Quantitative analysis of cell-free Epstein-Barr virus DNA in the plasma of patients with peripheral T-cell and NK-cell lymphomas and peripheral T-cell proliferative diseases.

PubMed

Suwiwat, Supaporn; Pradutkanchana, Jintana; Ishida, Takafumi; Mitarnun, Winyou

2007-12-01

The level of circulating EBV DNA is a prognostic marker in patients with some EBV-associated malignant diseases. To investigate the presence and nature of Epstein-Barr virus (EBV) DNA in the plasma and to evaluate the correlation of plasma concentrations of EBV DNA with the EBV genomic status in peripheral blood T-cells and neoplastic cells and with the clinical outcome of patients with peripheral T-cell and NK-cell lymphomas (PTCL) and peripheral T-cell proliferative diseases (PTPD). EBV DNA in the plasma of 45 patients and 45 controls was measured using real-time PCR. The presence of the EBV genome in the isolated peripheral blood lymphocytes (CD3+ and CD3- cells) was analysed by PCR. Detection of EBV-encoded early RNA (EBER) in corresponding tumor tissues was carried out using in situ hybridization. DNase I digestion was applied to plasma samples to detect naked EBV DNA. Cell-free EBV DNA was detected in 32/38 (84%) of PTCL patients and 5/7 (71%) of PTPD patients, but not in the controls. Patients with EBV genome in peripheral blood CD3+ cells and EBV genome (EBER) in the tumor cells, compared to those without these findings, had significantly higher plasma EBV DNA levels. The majority of circulating EBV DNA molecules was naked form. The plasma EBV DNA levels were not related to survival. The concentration of EBV DNA in the plasma was not a prognostic marker in PTCL and PTPD patients.

Effect of strenuous physical exercise on circulating cell-derived microparticles.

PubMed

Chaar, Vicky; Romana, Marc; Tripette, Julien; Broquere, Cédric; Huisse, Marie-Geneviève; Hue, Olivier; Hardy-Dessources, Marie-Dominique; Connes, Philippe

2011-01-01

Strenuous exercise is associated with an inflammatory response involving the activation of several types of blood cells. In order to document the specific activation of these cell types, we studied the effect of three maximal exercise tests conducted to exhaustion on the quantitative and qualitative pattern of circulating cell-derived microparticles and inflammatory molecules in healthy subjects. This study mainly indicated that the plasma concentration of microparticles from platelets and polymorphonuclear neutrophils (PMN) was increased immediately after the strenuous exercise. In addition, the increase in plasma concentration of microparticles from PMN and platelets was still observed after 2 hours of recovery. A similar pattern was observed for the IL-6 plasma level. In contrast, no change was observed for either soluble selectins or plasma concentration of microparticles from red blood cells, monocytes and endothelial cells. In agreement, sVCAM-1 and sICAM-1 levels were not changed by the exercise. We conclude that a strenuous exercise is accompanied by platelet- and PMN-derived microparticle production that probably reflects the activation of these two cell types.

Biosensors for liquid biopsy: circulating nucleic acids to diagnose and treat cancer.

PubMed

Bellassai, Noemi; Spoto, Giuseppe

2016-10-01

The detection of cancer biomarkers freely circulating in blood offers new opportunities for cancer early diagnosis, patient follow-up, and therapy efficacy assessment based on liquid biopsy. In particular, circulating cell-free nucleic acids released from tumor cells have recently attracted great attention also because they become detectable in blood before the appearance of other circulating biomarkers, such as circulating tumor cells. The detection of circulating nucleic acids poses several technical challenges that arise from their low concentration and relatively small size. Here, possibilities offered by innovative biosensing approaches for the detection of circulating DNA in peripheral blood and blood-derived products such as plasma and serum blood are discussed. Different transduction principles are used to detect circulating DNAs and great advantages are derived from the combined use of nanostructured materials.

Simultaneous determination of hemolysis and hematocrit in extracorporeal circulation by plasma surface reflectance spectroscopy.

PubMed

Sakota, Daisuke; Kani, Yuki; Kosaka, Ryo; Nishida, Masahiro; Maruyama, Osamu

2013-01-01

To achieve quantitative non-invasive optical diagnosis of blood abnormalities during extracorporeal circulation therapies, plasma surface reflectance spectroscopy was developed by implementing oblique-incidence optical fiber reflectometry on the surface of circulating blood. The reflected light in the wavelength range from 450 to 600 nm changed with respect to the plasma free hemoglobin level and could be used to quantify the free hemoglobin at an accuracy of 5.7 ± 3.5 mg/dL. In contrast, the spectrum did not changed by varying the hematocrit. In the wavelength range from 600 to 800 nm, the obtained spectrum was affected by both the hematocrit change and hemolysis. The linear correlation between the hematocrit value and the spectrum was confirmed at R(2) = 0.99. The feasibility of determining of the hematocrit of arbitrary hemolyzed blood was confirmed. The developed system permits the extraction of the optical characteristics of both plasma and red blood cells without centrifugation. The study establishes non-invasive optical diagnostics capable of analyzing the optical properties of both plasma and red blood cells.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.

PubMed

Jackson, Jennifer B; Choi, Daniel S; Luketich, James D; Pennathur, Arjun; Ståhlberg, Anders; Godfrey, Tony E

2016-03-01

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Levels of circulating CD45dimCD34+VEGFR2+ progenitor cells correlate with outcome in metastatic renal cell carcinoma patients treated with tyrosine kinase inhibitors

PubMed Central

Farace, F; Gross-Goupil, M; Tournay, E; Taylor, M; Vimond, N; Jacques, N; Billiot, F; Mauguen, A; Hill, C; Escudier, B

2011-01-01

Background: Predicting the efficacy of antiangiogenic therapy would be of clinical value in patients (pts) with metastatic renal cell carcinoma (mRCC). We tested the hypothesis that circulating endothelial cell (CEC), bone marrow-derived CD45dimCD34+VEGFR2+ progenitor cell or plasma angiogenic factor levels are associated with clinical outcome in mRCC pts undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty-five mRCC pts were prospectively monitored at baseline (day 1) and day 14 during treatment (46 pts received sunitinib and 9 pts received sorafenib). Circulating endothelial cells (CD45−CD31+CD146+7-amino-actinomycin (7AAD)− cells) were measured in 1 ml whole blood using four-color flow cytometry (FCM). Circulating CD45dimCD34+VEGFR2+7AAD− progenitor cells were measured in progenitor-enriched fractions by four-color FCM. Plasma VEGF, sVEGFR2, SDF-1α and sVCAM-1 levels were determined by ELISA. Correlations between baseline CEC, CD45dimCD34+VEGFR2+7AAD− progenitor cells, plasma factors, as well as day 1–day 14 changes in CEC, CD45dimCD34+VEGFR2+7AAD− progenitor, plasma factor levels, and response to TKI, progression-free survival (PFS) and overall survival (OS) were examined. Results: No significant correlation between markers and response to TKI was observed. No association between baseline CEC, plasma VEGF, sVEGFR-2, SDF-1α, sVCAM-1 levels with PFS and OS was observed. However, baseline CD45dimCD34+VEGFR2+7AAD− progenitor cell levels were associated with PFS (P=0.01) and OS (P=0.006). Changes in this population and in SDF-1α levels between day 1 and day 14 were associated with PFS (P=0.03, P=0.002). Changes in VEGF and SDF-1α levels were associated with OS (P=0.02, P=0.007). Conclusion: Monitoring CD45dimCD34+VEGFR2+ progenitor cells, plasma VEGF and SDF-1α levels could be of clinical interest in TKI-treated mRCC pts to predict outcome. PMID:21386843

High Levels of Peripheral Blood Circulating Plasma Cells as a Specific Risk Factor for Progression of Smoldering Multiple Myeloma

PubMed Central

Bianchi, Giada; Kyle, Robert A.; Larson, Dirk R.; Witzig, Thomas E.; Kumar, Shaji; Dispenzieri, Angela; Morice, William G.; Rajkumar, S. Vincent

2012-01-01

Smoldering multiple myeloma (SMM) carries a 50% risk of progression to multiple myeloma (MM) or related malignancy within the first 5 years following diagnosis. The goal of this study was to determine if high levels of circulating plasma cells (PCs) are predictive of SMM transformation within the first 2–3 years from diagnosis. Ninety-one patients diagnosed with SMM at Mayo Clinic from January 1994 through January 2007 who had testing for circulating PCs using an immunofluorescent assay and adequate follow up to ascertain disease progression, were studied. High level of circulating PCs was defined as absolute peripheral blood PCs >5000 ×106/L and/or > 5% cytoplasmic immunoglobulin (Ig) positive PCs per 100 peripheral blood mononuclear cells. Patients with high circulating PCs (14 of 91 patients, 15%) were significantly more likely to progress to active disease within 2 years compared with patients without high circulating PCs, 71% versus 25%, respectively, P=0.001. Corresponding rates for progression within 3 years were 86% versus 35%, respectively, P<0.001. Overall survival (OS) after both SMM diagnosis and MM diagnosis was also significantly different. High levels of circulating PCs identify SMM patients with an elevated risk of progression within the first 2 to 3 years following diagnosis. PMID:22902364

Isolation of Circulating Plasma Cells in Multiple Myeloma Using CD138 Antibody-Based Capture in a Microfluidic Device

NASA Astrophysics Data System (ADS)

Qasaimeh, Mohammad A.; Wu, Yichao C.; Bose, Suman; Menachery, Anoop; Talluri, Srikanth; Gonzalez, Gabriel; Fulciniti, Mariateresa; Karp, Jeffrey M.; Prabhala, Rao H.; Karnik, Rohit

2017-04-01

The necessity for bone marrow aspiration and the lack of highly sensitive assays to detect residual disease present challenges for effective management of multiple myeloma (MM), a plasma cell cancer. We show that a microfluidic cell capture based on CD138 antigen, which is highly expressed on plasma cells, permits quantitation of rare circulating plasma cells (CPCs) in blood and subsequent fluorescence-based assays. The microfluidic device is based on a herringbone channel design, and exhibits an estimated cell capture efficiency of ~40-70%, permitting detection of <10 CPCs/mL using 1-mL sample volumes, which is difficult using existing techniques. In bone marrow samples, the microfluidic-based plasma cell counts exhibited excellent correlation with flow cytometry analysis. In peripheral blood samples, the device detected a baseline of 2-5 CD138+ cells/mL in healthy donor blood, with significantly higher numbers in blood samples of MM patients in remission (20-24 CD138+ cells/mL), and yet higher numbers in MM patients exhibiting disease (45-184 CD138+ cells/mL). Analysis of CPCs isolated using the device was consistent with serum immunoglobulin assays that are commonly used in MM diagnostics. These results indicate the potential of CD138-based microfluidic CPC capture as a useful ‘liquid biopsy’ that may complement or partially replace bone marrow aspiration.

