMed2006

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

BACKGROUND: Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. METHODS: We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. RESULTS: The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. CONCLUSIONS: Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Fluorescence↗

PubMed1994

Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines.

The epidermal growth factor receptor (EGFR) is considered a tumor-related marker with potential diagnostic and prognostic value. In order to assess the sensitivity of flow cytometry to detect EGFR and to quantify receptors objectively, two human bladder carcinoma cell lines with different urothelial differentiation, RT4 and J82, were grown in vitro, and their membrane EGFR content was measured by flow cytometry. Exponential monolayers showed decrease of EGFR content after 20 min pulses with 10 ng/ml EGF in medium, as detected with the antibody EGFR1 in a double staining technique with propidium iodide for DNA evaluation. Further decrease of green fluorescence intensity was seen in cells constantly exposed to EGF. Absolute receptor numbers were determined by Scatchard analysis with radioactive EGF and resulted in relatively low receptor numbers for both cell lines (approximately 3-4 x 10(4) EGFR/cell), as well as one affinity class. These findings could be matched by absolute receptor quantification by flow cytometry, adding beads with defined antigenic sites (Quantum Simply Cellular, Microbead Corporation) to the cell suspension for staining. Our data suggest that flow cytometric EGFR detection and quantitation may be supplied to in vivo tumor samples and that measurements by multiparameter analysis may define subpopulations valuable for tumor diagnosis and judgment on tumor progression.

Biomarkers, Tumor↗

PubMed2001

Induction of interleukin-1beta and interleukin-6 mRNA by low doses of ionizing radiation in macrophages.

We have previously reported the antimetastatic effects and augmentation of immune responses, which would be a mechanism of the antimetastatic effects, of 0.1 to 0.2 Gy total body irradiation. To elucidate the cellular mechanisms of the augmentation of immune response, we investigated the effects of low-dose irradiation on gene expression of interleukin-1beta (IL-1beta) and IL-6 using mouse peritoneal macrophages in vitro. Absolute mRNA quantification was carried out using competitive polymerase chain reaction. Gene expression of IL-1beta and IL-6 was increased 1 to 2 hr after 2.0 Gy irradiation and then decreased to below the basal expression level 4 hr after irradiation. Irradiation with 0.1 Gy increased IL-6 expression 2 hr after irradiation, but it did not affect IL-1beta expression. Downregulation of IL-1beta and IL-6 observed 4 hr after 2.0 Gy irradiation was not observed with 0.1 Gy irradiation. The protein kinase C (PKC) inhibitor H7 and the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin inhibited induction of IL-1beta and IL-6 expression, which suggests that radiation-induced IL-1beta and IL-6 expression is achieved by PKC- and PI3-kinase-mediated signaling.

Androstadienes↗

PubMed2006

Toward quantitative short-echo-time in vivo proton MR spectroscopy without water suppression.

A methodological development for quantitative short-echo-time (TE) in vivo proton MR spectroscopy (MRS) without water suppression (WS) is described that integrates experimental and software approaches. Experimental approaches were used to eliminate frequency modulation sidebands and first-order phase errors. The dominant water signal was modeled and extracted by the matrix pencil method (MPM) and was used as an internal reference for absolute metabolite quantification. Spectral fitting was performed by combining the baseline characterization by a wavelet transform (WT)-based technique and time-domain (TD) parametric spectral analysis using full prior knowledge of the metabolite model spectra. The model spectra were obtained by spectral simulation instead of in vitro measurements. The performance of the methodology was evaluated by Monte Carlo (MC) studies, phantom measurements, and in vivo measurements on rat brains. More than 10 metabolites were quantified from spectra measured at TE = 20 ms on a 4.7 T system.

Algorithms↗

PubMed1993

Interpretation of the laser Doppler flow signal from the liver of the rat.