Strong IMF By-Related Plasma Convection in the Ionosphere and Cusp Field-Aligned Currents Under Northward IMF Conditions

NASA Technical Reports Server (NTRS)

Le, G.; Lu, G.; Strangeway, R. J.; Pfaff, R. F., Jr.; Vondrak, Richard R. (Technical Monitor)

2001-01-01

We present in this paper an investigation of IMF-By related plasma convection and cusp field-aligned currents using FAST data and AMIE model during a prolonged interval with large positive IMF By and northward Bz conditions (By/Bz much greater than 1). Using the FAST single trajectory observations to validate the global convection patterns at key times and key locations, we have demonstrated that the AMIE procedure provides a reasonably good description of plasma circulations in the ionosphere during this interval. Our results show that the plasma convection in the ionosphere is consistent with the anti-parallel merging model. When the IMF has a strongly positive By component under northward conditions, we find that the global plasma convection forms two cells oriented nearly along the Sun-earth line in the ionosphere. In the northern hemisphere, the dayside cell has clockwise convection mainly circulating within the polar cap on open field lines. A second cell with counterclockwise convection is located in the nightside circulating across the polar cap boundary, The observed two-cell convection pattern appears to be driven by the reconnection along the anti-parallel merging lines poleward of the cusp extending toward the dusk side when IMF By/Bz much greater than 1. The magnetic tension force on the newly reconnected field lines drives the plasma to move from dusk to dawn in the polar cusp region near the polar cap boundary. The field-aligned currents in the cusp region flow downward into the ionosphere. The return field-aligned currents extend into the polar cap in the center of the dayside convection cell. The field-aligned currents are closed through the Peterson currents in the ionosphere, which flow poleward from the polar cap boundary along the electric field direction.

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery.

PubMed

Emanueli, Costanza; Shearn, Andrew I U; Laftah, Abas; Fiorentino, Francesca; Reeves, Barnaby C; Beltrami, Cristina; Mumford, Andrew; Clayton, Aled; Gurney, Mark; Shantikumar, Saran; Angelini, Gianni D

2016-01-01

Exosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current "gold standard" surrogate biomarker of myocardial damage. The concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed (miR-1, miR-24, miR-133a/b, miR-208a/b, miR-210), non-cardiovascular (miR-122) and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs. The plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.

Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery

PubMed Central

Emanueli, Costanza; Fiorentino, Francesca; Reeves, Barnaby C.; Beltrami, Cristina; Mumford, Andrew; Clayton, Aled; Gurney, Mark; Shantikumar, Saran; Angelini, Gianni D.

2016-01-01

Introduction Exosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current “gold standard” surrogate biomarker of myocardial damage. Methods and Results The concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed (miR-1, miR-24, miR-133a/b, miR-208a/b, miR-210), non-cardiovascular (miR-122) and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs. Conclusions The plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients. PMID:27128471

Plasma processing conditions substantially influence circulating microRNA biomarker levels.

PubMed

Cheng, Heather H; Yi, Hye Son; Kim, Yeonju; Kroh, Evan M; Chien, Jason W; Eaton, Keith D; Goodman, Marc T; Tait, Jonathan F; Tewari, Muneesh; Pritchard, Colin C

2013-01-01

Circulating, cell-free microRNAs (miRNAs) are promising candidate biomarkers, but optimal conditions for processing blood specimens for miRNA measurement remain to be established. Our previous work showed that the majority of plasma miRNAs are likely blood cell-derived. In the course of profiling lung cancer cases versus healthy controls, we observed a broad increase in circulating miRNA levels in cases compared to controls and that higher miRNA expression correlated with higher platelet and particle counts. We therefore hypothesized that the quantity of residual platelets and microparticles remaining after plasma processing might impact miRNA measurements. To systematically investigate this, we subjected matched plasma from healthy individuals to stepwise processing with differential centrifugation and 0.22 µm filtration and performed miRNA profiling. We found a major effect on circulating miRNAs, with the majority (72%) of detectable miRNAs substantially affected by processing alone. Specifically, 10% of miRNAs showed 4-30x variation, 46% showed 30-1,000x variation, and 15% showed >1,000x variation in expression solely from processing. This was predominantly due to platelet contamination, which persisted despite using standard laboratory protocols. Importantly, we show that platelet contamination in archived samples could largely be eliminated by additional centrifugation, even in frozen samples stored for six years. To minimize confounding effects in microRNA biomarker studies, additional steps to limit platelet contamination for circulating miRNA biomarker studies are necessary. We provide specific practical recommendations to help minimize confounding variation attributable to plasma processing and platelet contamination.

Monitoring of organ transplants through genomic analyses of circulating cell-free DNA

NASA Astrophysics Data System (ADS)

de Vlaminck, Iwijn

Solid-organ transplantation is the preferred treatment for patients with end-stage organ diseases, but complications due to infection and acute rejection undermine its long-term benefits. While clinicians strive to carefully monitor transplant patients, diagnostic options are currently limited. My colleagues and I in the lab of Stephen Quake have found that a combination of next-generation sequencing with a phenomenon called circulating cell-free DNA enables non-invasive diagnosis of both infection and rejection in transplantation. A substantial amount of small fragments of cell-free DNA circulate in blood that are the debris of dead cells. We discovered that donor specific DNA is released in circulation during injury to the transplant organ and we show that the proportion of donor DNA in plasma is predictive of acute rejection in heart and lung transplantation. We profiled viral and bacterial DNA sequences in plasma of transplant patients and discovered that the relative representation of different viruses and bacteria is informative of immunosuppression. This discovery suggested a novel biological measure of a person's immune strength, a finding that we have more recently confirmed via B-cell repertoire sequencing. Lastly, our studies highlight applications of shotgun sequencing of cell-free DNA in the broad, hypothesis free diagnosis of infection.

High levels of circulating VEGFR2+ Bone marrow-derived progenitor cells correlate with metastatic disease in patients with pediatric solid malignancies.

PubMed

Taylor, Melissa; Rössler, Jochen; Geoerger, Birgit; Laplanche, Agnès; Hartmann, Olivier; Vassal, Gilles; Farace, Françoise

2009-07-15

Pediatric solid malignancies display important angiogenic potential, and blocking tumor angiogenesis represents a new therapeutic approach for these patients. Recent studies have evidenced rare circulating cells with endothelial features contributing to tumor neovascularization and have shown the pivotal role of bone marrow-derived (BMD) progenitor cells in metastatic disease progression. We measured these cells in patients with pediatric solid malignancies as a prerequisite to clinical trials with antiangiogenic therapy. Peripheral blood was drawn from 45 patients with localized (n = 23) or metastatic (n = 22) disease, and 20 healthy subjects. Subsets of circulating vascular endothelial growth factor receptor (VEGFR)2+-BMD progenitor cells, defined as CD45-CD34+VEGFR2(KDR)+7AAD- and CD45(dim)CD34+VEGFR2+7AAD- events, were measured in progenitor-enriched fractions by flow cytometry. Mature circulating endothelial cells (CEC) were measured in whole blood as CD31+CD146+CD45-7AAD- viable events. Data were correlated with VEGF and sVEGFR2 plasma levels. The CD45-CD34+VEGFR2(KDR)+7AAD- subset represented <0.003% of circulating BMD progenitor cells (< or =0.05 cells/mL). However, the median level (range) of the CD45(dim)CD34+VEGFR2+7AAD- subset was higher in patients compared with healthy subjects, 1.5% (0%-10.3%) versus 0.3% (0%-1.6%) of circulating BMD progenitors (P < 0.0001), and differed significantly between patients with localized and metastatic disease, 0.7% (0%-8.6%) versus 2.9% (0.6%-10.3%) of circulating BMD progenitors (P < 0.001). Median CEC value was 7 cells/mL (0-152 cells/mL) and similar in all groups. Unlike VEGFR2+-BMD progenitors, neither CECs, VEGF, or sVEGFR2 plasma levels correlated with disease status. High levels of circulating VEGFR2+-BMD progenitor cells correlated with metastatic disease. Our study provides novel insights for angiogenesis mechanisms in pediatric solid malignancies for which antiangiogenic targeting of VEGFR2+-BMD progenitors could be of interest.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

PubMed Central

Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified. PMID:25769545

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

PubMed

Saavedra, Paula; Girona, Josefa; Bosquet, Alba; Guaita, Sandra; Canela, Núria; Aragonès, Gemma; Heras, Mercedes; Masana, Lluís

2015-11-01

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

Evidence for functional heterogeneity of circulating B-type natriuretic peptide.

PubMed

Liang, Faquan; O'Rear, Jessica; Schellenberger, Ute; Tai, Lungkuo; Lasecki, Michael; Schreiner, George F; Apple, Fred S; Maisel, Alan S; Pollitt, N Stephen; Protter, Andrew A

2007-03-13

These studies describe molecular forms of circulating B-type natriuretic peptide (BNP) as well as their biological activity. Increased circulating levels of immunoreactive BNP correlate with the severity of heart failure and are considered a sensitive biomarker. However, little is known about the molecular forms of circulating BNP and their biological activity. Western blot analysis was used to characterize immunoreactive BNP species in heart failure plasma. Recombinant proBNP was assessed for reactivity in commercially available BNP assays and cell activity by cyclic guanosine monophosphate production in vascular cells. Heart failure plasma contained both low- (LMW-BNP) and high-molecular-weight (HMW-BNP) forms. The LMW-BNP migrated similarly to a 32-amino acid BNP standard, whereas HMW-BNP, when deglycosylated, was similar to deglycosylated recombinant proBNP. Recombinant proBNP and BNP were equally recognized by the Triage BNP assay (Biosite, San Diego, California). Furthermore, recombinant proBNP and BNP were both recognized by the Advia Centaur BNP test (Bayer Diagnostics, Tarrytown, New York), but only recombinant proBNP was recognized by the Elecsys NTproBNP assay (Roche Diagnostics, Indianapolis, Indiana). Recombinant proBNP exerted significantly less biological activity than BNP on human endothelial and vascular smooth muscle cells. Comparison of effective concentration (50%) values indicates that proBNP is 6- to 8-fold less potent than BNP in these human cells. This study demonstrates that proBNP, constituting a substantial portion of immunoreactive BNP in heart failure plasma, possesses significantly lower biological activity than the processed 32-amino acid hormone. These results implicate a discordance in heart failure between the high circulating levels of immunoreactive BNP and hormone activity, suggesting that some patients may be in a state of natriuretic peptide deficiency.

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; Skowronski, Elaine A.; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M.; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

2014-01-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 µL of CLL blood and 5 µL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). PMID:24723219

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma.

PubMed

Sonnenberg, Avery; Marciniak, Jennifer Y; Skowronski, Elaine A; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M; Widhopf, George F; Kipps, Thomas J; Heller, Michael J

2014-07-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of free circulating tumor DNA as a diagnostic marker for breast cancer.

PubMed

Catarino, Raquel; Ferreira, Maria M; Rodrigues, Helena; Coelho, Ana; Nogal, Ana; Sousa, Abreu; Medeiros, Rui

2008-08-01

To determine whether the amounts of circulating DNA could discriminate between breast cancer patients and healthy individuals by using real-time PCR quantification methodology. Our standard protocol for quantification of cell-free plasma DNA involved 175 consecutive patients with breast cancer and 80 healthy controls. We found increased levels of circulating DNA in breast cancer patients compared to control individuals (105.2 vs. 77.06 ng/mL, p < 0.001). We also found statistically significant differences in circulating DNA amounts in patients before and after breast surgery (105.2 vs. 59.0 ng/mL, p = 0.001). Increased plasma cell-free DNA concentration was a strong risk factor for breast cancer, conferring an increased risk for the presence of this disease (OR, 12.32; 95% CI, 2.09-52.28; p < 0.001). Quantification of circulating DNA by real-time PCR may be a good and simple tool for detection of breast cancer with a potential to clinical applicability together with other current methods used for monitoring the disease.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects

PubMed Central

Kriebardis, Anastasios G.; Antonelou, Marianna H.; Georgatzakou, Hara T.; Tzounakas, Vassilis L.; Stamoulis, Konstantinos E.; Papassideri, Issidora S.