It has been proposed that the laser Doppler flow (LDF) signal from the surface of the rat liver is almost exclusively a measure of hepatic arterial and not of total liver blood flow and therefore that LDF is not a suitable technique for the measurement of blood flow in the hepatic microcirculation. The objective of the present study was twofold: (i) to establish that liver blood flow is homogeneously distributed and (ii) to assess the behavior of the LDF signal during changes in hepatic perfusion. When 51Cr-labeled microspheres were injected into the portal vein (n = 12), no significant differences in the relative flow (cpm/lobe to cpm/liver) to each of the liver lobes were found nor was there any difference in the ratio of flow to the outer 1-2 mm of lobe as compared to that to the "core" of the liver. Temporary occlusion of the hepatic artery and the portal vein caused approximately 13% (n = 7, P < 0.001) and approximately 74% (n = 7, P < 0.001) fall in LDF signal, respectively. Diversion of flow from the anterior to the posterior lobes (n = 5) caused a 97.9 +/- 21.1% (SD, P < 0.001) rise in LDF signal in the posterior lobes. Zero-flow LDF signal was found to represent 13.0 +/- 4.1% of maximum. Hemorrhage (in 1.5-ml aliquots) was associated with a fall in mean arterial pressure (MAP) and LDF signals. A linear relationship between MAP and the LDF signal (r > 0.9) was found. Reinfusion of blood caused both MAP and the LDF signal to return to normal. We conclude that (i) blood flow in rat liver is homogeneously distributed; (ii) the LDF signal from the liver surface responds in a manner predicted by conventional theories of hepatic hemodynamics during alteration, either independent or combined, in hepatic arterial and portal venous blood flow; and (iii) LDF may be used to measure relative changes in hepatic perfusion but problems associated with zero-flow signal and intersite variability preclude its quantification in absolute flow units.

Animals↗

PubMed2001

Fetal hippocampal CA3 cell grafts transplanted to lesioned CA3 region of the adult hippocampus exhibit long-term survival in a rat model of temporal lobe epilepsy.

Intracerebroventricular administration of kainic acid in the adult rat, a widely used model for studying human temporal lobe epilepsy, results in widespread degeneration of CA3-pyramidal neurons. Transplantation of specific fetal hippocampal CA3 cell grafts into the lesioned CA3-region at a prolonged post lesion delay of 45-day leads to 31% graft cell survival at 1 month postgrafting and significantly facilitates appropriate recovery of the lesioned host hippocampus. However, the capability of hippocampal CA3 cell grafts for enduring survival in this model is unknown. We hypothesize that a significant fraction of fetal CA3 cells grafted into the lesioned CA3 region of the adult hippocampus at 45-days postlesion exhibit long-term survival. We measured the extent of cell survival within 5'-bromodeoxyuridine-labeled CA3 cell grafts at 1 year postgrafting, following their transplantation at 45 days postlesion into the lesioned CA3-region. Quantification of absolute graft cell survival using BrdU immunostaining and the optical fractionator counting method revealed survival of 36% of grafted cells at 1 year postgrafting. Thus, over a third of fetal hippocampal CA3 cells transplanted to the lesioned CA3-region at 45 days postlesion exhibit long-term survival. Further, the extent of cell survival in these grafts is highly analogous to the degree of cell survival in CA3 grafts analyzed earlier at 1 month postgrafting, suggesting that specific fetal cells that survive the first month of grafting into the lesioned CNS area are capable of exhibiting enduring survival.

Aging↗

PubMed1994

Developmental regulation of brain-derived neurotrophic factor messenger RNAs transcribed from different promoters in the rat brain.

During the development of the nervous system many types of neurons are produced in excess and die because they fail to obtain sufficient amounts of target-derived neurotrophic factors (for review, see Refs 1, 16). Brain-derived neurotrophic factor is a protein that belongs to the nerve growth factor family of neurotrophins (for review, see Ref. 17) which promotes the survival and differentiation of distinct neuronal populations that partially overlap with those affected by the other members of the family (for review, see Ref. 11). We have recently shown that four promoters direct tissue specific expression of the rat brain-derived neurotrophic factor gene. In the present study we have quantified the levels of brain-derived neurotrophic factor messenger RNAs transcribed from different promoters during the rat brain development. Our data show that different promoters have specific regulation patterns during embryonic development but are regulated coordinately in several regions of rat brain during postnatal development, suggesting that the four transcription units have both distinct and common regulatory elements. Quantification of absolute levels of brain-derived neurotrophic factor messenger RNA revealed that it is expressed significantly higher in the rat brain than previously estimated.

Aging↗

PubMed2005

Increased expression of MDR1 mRNAs and P-glycoprotein in placentas from HIV-1 infected women.

P-glycoprotein transports several compounds including protease inhibitors, actually used in the clinical management of HIV-1 infection. Since P-glycoprotein is expressed in placental trophoblasts, its efflux activity could interfere with placental transfer of antiretrovirals. The purpose of this study was to investigate the expression of P-gp-encoding MDR1 gene and P-gp itself in full-term placentas from uninfected (n=35) and HIV-1 infected women (n=24). MDR1 transcripts were quantified by real-time PCR using relative (MDR1 normalized upon 28S levels) and absolute (copy number) determinations. P-glycoprotein localization and expression were evaluated by immunohistochemistry and western blot analysis, respectively. Relative or absolute PCR quantification showed a significant 3.3-fold (p<0.0009) or 3.7-fold (p<0.0002) mean increase in MDR1 placental transcription in HIV-infected compared to non-infected women, respectively. Ratios of individual HIV-positive values to HIV-negative mean ranged from 0.1 to 21.8. Moreover a significant 2.5-fold increased expression of immunoreactive P-glycoprotein was evidenced in placentas from HIV-infected women (p<0.0001). This MDR1 overexpression was observed in a similar extent in placentas from pregnant women treated with Zidovudine alone or in combination with Nelfinavir and/or Lamivudine. Our findings suggest that P-glycoprotein in placentas from HIV-infected women would contribute to modulate the materno-fetal transport of antiretrovirals across the placental barrier and consequently diminish fetal exposure to these compounds.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR.

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.

Animals↗

PubMed1997

The rate of mitochondrial mutagenesis is faster in mice than humans.

We have investigated mitochondrial DNA (mtDNA) mutagenesis in the laboratory mouse. Using a nested PCR method for quantification, the absolute frequency, tissue distribution and rate of increase of mitochondrial deletion mutations was determined. Multiple deletions arise in brain, cardiac muscle and kidney tissues: deletions occur most frequently at regions of directly repeated mtDNA homology. Deletion frequencies rose by 2.5 x 10(5), 6300- and 4000-fold in heart, brain and kidney, respectively, between young and old mice. The rates of mtDNA mutation accumulation in mouse and human hearts are modeled well by exponential equations, with r-values of 0.96 and 0.97, and mutations rose much faster in mouse than human mtDNA per unit time. Thus, maintenance of the human mitochondrial genome is much better than that of mice, consistent with the higher rate and final extent of total DNA repair in humans than mice, that has been observed by others and consistent with the predictions of the disposable soma model of aging. A comparison of mtDNA mutagenesis from cardiocytes vs. whole heart tissue was undertaken. Deletion mutations were observed to be 100-fold lower in DNA prepared from isolated cardiocytes than from whole heart homogenates, consistent with a model of uneven mtDNA mutation accumulation.

Adolescent↗

PubMed2000

Analysis of input functions from different arterial branches with gamma variate functions and cluster analysis for quantitative blood volume measurements.

Regional cerebral blood volume (rCBV) provides valuable information about the nature and progress of diseases of the central nervous system. While relative rCBV maps can be derived directly from dynamic susceptibility contrast data, the arterial input function (AIF) has to be measured for absolute rCBV quantification. For determination of the AIF pixels located completely within a feeding artery must be selected. However, by using a region-of-interest (ROI) based selection some confounding effects can occur, especially if single shot echo planar imaging (EPI) with low spatial resolution is used. In this study we analyzed the influence of partial volume effects and spatial misregistration due to frequency shifts induced by paramagnetic contrast agents. We analyzed AIFs from the internal carotid artery (ICA), the vertebral artery (VA) and the middle cerebral artery (MCA) using gamma variate function based parameterization. The concentration time curves (CTC) of several pixels which were selected on the basis of strong signal drop appeared distorted during the bolus passage. Moreover, the amplitudes of input functions derived from the MCA were smaller by a factor of three as compared to those of the ICA and VA. Simulations revealed that these effects can be attributed to a spatial shift of the vessel along phase-encoding direction during the passage of the bolus. We therefore developed a procedure for a pixel selection based on cluster analysis which classifies pixels according to the parameters of the fitted gamma variate functions. This approach accounted for misregistration of the vessel and yielded very consistent results for a group of normal subjects.

Brain↗

PubMed2005

Whole-grain intake of British young people aged 4-18 years.