2016-01-01

Background Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Materials and methods Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors’ haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Results Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. Discussion The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles. PMID:27136430

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

PubMed

Kriebardis, Anastasios G; Antonelou, Marianna H; Georgatzakou, Hara T; Tzounakas, Vassilis L; Stamoulis, Konstantinos E; Papassideri, Issidora S

2016-05-01

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

What Is Blood?

MedlinePlus

... Blood / What is Blood? WHERE CAN I DONATE? Blood is the red fluid that circulates in our blood vessels, i. ... cells, white blood cells, and platelets. The remainder is a fluid called plasma. Blood cells are produced in bone marrow. Red cells, white cells and platelets are made in ...

Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR.

PubMed

Ye, Wei; Tang, Xiaojun; Liu, Chu; Wen, Chaowei; Li, Wei; Lyu, Jianxin

2017-04-01

To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ 0 ) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA. In the results, the ddPCR method showed high sensitivity and specificity. When the DNA was extracted from plasma prior to ddPCR, the ccf-mtDNA copy number was higher than that measured without extraction. This difference was not due to a PCR inhibitor, such as EDTA-Na 2 , an anti-coagulant in plasma, because standard EDTA-Na 2 concentration (5 mM) did not significantly inhibit ddPCR reactions. The difference might be attributable to plasma exosomal mtDNA, which was 4.21 ± 0.38 copies/μL of plasma, accounting for ∼19% of plasma ccf-mtDNA. Therefore, ddPCR can quickly and reliably detect ccf-mtDNA from plasma with a prior DNA extraction step, providing for a more accurate detection of ccf-mtDNA. The direct use of plasma as a template in ddPCR is suitable for the detection of exogenous cell-free nucleic acids within plasma, but not of nucleic acids that have a vesicle-associated form, such as exosomal mtDNA. Graphical Abstract Designs of the present work. *: Module 1, #: Module 2, &: Module 3.

PROTEIN METABOLISM AND EXCHANGE AS INFLUENCED BY CONSTRICTION OF THE VENA CAVA

PubMed Central

McKee, Frank W.; Hyatt, Robert E.; Wilt, William G.; Tishkoff, Garson H.; Whipple, George H.

1949-01-01

Further studies of ascitic fluid production and related factors in dogs with constriction of the vena cava above the diaphragm are reported. Whole dog plasma given intravenously to such animals produces a rise in circulating plasma protein to normal levels, but increases the output of ascitic fluid with a loss of protein via the ascites equivalent to 72, 76, and 65 per cent respectively, of the injected protein. Forced ingestion of water in excess of the test animal's normal needs and desires produces no significant changes in the circulating plasma protein level or in ascitic fluid production. Amino acid growth mixtures given intravenously in distilled water cause weight loss, elevation of circulating plasma proteins, a slightly negative nitrogen balance, but no ascitic fluid production. Amino acid growth mixtures given intravenously in normal saline cause depression of the circulating plasma proteins, negative nitrogen balance, and significant ascitic fluid production. Ascitic fluid given intravenously to the test animals causes a marked depression of circulating plasma proteins, a marked increase in ascitic fluid production containing the equivalent of 116 and 98 per cent of the injected protein, and a negative nitrogen balance. Ascitic fluid given orally produces a marked depression of circulating plasma proteins, and a marked increase in ascitic fluid secretion, containing the equivalent of 66, 66, and 54 per cent respectively, of the ingested protein. Sodium chloride is a dominant factor in some of these experiments where abundant ascites production is recorded. Protein levels and intake are important, but take second place to sodium. Ascitic fluids show electrophoretic patterns which are almost identical to the plasma patterns. The A/G ratios are often equal in ascitic fluid and plasma, sometimes even lower in the ascitic fluid. This emphasizes the ease with which globulins pass cell or other membrane barriers in these experiments. PMID:18143588

In–Depth Characterization of Viral Isolates from Plasma and Cells Compared with Plasma Circulating Quasispecies in Early HIV-1 Infection

PubMed Central

Erkizia, Itziar; Pino, Maria; Pou, Christian; Paredes, Roger; Clotet, Bonaventura; Martinez-Picado, Javier; Prado, Julia G.

2012-01-01

Background The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. Methodology/Principal Findings We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. Conclusion This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies. PMID:22393441

Increased numbers of circulating ICOS⁺ follicular helper T and CD38⁺ plasma cells in patients with newly diagnosed primary biliary cirrhosis.

PubMed

Wang, Li; Sun, Xiguang; Qiu, Jinpeng; Cai, Yanjun; Ma, Liang; Zhao, Pingwei; Jiang, Yanfang

2015-02-01

Aberrant activation of follicular helper T (TFH) and B cells is associated with the development of autoimmune diseases. However, little is known about the potential role of these cells in the development of primary biliary cirrhosis (PBC). This study aimed at characterizing the numbers of different subsets of circulating Tfh and B cells as well as evaluating their potential association with the levels of immunoglobulins and autoantibodies in newly diagnosed PBC patients. The numbers of circulating CD27(+), CD38(+), CD86(+) and CD95(+) B cells as well as inducible T cell costimulator (ICOS)(+) and programmed death-1 (PD-1)(+), IL-21(+) TFH cells were examined in 58 patients with newly diagnosed PBC and 30 matched healthy controls (HCs). The numbers of circulating CD38(+)CD19(+), CD86(+)CD19(+), and CD95(+)CD19(+) B cells; CD3(+)CD4(+)CXCR5(+)ICOS(+) and CD3(+)CD4(+)CXCR5(+)PD-1(+) Tfh cells; and the levels of serum IL-21 in the PBC patients were significantly greater, but the numbers of CD27(+)CD19(+) B cells were significantly less than those in the HCs (p < 0.05). The numbers of CD3(+)CD4(+)CXCR5(+)ICOS(+) Tfh cells were positively correlated with the numbers of CD38(+)CD19(+) and CD86(+)CD38(+)CD19(+) B cells and the levels of serum anti-mitochondrial antibodies against M2 antigen (AMA-M2), AMA and immunolgubin M (IgM) in the PBC patients. The levels of serum IL-21 were positively correlated with the levels of serum AMA-M2, AMA, IgG and IgM, but negatively with the numbers of CD27(+)CD19(+) B cells in the PBC patients. Increased numbers of circulating ICOS(+) and IL-21(+) Tfh and CD38(+) plasma cells may be exhibited by patients with recent diagnoses of PBC.

Red blood cell and iron metabolism during space flight

NASA Technical Reports Server (NTRS)

Smith, Scott M.

2002-01-01

Space flight anemia is a widely recognized phenomenon in astronauts. Reduction in circulating red blood cells and plasma volume results in a 10% to 15% decrement in circulatory volume. This effect appears to be a normal physiologic adaptation to weightlessness and results from the removal of newly released blood cells from the circulation. Iron availability increases, and (in the few subjects studied) iron stores increase during long-duration space flight. The consequences of these changes are not fully understood.

Incorporation of a circulating protein into megakaryocyte and platelet granules

NASA Technical Reports Server (NTRS)

Handagama, P. J.; George, J. N.; Shuman, M. A.; McEver, R. P.; Bainton, D. F.

1987-01-01

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

Plasma exosome microRNAs are indicative of breast cancer.

PubMed

Hannafon, Bethany N; Trigoso, Yvonne D; Calloway, Cameron L; Zhao, Y Daniel; Lum, David H; Welm, Alana L; Zhao, Zhizhuang J; Blick, Kenneth E; Dooley, William C; Ding, W Q

2016-09-08

microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.

PubMed

Liao, Gary J W; Gronowski, Ann M; Zhao, Zhen

2014-01-20

The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made non-invasive prenatal testing (NIPT) possible. Maternal plasma cell free DNA is a mixture of maternal and fetal DNA, of which, fetal DNA represents a minor population in maternal plasma. Therefore, methods with high sensitivity and precision are required to detect and differentiate fetal DNA from the large background of maternal DNA. In recent years, technical advances in the molecular analysis of fetal DNA (e.g., digital PCR and massively parallel sequencing (MPS)) has enabled the successful implementation of noninvasive testing into clinical practice, such as fetal sex assessment, RhD genotyping, and fetal chromosomal aneuploidy detection.With the ability to decipher the entire fetal genome from maternal plasma DNA, we foresee that an increased number of non-invasive prenatal tests will be available for detecting many single-gene disorders in the near future. This review briefly summarizes the technical aspects of the NIPT and application of NIPT in clinical practice.

Endothelial cell expression of adhesion molecules is induced by fetal plasma from pregnancies with umbilical placental vascular disease.

PubMed

Wang, Xin; Athayde, Neil; Trudinger, Brian

2002-07-01

To test the hypothesis that local production with spill into the fetal circulation of factor(s) injurious to endothelium is responsible for the vascular pathology present when the umbilical artery Doppler study is abnormal. Expression of adhesion molecules is a feature of endothelial cell activation. Case-control study. University teaching hospital. Fetal plasma was collected from 27 normal pregnancies, 39 pregnancies with umbilical placental vascular disease defined by abnormal umbilical artery Doppler and 11 pregnancies with pre-eclampsia and normal umbilical artery Doppler. Isolated and cultured human umbilical vein endothelial cells from normal pregnancies were incubated with fetal plasma from three study groups. mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were assessed by reverse transcription-polymerase chain reaction. To confirm the occurrence of this in vivo, we measured the levels of soluble fractions of sICAM-1, sVCAM-1 and sPECAM-1 in the fetal circulation in the fetal plasma used for endothelial cell incubation. The mRNA expression of ICAM-1 [median 1.1 (interquartile range 0.5-1.9) vs 0.7 (0.3-1.2), P < 0.05] and PECAM-1 [2.1 (1.2-3.0) vs 1.5 (0.7-2.1), P < 0.05] was significantly higher following incubation with fetal plasma from umbilical placental vascular disease compared with the normal group. There was no difference in the expression of VCAM-1 [1.2 (0.9-1.8) vs 1.1 (0.8-1.6), ns]. The group with maternal pre-eclampsia and normal umbilical artery Doppler did not differ from the normal group. In the umbilical placental vascular disease group, the results were similar in the presence or absence of pre-eclampsia. For soluble fractions of the adhesion molecules released into the fetal circulation, we found the levels (ng/mL) of sICAM- I [median 248.5 (interquartile range 197.3-315.7) vs 174.2 (144.5-212.9), P < 0.05] and sPECAM-1 [9.3 (6.2-11.1) vs 6.1 (5.4-7.7), P < 0.05] in fetal plasma to be significantly increased in the presence of umbilical placental vascular disease compared with the normal. Vascular disease in the fetal umbilical placental circulation is associated with an elevation in mRNA expression by endothelial cells of ICAM-1 and PECAM-1. Our study provides evidence for endothelial cell activation and dysfunction in umbilical placental vascular disease. We speculate that the plasma factor(s) affecting the vessels of the umbilical villous tree is locally released by the trophoblast. The occurrence of the maternal syndrome of pre-eclampsia appears to be independent of this.