Inverse associations between whole-grain food consumption and risk of CVD, some cancers and type 2 diabetes have been reported. However, there are few reports of whole-grain intake, particularly among young people. The objective of the present study was to estimate whole-grain intake in a nationally representative sample of young people aged 4-18 years living in Great Britain. Whole-grain intake was estimated using 7 d weighed dietary records from 1583 young people who participated in the cross-sectional National Diet and Nutrition Survey in 1997. Whole-grain intake was quantified from the consumption of all foods containing > or =10 % whole-grain content. Median whole-grain intake was 7 g/d (interquartile range 0-19 g/d), with a corresponding mean of 13 (sd 18) g/d. Intake was significantly lower among young people whose head of household had a manual occupation, but did not differ significantly by sex, age, region or season. There was no whole-grain intake for 27 % of participants. The percentages for less than one and less than three 16 g amounts of whole-grain intake per d were 70 and 94, respectively, while corresponding percentages based on 20 g amounts were 76 and 97. Foods with <51 % whole-grain content provided 28 % of whole-grain intake overall, with a higher percentage in older adolescents. The main sources of whole-grain intake were breakfast cereals (56 %) and bread (25 %). The present study provides the first quantification of absolute whole-grain intake from all significant food sources in any representative age group in the UK. Although there is some debate regarding the quantity of whole grains required for good health, whole-grain intake among British young people is low.

Adolescent↗

PubMed2004

Quantification of prostate specific antigen mRNA levels in circulation after prostatic surgery and endocrine treatment by quantitative reverse transcription-polymerase chain reaction.

Various methods to detect prostatic cells in circulation have given conflicting results. This is probably because qualitative rather than quantitative methods have been used to detect mRNA from prostatic cells. A quantitative method has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) for detection of prostate specific antigen (PSA) mRNA in peripheral blood. A competitive internal mRNA standard was used for quantification of absolute amounts of PSA mRNA. The detection limit of the assay was 7 copies of mRNA, and the highest level of circulating PSA mRNA in 88 control subjects was 25 copies per milliliter of blood. This method was used to study the influence of prostatic surgery and endocrine treatment on prostatic cells in the circulation of 56 patients undergoing biopsy, radical prostatectomy, transurethral resection of the prostate (TURP), orchiectomy, or androgen blockade. Blood samples were drawn before, during and up to 26 weeks after these procedures had been carried out. The highest level of PSA mRNA in controls was 25 copies per milliliter of blood. After RP, TURP or orchiectomy, PSA mRNA levels increased above this level in 27%, 29%, and 25% of the samples, respectively. After prostate biopsy, two out of 15 patients became positive. PSA mRNA levels that were elevated by surgery became undetectable within 1-3 days. No significant correlation was found between PCR positivity and the clinical characteristics of the patients. It is concluded that the level of PSA mRNA in peripheral blood increases after prostatic surgery, indicating temporary dissemination of prostatic cells. However, preoperative levels do not correlate with serum PSA, stage or grade.

Cell Line↗

PubMed2005

Quantitative measurement of regional lung gas volume by synchrotron radiation computed tomography.

The aim of this study was to assess the feasibility of a novel respiration-gated spiral synchrotron radiation computed tomography (SRCT) technique for direct quantification of absolute regional lung volumes, using stable xenon (Xe) gas as an inhaled indicator. Spiral SRCT with K-edge subtraction using two monochromatic x-ray beams was used to visualize and directly quantify inhaled Xe concentrations and airspace volumes in three-dimensional (3D) reconstructed lung images. Volume measurements were validated using a hollow Xe-filled phantom. Spiral images spanning 49 mm in lung height were acquired following 60 breaths of an 80% Xe-20% O2 gas mixture, in two anaesthetized and mechanically ventilated rabbits at baseline and after histamine aerosol inhalation. Volumetric images of 20 mm lung sections were obtained at functional residual capacity (FRC) and at end-inspiration. 3D images showed large patchy filling defects in peripheral airways and alveoli following histamine provocation. Local specific lung compliance was calculated based on FRC/end-inspiration images in normal lung. This study demonstrates spiral SRCT as a new technique for direct determination of regional lung volume, offering possibilities for non-invasive investigation of regional lung function and mechanics, with a uniquely high spatial resolution. An example of non-uniform volume distribution in rabbit lung following histamine inhalation is presented.

Animals↗

PubMed2006

SPECT Imaging for Radioaerosol Deposition and Clearance Studies.