Increased Levels of Cell-Free miR-517a and Decreased Levels of Cell-Free miR-518b in Maternal Plasma Samples From Placenta Previa Pregnancies at 32 Weeks of Gestation.

PubMed

Hasegawa, Yuri; Miura, Kiyonori; Higashijima, Ai; Abe, Shuhei; Miura, Shoko; Yoshiura, Koh-ichiro; Masuzaki, Hideaki

2015-12-01

The aim of this study was to clarify the association between placenta previa and circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miRNAs) in maternal plasma. Twenty singleton pregnancies with placenta previa (placenta previa group) and 26 uncomplicated pregnancies (control group) were recruited. Blood sampling was performed at 32 weeks of gestation, and cesarean delivery in all cases of placenta previa was performed at a mean gestational age of 37 weeks. The maternal plasma concentrations of cell-free pregnancy-associated placenta-specific miRNAs (miR-517a and miR-518b) were measured by absolute quantitative real-time reverse transcription-polymerase chain reaction. Plasma concentrations of cell-free miR-517a were significantly higher in the placenta previa group than that in the control group (P = .011), while the plasma concentration of cell-free miR-518b was significantly lower in the placenta previa group than that in the control group (P = .004). Plasma concentrations of cell-free miR-517a in placenta previa were significantly higher in placenta previa with alert bleeding later group than those in placenta previa without alert bleeding group or control group (P = .030 or .047, respectively) and correlated with the volume of hemorrhage at delivery (R and P value: .512 and .025). Plasma concentrations of cell-free miR-517a and miR-518b at 32 weeks of gestation were altered in pregnant women with placenta previa, and the circulating level of cell-free miR-517a in placenta previa may be a predictive marker for the risks of alert bleeding later and massive hemorrhage at delivery. © The Author(s) 2015.

Circulating miR-1, miR-133a, and miR-206 levels are increased after a half-marathon run.

PubMed

Gomes, Clarissa P C; Oliveira, Getúlio P; Madrid, Bibiano; Almeida, Jeeser A; Franco, Octávio L; Pereira, Rinaldo W

2014-11-01

Circulating miRNAs are potential biomarkers that can be important molecules driving cell-to-cell communication. To investigate circulating muscle-specific miRNAs in recreational athletes. Three miRNAs from whole plasma before and after a half-marathon were analyzed by qPCR. MiR-1, -133a, and -206 significantly increased after the race. Increased levels of miRNAs after exercise point to potential biomarkers and to the possibility of being functional players following endurance training. These miRNAs are potential biomarkers of muscle damage or adaptation to exercise.

Body composition changes in men and women after 2-3 weeks of bed rest

NASA Technical Reports Server (NTRS)

Pace, N.; Kodama, A. M.; Grunbaum, B. W.; Rahlmann, D. F.; Price, D. C.; Newsom, B. D.

1976-01-01

Several parameters of body composition were measured in eight men before and after 14 days of continuous recumbency, and in eight women before and after 17 days of recumbency. The parameters measured included body weight, body water, body potassium, plasma volume, and plasma protein concentrations. From these, values were derived for body fat content, lean body mass, body mass, and circulating plasma proteins. In general, the men and women responded similarly to continuous recumbency. Characteristically, there was significant reduction of plasma volume and body potassium in both groups. The women showed a significant reduction in circulating plasma protein, entirely in the albumin fraction; a similar change was observed in the men. The women, but not the men, showed a significant increase in circulating fibrinogen. Both men and women lost body cell mass, while body fat content remained the same or tended to increase slightly. It is expected that similar changes would occur in weightlessness. It is further concluded that women should tolerate the weightlessness of space flight physiologically as well as men.

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

PubMed Central

Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

2015-01-01

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.

PubMed

Shih, Chun-Liang; Chong, Kowit-Yu; Hsu, Shih-Che; Chien, Hsin-Jung; Ma, Ching-Ting; Chang, John Wen-Cheng; Yu, Chia-Jung; Chiou, Chiuan-Chian

2016-01-25

Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules. Magnetic beads coated with annexin A5 (ANX-beads), which bound to phosphatidylserine moieties on the surfaces of most EVs, were tested for their ability to capture induced apoptotic bodies in vitro and other phosphatidylserine-presenting vesicles in body fluids. Our results show that up to 60% of induced apoptotic bodies could be captured by the ANX-beads. The vesicles captured from cultured media or plasma contained amplifiable RNA. Suitable blood samples for EV collection included EDTA-plasma and serum but not heparin-plasma. In addition, EVs in plasma were labile to freeze-and-thaw cycles. In rodents xenografted with human cancer cells, tumor-derived mRNA could be detected in EVs captured from serum samples. Active proteins could be detected in EVs captured from ascites but not from plasma. In conclusion, we have developed a magnetic bead-based procedure for the collection of EVs from body fluids and proved that captured EVs contain biomolecules from their parent cells, and therefore have great potential for disease diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Plasma Cell Depletion Attenuates Hypertension in an Experimental Model of Autoimmune Disease.

PubMed

Taylor, Erin B; Barati, Michelle T; Powell, David W; Turbeville, Hannah R; Ryan, Michael J

2018-04-01

Numerous studies show a direct relation between circulating autoantibodies, characteristic of systemic autoimmune disorders, and primary hypertension in humans. Whether these autoantibodies mechanistically contribute to the development of hypertension remains unclear. Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by aberrant immunoglobulin production, notably pathogenic autoantibodies, and is associated with prevalent hypertension, renal injury, and cardiovascular disease. Because plasma cells produce the majority of serum immunoglobulins and are the primary source of autoantibodies in SLE, we hypothesized that plasma cell depletion using the proteasome inhibitor bortezomib would lower autoantibody production and attenuate hypertension. Thirty-week-old female SLE (NZBWF1) and control (NZW [New Zealand White]) mice were injected IV with vehicle (0.9% saline) or bortezomib (0.75 mg/kg) twice weekly for 4 weeks. Bortezomib treatment significantly lowered the percentage of bone marrow plasma cells in SLE mice. Total plasma IgG and anti-dsDNA IgG levels were higher in SLE mice compared with control mice but were lowered by bortezomib treatment. Mean arterial pressure (mm Hg) measured in conscious mice by carotid artery catheter was higher in SLE mice than in control mice, but mean arterial pressure was significantly lower in bortezomib-treated SLE mice. Bortezomib also attenuated renal injury, as assessed by albuminuria and glomerulosclerosis, and reduced glomerular immunoglobulin deposition and B and T lymphocytes infiltration into the kidneys. Taken together, these data show that the production of autoantibodies by plasma cells mechanistically contributes to autoimmune-associated hypertension and suggests a potential role for patients with primary hypertension who have increased circulating immunoglobulins. © 2018 American Heart Association, Inc.

The metabolites in peripheral blood mononuclear cells showed greater differences between patients with impaired fasting glucose or type 2 diabetes and healthy controls than those in plasma.

PubMed

Kim, Minjoo; Kim, Minkyung; Han, Ji Yun; Lee, Sang-Hyun; Jee, Sun Ha; Lee, Jong Ho

2017-03-01

To determine differences between peripheral blood mononuclear cells and the plasma metabolites in patients with impaired fasting glucose or type 2 diabetes and healthy controls. In all, 65 nononobese patients (aged 30-70 years) with impaired fasting glucose or type 2 diabetes and 65 nonobese sex-matched healthy controls were included, and fasting peripheral blood mononuclear cell and plasma metabolomes were profiled. The diabetic or impaired fasting glucose patients showed higher circulating and peripheral blood mononuclear cell lipoprotein phospholipase A 2 activities, high-sensitivity C-reactive protein and tumour necrosis factor-α than controls. Compared with controls, impaired fasting glucose or diabetic subjects showed increases in 11 peripheral blood mononuclear cell metabolites: six amino acids (valine, leucine, methionine, phenylalanine, tyrosine and tryptophan), l-pyroglutamic acid, two fatty acid amides containing palmitic amide and oleamide and two lysophosphatidylcholines. In impaired fasting glucose or diabetic patients, peripheral blood mononuclear cell lipoprotein phospholipase A 2 positively associated with peripheral blood mononuclear cell lysophosphatidylcholines and circulating inflammatory markers, including tumour necrosis factor-α, high-sensitivity C-reactive protein and lipoprotein phospholipase A 2 activities. In plasma metabolites between patients and healthy controls, we observed significant increases in only three amino acids (proline, valine and leucine) and decreases in only five lysophosphatidylcholines. This study demonstrates significant differences in the peripheral blood mononuclear cell metabolome in patients with impaired fasting glucose or diabetes compared with healthy controls. These differences were greater than those observed in the plasma metabolome. These data suggest peripheral blood mononuclear cells as a useful tool to better understand the inflammatory pathophysiology of diabetes.

Associations of circulating plasma microRNAs with age, body mass index and sex in a population-based study.

PubMed

Ameling, Sabine; Kacprowski, Tim; Chilukoti, Ravi Kumar; Malsch, Carolin; Liebscher, Volkmar; Suhre, Karsten; Pietzner, Maik; Friedrich, Nele; Homuth, Georg; Hammer, Elke; Völker, Uwe

2015-10-14

Non-cellular blood circulating microRNAs (plasma miRNAs) represent a promising source for the development of prognostic and diagnostic tools owing to their minimally invasive sampling, high stability, and simple quantification by standard techniques such as RT-qPCR. So far, the majority of association studies involving plasma miRNAs were disease-specific case-control analyses. In contrast, in the present study, plasma miRNAs were analysed in a sample of 372 individuals from a population-based cohort study, the Study of Health in Pomerania (SHIP). Quantification of miRNA levels was performed by RT-qPCR using the Exiqon Serum/Plasma Focus microRNA PCR Panel V3.M covering 179 different miRNAs. Of these, 155 were included in our analyses after quality-control. Associations between plasma miRNAs and the phenotypes age, body mass index (BMI), and sex were assessed via a two-step linear regression approach per miRNA. The first step regressed out the technical parameters and the second step determined the remaining associations between the respective plasma miRNA and the phenotypes of interest. After regressing out technical parameters and adjusting for the respective other two phenotypes, 7, 15, and 35 plasma miRNAs were significantly (q < 0.05) associated with age, BMI, and sex, respectively. Additional adjustment for the blood cell parameters identified 12 and 19 miRNAs to be significantly associated with age and BMI, respectively. Most of the BMI-associated miRNAs likely originate from liver. Sex-associated differences in miRNA levels were largely determined by differences in blood cell parameters. Thus, only 7 as compared to originally 35 sex-associated miRNAs displayed sex-specific differences after adjustment for blood cell parameters. These findings emphasize that circulating miRNAs are strongly impacted by age, BMI, and sex. Hence, these parameters should be considered as covariates in association studies based on plasma miRNA levels. The established experimental and computational workflow can now be used in future screening studies to determine associations of plasma miRNAs with defined disease phenotypes.

PATHOGENESIS OF FEVER: EFFECTS OF VARIOUS ENDOGENOUS PYROGENS UPON THE LEVEL OF CIRCULATING GRANULOCYTES IN NORMAL RABBITS

PubMed Central

King, M. K.

1960-01-01

The endogenous pyrogen in the serum or plasma of rabbits 2 hours after the intravenous injection of typhoid vaccine had a marked effect on the circulating leucocytes of normal rabbits. Immediately following intravenous injection there was a brief, but marked, granulocytopenia which was quickly followed by a granulocytosis. Under the same circumstances pooled heterologous serum or plasma from normal rabbits produced no fever or significant change in the level of circulating leucocytes. The cell-free fluid of sterile peritoneal exudates produced a marked leucocytosis without a preceding leucopenia when injected intravenously into normal rabbits. When comparably pyrogenic doses of typhoid vaccine were injected in the same manner no significant change in the level of circulating leucocytes occurred. The relevance of these findings to the pathogenesis of fever is discussed. PMID:13756095

Pathogenesis of fever: effects of various endogenous pyrogens upon the level of circulating granulocytes in normal rabbits.

PubMed

KING, M K

1960-11-01

The endogenous pyrogen in the serum or plasma of rabbits 2 hours after the intravenous injection of typhoid vaccine had a marked effect on the circulating leucocytes of normal rabbits. Immediately following intravenous injection there was a brief, but marked, granulocytopenia which was quickly followed by a granulocytosis. Under the same circumstances pooled heterologous serum or plasma from normal rabbits produced no fever or significant change in the level of circulating leucocytes. The cell-free fluid of sterile peritoneal exudates produced a marked leucocytosis without a preceding leucopenia when injected intravenously into normal rabbits. When comparably pyrogenic doses of typhoid vaccine were injected in the same manner no significant change in the level of circulating leucocytes occurred. The relevance of these findings to the pathogenesis of fever is discussed.

Liquid biopsy: a step forward towards precision medicine in urologic malignancies.

PubMed

Di Meo, Ashley; Bartlett, Jenni; Cheng, Yufeng; Pasic, Maria D; Yousef, George M

2017-04-14

There is a growing trend towards exploring the use of a minimally invasive "liquid biopsy" to identify biomarkers in a number of cancers, including urologic malignancies. Multiple aspects can be assessed in circulating cell-free DNA, including cell-free DNA levels, integrity, methylation and mutations. Other prospective liquid biopsy markers include circulating tumor cells, circulating RNAs (miRNA, lncRNAs and mRNAs), cell-free proteins, peptides and exosomes have also emerged as non-invasive cancer biomarkers. These circulating molecules can be detected in various biological fluids, including blood, urine, saliva and seminal plasma. Liquid biopsies hold great promise for personalized medicine due to their ability to provide multiple non-invasive global snapshots of the primary and metastatic tumors. Molecular profiling of circulating molecules has been a stepping-stone to the successful introduction of several non-invasive multi-marker tests into the clinic. In this review, we provide an overview of the current state of cell-free DNA-based kidney, prostate and bladder cancer biomarker research and discuss the potential utility other circulating molecules. We will also discuss the challenges and limitations facing non-invasive cancer biomarker discovery and the benefits of this growing area of translational research.

THE LOCALIZATION OF HOMOLGOUS PLASMA PROTEINS IN THE TISSUES OF YOUNG HUMAN BEINGS AS DEMONSTRATED WITH FLUORESCENT ANTIBODIES

PubMed Central

Gitlin, David; Landing, Benjamin H.; Whipple, Ann

1953-01-01

Employing fluorescent antibodies for the detection of homologous plasma proteins in tissue sections, the distribution of plasma albumin, γ-globulin, β-lipoprotein, β1-metal-combining globulin, and fibrinogen has been studied in the tissues of infants and children. Plasma albumin, γ-globulin, and β1-metal-combining globulin were found in many cells and particularly cell nuclei, connective tissues and interstitial spaces, lymphatics, and blood vessels. β-Lipoprotein was found mostly in the nuclei of all cell types while fibrinogen was restricted largely to the lymphatic and vascular channels, connective tissues and the interstitial spaces. The widespread distribution of these plasma proteins in cells and connective tissues indicates the magnitude of the extravascular plasma protein pool which is in equilibrium with circulating plasma. Unfortunately, these results do not permit accurate localization of the sites of production of these plasma proteins, but do give some idea of their intimate relationship to the tissues. PMID:13022871

[The Relevance of Hemolysis in Anesthesia and Intensive Care Medicine].

PubMed

Graw, Jan A; Baron, David M; Francis, Roland C E

2018-04-01

Hemolysis leads to an increase of circulating intravascular cell-free hemoglobin. Increased plasma concentrations of cell-free hemoglobin are relevant in critically ill patients because cell-free hemoglobin causes vasoconstriction by depletion of endothelial nitric oxide, oxidative stress, and inflammation. Furthermore, cell-free hemoglobin contributes to tissue injuries such as renal failure and intestinal mucosa damage after cardiac surgery. High concentrations of cell-free hemoglobin are associated with an increased mortality in patients with sepsis. Currently, it is unclear if hemolysis associated with transfusion of packed red blood cells that have been stored for prolonged periods of time is relevant for the clinical outcome. However, humans possess plasma proteins haptoglobin and hemopexin which bind to plasma hemoglobin and cell-free heme, respectively. The haptoglobin-hemoglobin and hemopexin-heme complexes are then eliminated from the plasma by hepatic or splenic uptake. Georg Thieme Verlag KG Stuttgart · New York.

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

PubMed Central

Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

2016-01-01

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer.

PubMed

Hrebien, Sarah; O'Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Garcia-Murillas, Isaac; Turner, Nicholas

2016-01-01

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48-72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77-0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.

Extracellular vesicle RNAs reflect placenta dysfunction and are a biomarker source for preterm labour.

PubMed

Fallen, Shannon; Baxter, David; Wu, Xiaogang; Kim, Taek-Kyun; Shynlova, Oksana; Lee, Min Young; Scherler, Kelsey; Lye, Stephen; Hood, Leroy; Wang, Kai

2018-05-01

Preterm birth (PTB) can lead to lifelong complications and challenges. Identifying and monitoring molecular signals in easily accessible biological samples that can diagnose or predict the risk of preterm labour (PTL) in pregnant women will reduce or prevent PTBs. A number of studies identified putative biomarkers for PTL including protein, miRNA and hormones from various body fluids. However, biomarkers identified from these studies usually lack consistency and reproducibility. Extracellular vesicles (EVs) in circulation have gained significant interest in recent years as these vesicles may be involved in cell-cell communication. We have used an improved small RNA library construction protocol and a newly developed size exclusion chromatography (SEC)-based EV purification method to gain a comprehensive view of circulating RNA in plasma and its distribution by analysing RNAs in whole plasma and EV-associated and EV-depleted plasma. We identified a number of miRNAs in EVs that can be used as biomarkers for PTL, and these miRNAs may reflect the pathological changes of the placenta during the development of PTL. To our knowledge, this is the first study to report a comprehensive picture of circulating RNA, including RNA in whole plasma, EV and EV-depleted plasma, in PTL and reveal the usefulness of EV-associated RNAs in disease diagnosis. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Isolation and characterization of circulating micro(nano)vesicles in the plasma of colorectal cancer patients and their interactions with tumor cells.

PubMed

Stec, Małgorzata; Baj-Krzyworzeka, Monika; Baran, Jarosław; Węglarczyk, Kazimierz; Zembala, Maria; Barbasz, Jakub; Szczepanik, Antoni; Zembala, Marek

2015-11-01

Micro(nano)vesicles (MV) are regarded as important messengers in cell-to-cell communication. There is also evidence for their pivotal role in cancer progression. Circulating MV are of different body cells origin, including tumor cell‑derived MV (TMV) in cancer patients. Determination of circulating TMV is of importance because of their potential diagnostic and therapeutic applications. In the present study, an analysis of circulating MV in colorectal cancer (CRC) patients was undertaken. Plasma from healthy donors was used as the control. In order to define MV characteristics, two plasma fractions: obtained by sequential centrifugation at 15,000 x g (MV15) and 50,000 x g (MV50) were used for analysis. The two fractions possessed a large range of sizes: 70(80)-1,300(1,400) nm and the most common particles with sizes 70-90 nm, both in patients and controls. Atomic force microscopy images of MV50 revealed a heterogeneous population of particles with different shapes and sizes. MV15 contained an increased level of CD41+ and CD61+ particles, suggesting their platelet origin. No difference between patients and controls was observed. A more precise analysis of MV50 showed the increased level of particles expressing EGFR (HER-1/Erb B1), HER-2/neu and Mucin1 (MUC1), suggesting their tumor origin. The total level of MV50‑expressing EGFR, HER-2/neu and MUC1 was enhanced in CRC patients. MV50 both of patients and controls attached to a colon cancer cell line (SW480) and to isolated blood monocytes at 2 h and were engulfed at 24 h. This uptake showed the lack of specificity. Thus, apart from the direct delivery of MV to the tumor site by plasma, monocytes carrying MV may also be involved in their transportation. Taken together, the presented data indicate that MV15 contain mainly platelet‑derived particles, while MV50 from CRC patients are enriched in TMV. Interaction of MV with cancer cells may pin-point their role in communication between tumor cells, resulting in molecular cargo exchange between them.

SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer.

PubMed

Balgkouranidou, Ioanna; Chimonidou, Maria; Milaki, Georgia; Tsaroucha, Emily; Kakolyris, Stylianos; Georgoulias, Vasilis; Lianidou, Evi

2016-08-01

SOX17 belongs to the high-mobility group-box transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p=0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.

Peripheral leukocyte subpopulations and catecholamine levels in astronauts as a function of mission duration

NASA Technical Reports Server (NTRS)

Mills, P. J.; Meck, J. V.; Waters, W. W.; D'Aunno, D.; Ziegler, M. G.

2001-01-01

OBJECTIVE: The objective of this study was to determine the effects of spaceflight duration on immune cells and their relationship to catecholamine levels. METHODS: Eleven astronauts who flew aboard five different US Space Shuttle flights ranging in duration from 4 to 16 days were studied before launch and after landing. RESULTS: Consistent with prior studies, spaceflight was associated with a significant increase in the number of circulating white blood cells (p <.01), including neutrophils (p <.01), monocytes (p <.05), CD3+CD4+ T-helper cells (p <.05), and CD19+ B cells (p <.01). In contrast, the number of CD3-CD16+56+ natural killer cells was decreased (p <.01). Plasma norepinephrine levels were increased at landing (p <.01) and were significantly correlated with the number of white blood cells (p <.01), neutrophils (p <.01), monocytes (p <.01), and B cells (p <.01). Astronauts who were in space for approximately 1 week showed a significantly larger increase on landing in plasma norepinephrine (p =.02) and epinephrine (p =.03) levels, as well as number of circulating CD3+CD4+ T-helper cells (p <.05) and CD3+CD8+ T-cytotoxic cells (p <.05) as compared with astronauts in space for approximately 2 weeks. CONCLUSIONS: The data suggest that the stress of spaceflight and landing may lead to a sympathetic nervous system-mediated redistribution of circulating leukocytes, an effect potentially attenuated after longer missions.