Planar gamma camera scintigraphy is well established for measuring the deposition and clearance of radioaerosols. Single photon emission computed tomography (SPECT) provides threedimensional (3D) reconstructions of the radioactivity distribution, thus avoiding the compression of 3D data into two-dimensional (2D) images and potentially offering superior assessment of aerosol deposition patterns. However, SPECT has traditionally been associated with long imaging times, making it unsuitable for measuring deposition and clearance of radioaerosols with fast clearance. Multi-detector SPECT systems can collect complete SPECT studies in <1 min, allowing both initial deposition and clearance over time to be assessed by dynamic SPECT. Simultaneous transmission measurement with an external source provides attenuation correction for absolute activity quantification as well as aiding in the definition of the lung volume of interest. A dynamic SPECT imaging protocol has been developed to allow fast imaging from the oropharynx to the abdomen using gamma cameras with limited axial field of views. This allows activity quantification not only in the lungs, but also in areas outside the thorax. However, fast dynamic SPECT imaging is technically and computationally more demanding and provides less scope for reducing the radioactivity administered to the subjects. It has been shown that dynamic SPECT, compared to planar imaging, is more sensitive in detecting changes in deposition as measured by the Penetration Index (PI). Thus, SPECT can better differentiate between large and small airways, which is important for lung regional analysis.

Administration, Inhalation↗

PubMed1990

Quantitative dual-energy coronary arteriography.

Subtraction techniques for digital cardiac imaging have been hampered by misregistration artifacts. The use of dual-energy imaging is being evaluated as a means for reducing these artifacts. Results reported previously indicate that the dual-energy technique may be useful for applications such as exercise ventriculography and general quantification tasks. The purpose of the current study is to investigate the use of dual-energy subtraction imaging for quantitative coronary arteriography. In vivo coronary vessel phantoms (0.2 to 7 mm2 in cross-sectional area) were used to study the potential advantages of tissue suppressed energy subtracted images over unsubtracted images for quantification of absolute vessel cross-sectional area when cardiac motion is present. Estimates of lumen cross-sectional area (N = 20) were determined using videodensitometric analysis of selected energy subtracted and unsubtracted images. Linear regression analysis of measured and actual cross-sectional area showed energy subtracted image data (slope = 1.06, intercept = 0.48 mm2, r = 0.99) to have improved accuracy (P less than .05) and precision (P less than .05) over unsubtracted image data (slope = 1.24, intercept = 1.07 mm2, r = 0.95).

Angiography, Digital Subtraction↗

PubMed1989

Comparison of two interpolative background subtraction methods using phantom and clinical data.

Two interpolative background subtraction methods used in scintigraphy are tested using both phantom and clinical data. Cauchy integral subtraction was found to be relatively free of artefacts but required more computing time than bilinear interpolation. Both methods may be used with reasonable confidence for the quantification of relative measurements such as left ventricular ejection fraction and myocardial perfusion index but should be avoided if at all possible in the quantification of absolute measurements such as glomerular filtration rate.

Glomerular Filtration Rate↗

PubMed2003

A new radionuclide approach for the quantification of left ventricular volumes: the 'geometric count based' method.

A new radionuclide method, called the 'geometric count based' (GCB) method, has been developed for the quantification of absolute left ventricular volume. As the method is based on planar radionuclide ventriculography, it is non-invasive and simple, and avoids the relatively cumbersome and longer lasting, dynamic procedure using single photon computed emission tomography, which can be used for achieving the same goal. The purpose of this study was to describe the exactness of the theoretical approach to the method and validate its accuracy both by physical experiments and the initial clinical trial, as compared to contrast ventriculography. Count based data were combined with the geometric based data assuming an ellipsoid left ventricular shape with identical short axes. The following equation for computing left ventricular end diastolic volume, EDV (in ml) was developed: EDV=2cMCtot/Cmax, where c is the manually drawn short axis (one row pixel ROI) of the prolate ellipsoid in LAO 45 degrees (cm), M is the calibrated pixel size (in cm2), Ctot is the total counts in LV ROI, and Cmax is the maximum pixel counts in the LV ROI. Physical experiments with two different 'heart shaped' phantoms were used to compare the results obtained by the GCB method with the true phantom volumes and with the method assuming LV ball shape (BLV), developed by other authors. The true volumes of cylindrical and ellipsoid phantoms of 112.5 ml and 190.5 ml were computed to be 114 ml and 196 ml by the GCB and 168 ml and 180 ml by the BLV methods, respectively. In a clinical study, GCB volumes were compared to volumes measured by using single plane contrast ventriculography in 38 coronary patients. A good correlation between the GCB method and contrast ventriculography was obtained both for EDV and end systolic ventricular volumes (r=0.94, r=0.90). Both phantom and initial clinical studies indicate that the GCB method is an accurate, non-invasive and simple radionuclide method for measuring left ventricular volumes. Additionally, it could be used even in the smallest nuclear medicine units, for example in intensive care units where there are mobile cameras.

Algorithms↗