Circulating factors induce coronary endothelial cell activation following exposure to inhaled diesel exhaust and nitrogen dioxide in humans: Evidence from a novel translational in vitro model**

EPA Science Inventory

The vascular toxicity of inhaled agents may be caused by soluble factors that are released into the systemic circulation. To confirm this in a straightforward manner, we obtained plasma from healthy human volunteers before and after exposure to diesel exhaust (DE) and nitrogen di...

Circulating S100A8 and S100A9 protein levels in plasma of patients with acquired aplastic anemia and myelodysplastic syndromes.

PubMed

Giudice, Valentina; Wu, Zhijie; Kajigaya, Sachiko; Fernandez Ibanez, Maria Del Pilar; Rios, Olga; Cheung, Foo; Ito, Sawa; Young, Neal S

2018-06-26

The alarmin family members S100A8 and S100A9 are acute phase inflammation proteins, but they also have been proposed as biomarkers in many malignant and non-malignant diseases. In this study, circulating S100A8 and S100A9 homodimers and S100A8/A9 heterodimers in plasma were systematically investigated by ELISA in aplastic anemia (AA) and myelodysplastic syndromes (MDS). Plasma was obtained from 58 severe AA (SAA) and 30 MDS patients, and from 47 age- and sex-matched healthy donors. In 40 out of the 58 AA subjects, S100A protein levels were measured before and 6 months after immunosuppressive therapy (IST). No differences were observed in AA patients at diagnosis compared to healthy controls for circulating S100A homodimers and heterodimers. After therapy, SAA-responders showed significantly increased circulating S100A8. Non-responding patients had significantly higher levels of circulating S100A8/A9 compared to responders and healthy controls, but without variations of S100A8 and S100A9 homodimers. In MDS patients, circulating S100A8 was significantly elevated compared to those of AA and/or healthy controls. By Pearson correlation analysis of protein levels and blood counts, multiple correlations were found. However, as S100A8 and S100A9 are abundantly present in white blood cells and platelets, correlations with blood counts likely mirror the higher number of cells in the blood of some patients. In conclusion, our findings indicate that circulating S100A8 is increased in MDS but not in AA, and that may be useful to distinguish these diseases in the differential diagnosis of bone marrow failure syndromes. Clinicaltrials.gov identifiers: NCT00260689, NCT00604201, NCT01328587, NCT01623167, NCT00001620, NCT00001397. Published by Elsevier Ltd.

Kinetics of plasma heat shock protein HSP-70 release in coronary artery surgery: on-pump versus off-pump.

PubMed

Pizon, M T; Gburek, T; Pizon, M; Sztefko, K

2006-12-01

Heat shock protein HSP-70 is known as protective chaperone molecule synthetized in response following ischemia and stress agents. It is detected in the myocardium and endothelium as well as in the circulation. Damaged as well as viable but exposed to stress cells contribute to the release of HSP-70 into the circulation. The aim of the study was to investigate if cardiopulmonary bypss (CPB) leads to more circulating HSP-70, on the basis of comparison dynamics of plasma concentration HSP-70 in 8 men undergoing procedures with the use of CPB (coronary artery bypass grafting, CABG group) and 8 men undergoing off-pump surgery (OPCAB group). Blood samples were taken preoperatively, twice intraoperatively, immediately after surgical procedure (1 h) and 24-hours thereafter. The concentration of plasma HSP-70 was measured by means of immunoassay. The derived results were compared statistically with the frequency of incidence postoperative atrial fibrillation (AF). In CABG group was observed continuous gradual increase of plasma HSP-70 concentration during the operation with the peak 1 h after surgery (P<0.01), in striking contrast to OPCAB group, in which was detected small, but non statistically significant increase of HSP-70 1 h after operation. Significantly more of circulating HSP-70 it was detected in CABG group during the operation and 1 h after surgery (CABG vs OPCAB, respectively P<0.015 and P<0.028). In both groups among patients witch AF it was found higher postoperative values of circulating HSP-70 compared with the non-AF group (P=0.0415). The use of CPB leads to significant more release of HSP-70 into the circulation. According to our findings high plasma concentration of HSP-70 may be the measure of operative cellular stress, ischemia or injury and may be related with greater onset of postoperative AF. High circulating HSP-70 levels is connected with higher incidence of postoperative AF after open heart surgery.

Selenium glutathione peroxidase activities and thyroid functions in human individuals

NASA Astrophysics Data System (ADS)

Bellisola, G.; Calza Contin, M.; Ceccato, D.; Cinque, G.; Francia, G.; Galassini, S.; Liu, N. Q.; Lo Cascio, C.; Moschini, G.; Sussi, P. L.

1996-04-01

At least two enzymes are involved in metabolism of thyroid hormones. GSHPx protects thyrocyte from high H 2O 2 levels that are required for iodination of prohormones to form T4 in thyroid cell. Type I iodothyronine 5'-deiodinase (5'-D) catalyzes the deiodination of L-thyroxin (T4) to the biologically active thyroid hormone 3,3'-5-triiodothyronine (T 3) in liver, in kidney and in thyroid tissues. Circulating thyroid hormones, plasma Se levels, GSHPx activities in platelets and in plasma were investigated in 29 human individuals with increased thyroid mass. PIXE was applied to measure Se in 1 ml of plasma because we supposed patients were in a marginal carential status for Se. Plasma Se concentrations were compared with those of normal individuals. Correlation studies between plasma Se level and both GSHPx activities were carried out as well as between platelets and plasma GSHPx activities to verify the hypothesis of a marginal Se deficiency in patients. Significance of circulating thyroid hormones levels will be discussed.

[Circulating endothelial progenitor cell levels in treated hypertensive patients].

PubMed

Maroun-Eid, C; Ortega-Hernández, A; Abad, M; García-Donaire, J A; Barbero, A; Reinares, L; Martell-Claros, N; Gómez-Garre, D

2015-01-01

Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP<140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

Major surgical trauma induces proteolysis of insulin-like growth factor binding protein-3 in transgenic mice and is associated with a rapid increase in circulating levels of matrix metalloproteinase-9.

PubMed

Belizon, A; Kirman, I; Balik, E; Karten, M; Jain, S; Whelan, R L

2007-04-01

The authors previously demonstrated a significant decrease in plasma levels of intact insulin-like growth factor binding protein-3 (IGFBP-3) after major open but not after laparoscopic-assisted surgery in humans. They postulated that this decrease may have an effect on postoperative tumor growth. It also has been shown that plasma levels of matrix metalloproteinase-9 (MMP-9), a protease capable of degrading IGFBP-3, are transiently increased after open colectomy in humans. The authors aimed to develop an animal model that would allow further study of the effect that surgical trauma has on plasma levels IGFBP-3 and MMP-9. In addition, they set out to assess the concentration of MMP-9 in circulating monocytes before and after surgery. The 30 mice included in this study were divided into three groups: sham laparotomy, carbon dioxide (CO2) pneumoperitoneum, and anesthesia control. All mice were IGFBP-3 transgenics (overexpressing human IGFBP-3) on a CD1 background. The mice were anesthetized using ketamine and xylazine. Blood was drawn retroorbitally 48 h before the procedure. The duration of the procedure was 30 min. The animals were killed 24 h postoperatively and blood was drawn. Intact IGFBP-3 levels were measured using a combination of Western blot analysis and enzyme-linked immunoassay (ELISA) at the two time points: before and after the operation. Plasma and peripheral blood mononuclear cell levels of MMP-9 were measured at each time point using zymography. Mononuclear cell lysates were used to determine intracellular MMP-9 levels. Plasma levels of intact IGFBP-3 were significantly lower than preoperative levels after sham laparotomy. A mean decrease of 76.6% was noted (p < 0.05). Zymography demonstrated significantly higher plasma MMP-9-related proteolytic activity than observed preoperatively after sham laparotomy (78.5 vs 42.3 Relative Units [RU]; p < 0.05). In the pneumoperitoneum group, no significant decrease was found between the pre- and postoperative levels of intact IGFBP-3. A nonsignificant increase in MMP-9 was noted after CO2 pneumoperitoneum (38 RU preoperatively vs. 46.4 RU postoperatively; p > 0.05). The anesthesia control group did not demonstrate a significant change in either circulating intact IGFBP-3 levels or MMP-9 levels. Mononuclear intracellular levels of MMP-9 were significantly lower after laparotomy than the preoperative levels (3 vs 37 RU). The postprocedure intracellular levels of MMP-9 were not significantly decreased in the pneumoperitoneum or anesthesia control group. Plasma levels of intact IGFBP-3, a cell growth regulating factor, were found to be decreased significantly after laparotomy. This decrease was not seen after pneumoperitoneum. Depletion of intact IGFBP-3 after laparotomy correlated with a rapid release of MMP-9 from mononuclear cells and an increase in circulating plasma MMP-9 levels. Matrix metalloproteinase-9 may play an important role in IGFBP-3 proteolysis after surgical trauma. Furthermore, circulating mononuclear cells are one source of MMP-9 after surgery. Finally, the model used reproduces events in humans after surgery, and thus should permit further study on the mechanism of IGFBP-3 proteolysis after surgical trauma.

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

PubMed

Mazurek, Agnieszka M; Fiszer-Kierzkowska, A; Rutkowski, T; Składowski, K; Pierzyna, M; Scieglińska, D; Woźniak, G; Głowacki, G; Kawczyński, R; Małusecka, E

2013-01-01

The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.

Tenascin-C Deficiency in Apo E−/− Mouse Increases Eotaxin Levels: Implications for Atherosclerosis

PubMed Central

Wang, Lai; Shah, Prediman K.; Wang, Wei; Song, Lei; Yang, Mingjie; Sharifi, Behrooz G.

2013-01-01

Aim To investigate the potential role of inflammatory cytokines in apo E−/− mouse in response to deletion of Tenascin-C (TNC) gene. Methods and results We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E−/− mice and apo E−/− TNC−/− double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E−/− mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E−/− mouse group. Inbreeding of apo E−/− mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E−/− mice had low level of eotaxin expression, cells derived from apo E−/−TNC−/− mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E−/−TNC−/− mice markedly stimulated mast cell migration whereas plasma from the apo E−/− mice had no such effect. Conclusion These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E−/− mice and that this chemokine plays a key role in the development of atherosclerosis. PMID:23433402

Cell-free circulating tumour DNA as a liquid biopsy in breast cancer.

PubMed

De Mattos-Arruda, Leticia; Caldas, Carlos

2016-03-01

Recent developments in massively parallel sequencing and digital genomic techniques support the clinical validity of cell-free circulating tumour DNA (ctDNA) as a 'liquid biopsy' in human cancer. In breast cancer, ctDNA detected in plasma can be used to non-invasively scan tumour genomes and quantify tumour burden. The applications for ctDNA in plasma include identifying actionable genomic alterations, monitoring treatment responses, unravelling therapeutic resistance, and potentially detecting disease progression before clinical and radiological confirmation. ctDNA may be used to characterise tumour heterogeneity and metastasis-specific mutations providing information to adapt the therapeutic management of patients. In this article, we review the current status of ctDNA as a 'liquid biopsy' in breast cancer. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer

PubMed Central

Madic, Jordan; Remon, Jordi; Honoré, Aurélie; Girard, Romain; Rouleau, Etienne; André, Barbara; Besse, Benjamin; Droniou, Magali; Lacroix, Ludovic

2017-01-01

Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results. PMID:28829811

Biomarkers in Immunoglobulin Light Chain Amyloidosis.

PubMed

Kufová, Z; Sevcikova, T; Growkova, K; Vojta, P; Filipová, J; Adam, Z; Pour, L; Penka, M; Rysava, R; Němec, P; Brozova, L; Vychytilova, P; Jurczyszyn, A; Grosicki, S; Barchnicka, A; Hajdúch, M; Simicek, M; Hájek, R

2017-01-01

Immunoglobulin light chain amyloidosis (AL amyloidosis - ALA) is a monoclonal gammopathy characterized by presence of aberrant plasma cells producing amyloidogenic immunoglobulin light chains. This leads to formation of amyloid fibrils in various organs and tissues, mainly in heart and kidney, and causes their dysfunction. As amyloid depositing in target organs is irreversible, there is a big effort to identify biomarker that could help to distinguish ALA from other monoclonal gammopathies in the early stages of disease, when amyloid deposits are not fatal yet. High throughput technologies bring new opportunities to modern cancer research as they enable to study disease within its complexity. Sophisticated methods such as next generation sequencing, gene expression profiling and circulating microRNA profiling are new approaches to study aberrant plasma cells from patients with light chain amyloidosis and related diseases. While generally known mutation in multiple myeloma patients (KRAS, NRAS, MYC, TP53) were not found in ALA, number of mutated genes is comparable. Transcriptome of ALA patients proves to be more similar to monoclonal gammopathy of undetermined significance patients, moreover level of circulating microRNA, that are known to correlate with heart damage, is increased in ALA patients, where heart damage in ALA typical symptom.Key words: amyloidosis - plasma cell - genome - transcriptome - microRNA.

Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer.

PubMed

Chudziak, Jakub; Burt, Deborah J; Mohan, Sumitra; Rothwell, Dominic G; Mesquita, Bárbara; Antonello, Jenny; Dalby, Suzanne; Ayub, Mahmood; Priest, Lynsey; Carter, Louise; Krebs, Matthew G; Blackhall, Fiona; Dive, Caroline; Brady, Ged

2016-01-21

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

Decreased Circulating mtDNA Levels in Professional Male Volleyball Players.

PubMed

Nasi, Milena; Cristani, Alessandro; Pinti, Marcello; Lamberti, Igor; Gibellini, Lara; De Biasi, Sara; Guazzaloca, Alessandro; Trenti, Tommaso; Cossarizza, Andrea

2016-01-01

Exercise exerts various effects on the immune system, and evidence is emerging on its anti-inflammatory effects; the mechanisms on the basis of these modifications are poorly understood. Mitochondrial DNA (mtDNA) released from damaged cells acts as a molecule containing the so-called damage-associated molecular patterns and can trigger sterile inflammation. Indeed, high plasma levels of mtDNA are associated to several inflammatory conditions and physiological aging and longevity. The authors evaluated plasma mtDNA in professional male volleyball players during seasonal training and the possible correlation between mtDNA levels and clinical parameters, body composition, and physical performance. Plasma mtDNA was quantified by real-time PCR every 2 mo in 12 professional volleyball players (PVPs) during 2 consecutive seasons. As comparison, 20 healthy nonathlete male volunteers (NAs) were analyzed. The authors found lower levels of mtDNA in plasma of PVPs than in NAs. However, PVPs showed a decrease of circulating mtDNA only in the first season, while no appreciable variations were observed during the second season. No correlation was observed among mtDNA, hematochemical, and anthropometric parameters. Regular physical activity appeared associated with lower levels of circulating mtDNA, further confirming the protective, anti-inflammatory effect of exercise.

Plasma serpinB1 is related to insulin sensitivity but not pancreatic β-Cell function in non-diabetic adults.

PubMed

Glicksman, Michael; Asthana, Asha; Abel, Brent S; Walter, Mary F; Skarulis, Monica C; Muniyappa, Ranganath

2017-03-01

Pancreatic β -cell dysfunction because of reduced β -cell mass and function is a primary determinant in the progression of diabetes. Increase in β -cell mass and compensatory hyperinsulinaemia is frequently associated with insulin-resistant states. Although the humoral factors mediating this compensatory response are unknown, serpinB1, a protease inhibitor, has recently been proposed to be one such factor. In this study, we examine the relationships between plasma serpinB1, insulin sensitivity, and pancreatic β -cell function in non-diabetic individuals. 117 subjects (women, n  = 50, men, n  = 67; age= 37.6 ± 10.8; BMI=31.1 ± 7.7 kg/m 2 ) underwent an insulin-modified frequently sampled intravenous glucose tolerance test (FSIVGTT) at the NIH Clinical Research Center. Acute insulin response (AIR) and insulin sensitivity index (SI) were obtained from the FSIVGTT with MINMOD analysis. The Quantitative Insulin Sensitivity Check Index (QUICKI) was calculated from fasting insulin and glucose values. Plasma serpinB1 levels were measured using an ELISA assay. Simple linear correlation analyses were performed to evaluate the relationship between serpinB1 and measures of insulin sensitivity and β -cell function. Circulating serpinB1 levels were unrelated to age, sex, race, BMI, or percent body fat. SI but not AIR significantly correlated with circulating serpinB1 levels ( r  = 0.23, P  < 0.05). QUICKI tended to positively correlate with serpinB1 ( r  = 0.16, P  = 0.09). Circulating serpinB1 is directly associated with insulin sensitivity but not β -cell function in non-diabetic adults. Whether this modest association plays a role in insulin sensitivity in humans remains to be clarified. Published [2017]. This article is a U.S. Government work and is in the public domain in the USA.

Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma.

PubMed

Camus, Vincent; Stamatoullas, Aspasia; Mareschal, Sylvain; Viailly, Pierre-Julien; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Picquenot, Jean Michel; Ruminy, Philippe; Maingonnat, Catherine; Bertrand, Philippe; Cornic, Marie; Tallon-Simon, Valérie; Becker, Stéphanie; Veresezan, Liana; Frebourg, Thierry; Vera, Pierre; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

2016-09-01

Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1-100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8-100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma. Copyright© Ferrata Storti Foundation.

Diabetic ketoacidosis elicits systemic inflammation associated with cerebrovascular endothelial cell dysfunction.

PubMed

Close, Taylor E; Cepinskas, Gediminas; Omatsu, Tatsushi; Rose, Keeley L; Summers, Kelly; Patterson, Eric K; Fraser, Douglas D

2013-08-01

To determine if the DKA-induced inflammation in juvenile mice provokes activation and dysfunction of CVECs. DKA in juvenile mice was induced with administration of STZ and ALX. Blood from DKA mice was assessed for cytokines and soluble cell adhesion proteins, and either DKA plasma or exogenous compounds were applied to immortalized bEND3. DKA increased circulating levels of IL-6, IL-8(KC), MCP-1, IL-10, sE-selectin, sICAM-1, and sVCAM-1. Stimulation of bEND3 with DKA plasma caused cellular activation (increased ROS and activation of NF-κΒ), upregulation of a proadhesive phenotype (E-selectin, ICAM-1, and VCAM-1), and increased leukocyte-bEND3 interaction (leukocyte rolling/adhesion). TEER, a measure of bEND3 monolayer integrity, was decreased by DKA plasma. Activation and dysfunction of bEND3 with DKA plasma were suppressed by plasma heat treatment (56°C, 1 hour) and replicated with the application of DKA recombinant cytomix (IL-6, IL-8[KC], MCP-1, and IL-10), implicating circulating inflammatory protein(s) as mediators. Treatment of bEND3 with β-OH-butyrate, the main ketone elevated in DKA, failed to mimic the DKA plasma-induced activation and dysfunction of bEND3. DKA elicits systemic inflammation associated with CVEC activation and dysfunction, possibly contributing to DKA-associated intracranial microvascular complications. © 2013 John Wiley & Sons Ltd.

Liquid Biopsy in Lung Cancer: A Perspective From Members of the Pulmonary Pathology Society.

PubMed

Sholl, Lynette M; Aisner, Dara L; Allen, Timothy Craig; Beasley, Mary Beth; Cagle, Philip T; Capelozzi, Vera L; Dacic, Sanja; Hariri, Lida P; Kerr, Keith M; Lantuejoul, Sylvie; Mino-Kenudson, Mari; Raparia, Kirtee; Rekhtman, Natasha; Roy-Chowdhuri, Sinchita; Thunnissen, Eric; Tsao, Ming; Vivero, Marina; Yatabe, Yasushi

2016-08-01

Liquid biopsy has received extensive media coverage and has been called the holy grail of cancer detection. Attempts at circulating tumor cell and genetic material capture have been progressing for several years, and recent financially and technically feasible improvements of cell capture devices, plasma isolation techniques, and highly sensitive polymerase chain reaction- and sequencing-based methods have advanced the possibility of liquid biopsy of solid tumors. Although practical use of circulating RNA-based testing has been hindered by the need to fractionate blood to enrich for RNAs, the detection of circulating tumor cells has profited from advances in cell capture technology. In fact, the US Food and Drug Administration has approved one circulating tumor cell selection platform, the CellSearch System. Although the use of liquid biopsy in a patient population with a genomically defined solid tumor may potentially be clinically useful, it currently does not supersede conventional pretreatment tissue diagnosis of lung cancer. Liquid biopsy has not been validated for lung cancer diagnosis, and its lower sensitivity could lead to significant diagnostic delay if liquid biopsy were to be used in lieu of tissue biopsy. Ultimately, notwithstanding the enthusiasm encompassing liquid biopsy, its clinical utility remains unproven.

Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia.

PubMed

Tanisawa, Kumpei; Taniguchi, Hirokazu; Sun, Xiaomin; Ito, Tomoko; Kawakami, Ryoko; Sakamoto, Shizuo; Higuchi, Mitsuru

2017-01-01

Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine linking obesity to skeletal muscle insulin resistance. Although previous studies reported that obesity was associated with high levels of circulating LECT2 in human, the associations of detailed body fat distribution with LECT2 levels have not been examined. Furthermore, although animal study suggested that exercise decreased circulating LECT2 levels, it remains unknown whether physical fitness is associated with LECT2 levels in human. We therefore examined the relationship of plasma LECT2 levels with various adiposity indices and cardiorespiratory fitness (CRF) in middle-aged and elderly Japanese men. Furthermore, we examined the relationship of LECT2 levels with the presence of metabolic syndrome, hypertension, insulin resistance and dyslipidemia to determine the clinical significance of measuring circulating LECT2. This was a cross-sectional study of 143 Japanese men (age: 30-79 years). Participants' plasma LECT2 levels were measured by an enzyme-linked immunosorbent assay. To assess their abdominal fat distributions, visceral fat area (VFA) and subcutaneous fat area (SFA) were measured using magnetic resonance imaging. CRF was assessed by measuring peak oxygen uptake ([Formula: see text]). All adiposity indices measured in this study were positively correlated with plasma LECT2 levels, while [Formula: see text] was negatively correlated with LECT2 levels after adjustment for age. The correlations, except for VFA were no longer significant with further adjustment for VFA. Stepwise multiple linear regression analysis revealed that VFA was the strongest predictor of plasma LECT2 levels. Plasma LECT2 levels differed based on the presence of metabolic syndrome and dyslipidemia, but not hypertension and insulin resistance. Logistic regression analyses revealed that plasma LECT2 levels were significantly associated with dyslipidemia independently of VFA; VFA was not significantly associated with dyslipidemia after adjustment for LECT2. VFA was the strongest predictor of plasma LECT2 that is a potential biomarker linking visceral obesity to dyslipidemia.

Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

PubMed

Leong, Sai Mun; Tan, Karen Mei-Ling; Chua, Hui Wen; Huang, Mo-Chao; Cheong, Wai Chye; Li, Mo-Huang; Tucker, Steven; Koay, Evelyn Siew-Chuan

2017-03-01

Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA) ® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses. © 2016 American Association for Clinical Chemistry.

Low-density lipoprotein receptor-related protein 1: a physiological Aβ homeostatic mechanism with multiple therapeutic opportunities

PubMed Central

Sagare, Abhay P.; Deane, Rashid; Zlokovic, Berislav V.

2012-01-01

Low-density lipoprotein receptor-related protein-1 (LRP1) is the main cell surface receptor involved in brain and systemic clearance of the Alzheimer's disease (AD) toxin amyloid-beta (Aβ). In plasma, a soluble form of LRP1 (sLRP1) is the major transport protein for peripheral Aβ. LRP1 in brain endothelium and mural cells mediates Aβ efflux from brain by providing a transport mechanism for A across the blood-brain barrier (BBB). sLRP1 maintains a plasma ‘sink’ activity for Aβ through binding of peripheral Aβ which in turn inhibits re-entry of free plasma Aβ into the brain. LRP1 in the liver mediates systemic clearance of Aβ. In AD, LRP1 expression at the BBB is reduced and Aβ binding to circulating sLRP1 is compromised by oxidation. Cell surface LRP1 and circulating sLRP1 represent druggable targets which can be therapeutically modified to restore the physiological mechanisms of brain Aβ homeostasis. In this review, we discuss how increasing LRP1 expression at the BBB and liver with lifestyle changes, statins, plant-based active principles and/or gene therapy on one hand, and how replacing dysfunctional plasma sLRP1 on the other regulate Aβ clearance from brain ultimately controlling the onset and/or progression of AD. PMID:22820095

Post-Effect of Air Quality Improvement on Biomarkers for Systemic Inflammation and Microparticles in Asthma Patients After the 2008 Beijing Olympic Games: a Pilot Study.

PubMed

Gao, Jinming; Xu, Xiaohua; Ying, Zhekang; Jiang, Lei; Zhong, Mianhua; Wang, Aixia; Chen, Lung-Chi; Lu, Bo; Sun, Qinghua

2017-08-01

This study's aim was to investigate the post-effect of an air quality improvement on systemic inflammation and circulating microparticles in asthmatic patients during, and 2 months after, the Beijing Olympics 2008. We measured the levels of circulating inflammatory cytokines and microparticles in the peripheral blood from asthma patients and healthy controls during (phase 1), and 2 months after (phase 2) the Beijing 2008 Olympic Games. The concentrations of circulating cytokines (including TNFα, IL-6, IL-8, and IL-10) were still seen reduced in phase 2 when compared with those in phase 1. The number of circulating endothelial cell-derived microparticles was significantly lower during the phase 2 than that during phase 1 in asthma patients. The level of plasma lipopolysaccharide-binding protein (LBP) was significantly decreased in asthmatics in phase 2. The level of norepinephrine was significantly higher in phase 2 than that in phase 1 in plasma from both asthma patients and healthy subjects. There were no significant differences in the gene profile for the toll-like receptor (TLR) signaling from peripheral blood mononuclear cells. In vitro, microvesicles from patients with asthma impaired the relaxation to bradykinin and contraction to acetylcholine, whereas microparticles from healthy subjects did not. These data suggested that reduction in systemic pro-inflammatory responses and circulating LBP and increased level of norepinephrine in asthma patients persisted even after 2 months of the air pollution intervention. These changes were independent of the TLR signaling pathway. Circulating microparticles might be associated with airway smooth muscle dysfunction.

Plasma cell treatment device Plasma-on-Chip: Monitoring plasma-generated reactive species in microwells

PubMed Central

Oh, Jun-Seok; Kojima, Shinya; Sasaki, Minoru; Hatta, Akimitsu; Kumagai, Shinya

2017-01-01

We have developed a plasma cell treatment device called Plasma-on-Chip that enables the real-time monitoring of a single cell culture during plasma treatment. The device consists of three parts: 1) microwells for cell culture, 2) a microplasma device for generating reactive oxygen and nitrogen species (RONS) for use in cell treatment, and 3) through-holes (microchannels) that connect each microwell with the microplasma region for RONS delivery. Here, we analysed the delivery of the RONS to the liquid culture medium stored in the microwells. We developed a simple experimental set-up using a microdevice and applied in situ ultraviolet absorption spectroscopy with high sensitivity for detecting RONS in liquid. The plasma-generated RONS were delivered into the liquid culture medium via the through-holes fabricated into the microdevice. The RONS concentrations were on the order of 10–100 μM depending on the size of the through-holes. In contrast, we found that the amount of dissolved oxygen was almost constant. To investigate the process of RONS generation, we numerically analysed the gas flow in the through-holes. We suggest that the circulating gas flow in the through-holes promotes the interaction between the plasma (ionised gas) and the liquid, resulting in enhanced RONS concentrations. PMID:28176800

A novel strategy for highly efficient isolation and analysis of circulating tumor-specific cell-free DNA from lung cancer patients using a reusable conducting polymer nanostructure.

PubMed

Lee, HyungJae; Jeon, SeungHyun; Seo, Jin-Suck; Goh, Sung-Ho; Han, Ji-Youn; Cho, Youngnam

2016-09-01

We have developed a reusable nanostructured polypyrrole nanochip and demonstrated its use in the electric field-mediated recovery of circulating cell-free DNA (cfDNA) from the plasma of lung cancer patients. Although cfDNA has been recognized and widely studied as a versatile and promising biomarker for the diagnosis and prognosis of cancers, the lack of efficient strategies to directly isolate cfDNA from the plasma has become a great hindrance to its potential clinical use. As a proof-of-concept study, we demonstrated a technique for the rapid and efficient isolation of cfDNA with high yield and purity. In particular, the synergistic effects of the electro-activity and the nanostructured features of the polypyrrole polymer enabled repeated retrieval of cfDNA using a single platform. Moreover, polypyrrole nanochip facilitated the amplification of tumor-specific DNA fragments from the plasma samples of patients with lung cancer characterized by mutations in exons 21 of the epidermal growth factor receptor gene (EGFR). Overall, the proposed polypyrrole nanochip has enormous potential for industrial and clinical applications with significantly enhanced efficiency in the recovery of tumor-associated circulating cfDNA. This may ultimately contribute to more robust and reliable evaluation of gene mutations in peripheral blood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

PubMed

Camus, Stéphane M; De Moraes, João A; Bonnin, Philippe; Abbyad, Paul; Le Jeune, Sylvain; Lionnet, François; Loufrani, Laurent; Grimaud, Linda; Lambry, Jean-Christophe; Charue, Dominique; Kiger, Laurent; Renard, Jean-Marie; Larroque, Claire; Le Clésiau, Hervé; Tedgui, Alain; Bruneval, Patrick; Barja-Fidalgo, Christina; Alexandrou, Antigoni; Tharaux, Pierre-Louis; Boulanger, Chantal M; Blanc-Brude, Olivier P

2015-06-11

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. © 2015 by The American Society of Hematology.

In vitro granulocyte adherence and in vivo margination: two associated complement-dependent functions. Studies based on the acute neutropenia of filtration leukophoresis.

PubMed

Fehr, J; Jacob, H S

1977-09-01

To study mechanisms and mediators regulating the distribution of intravascular granulocytes between circulating and marginated pools, a human model with extreme transient margination, the neutropenia of continuous flow filtration leukophoresis, was analyzed. Studies in animals demonstrated the existence of a complement (C)-derived granulocytopenia-inducing factor. Thus, autologous plasma, exposed to nylon fibers (NF) of the filtration system, produced an acute selective decrement of circulating granulocytes and monocytes. This phenomenon was blocked by decomplementing plasma, by pretreatment of plasma with EDTA or hydrazine, and by preheating at 56 degrees C, but did occur after recombination of heat-inactivated and hydrazine-treated plasma before NF exposure. Preheating plasma at 50 degrees C did not inhibit the neutropenic response, suggesting involvement of the classical pathway of C activation. Ultrafiltration studies indicated that the NF-provoked neutropenia-inducing factor has a mol wt in the range of 10,000-30,000, and is heat stable (56 degrees C). To analyze the hypothesis that C- induced neutrophil margination might be consequent to increased cell adhesiveness to endothelial surfaces, the role of C in promoting granulocyte adherence was evaluated in vitro. Measured with a plastic Petridish assay, granulocyte adherence was significantly reduced in heat- inactivated (56 degrees C) and hydrazine-treated plasma, but adherence promoting capacity was restored by mixing the two plasmas, or by adding purified C3 to hydrazine-treated plasma. After exposure to activated C, neutrophils showed significantly increased adhesiveness which was maintained when cells were resuspended in heat-inactivated plasma, but progressively lost when resuspended in fresh plasma. On the basis of these results we conclude that granulocyte adhesiveness in vitro and margination in vivo are closely associated, C-dependent phenomena.

Rituximab associated neutropenia: Description of three cases and an insight into the underlying pathogenesis

PubMed Central

Weissmann-Brenner, Alina; Brenner, Baruch; Belyaeva, Inessa; Lahav, Meir; Rabizadeh, Esther

2011-01-01

Summary Background To describe Rituximab associated neutropenia (RAN), and to explore its underlying mechanism. Case Report We describe three patients with RAN. The effect of patient’s plasma on colony forming unit, Granulocyte-Monocyte (CFU-GM) was measured by the addition of plasma to the culture of a healthy bone-marrow. Repeated tests were performed after recovery of white count. In the leukopenic period the patient’s plasma inhibited CFU growth completely. Control plasma did not have such an effect. Addition of patient’s cell supernatant to bone marrow cells did not change the number of CFU. The same effect was demonstrated in normal control. After recovery the patient’s plasma did not inhibit colony formation, similar to control. Conclusions RAN is a clinically significant side effect. It may take place during treatment or several months afterwards. Circulating antibodies in the plasma may be responsible for this unique BM toxicity. PMID:22037749

Plasma enteroglucagon and CCK levels and cell proliferation in defunctioned small bowel in the rat.

PubMed

Gornacz, G E; Ghatei, M A; Al-Mukhtar, M Y; Yeats, J C; Adrian, T E; Wright, N A; Bloom, S R

1984-11-01

Luminal nutrients exert a powerful trophic effect on small bowel mucosa. Recent evidence suggests that a circulating factor, possibly enteroglucagon, is also growth-promoting. In order to study the isolated effect of nonluminal influences on bowel mucosa, Thiry-Vella fistulae (TVF) were constructed in rats. Circulating enteric hormone concentrations were manipulated by resecting different lengths of remaining gut. Thirty-two male Wistar rats had either 25%, 50%, 75%, or 90% proximal small bowel resection. In each animal the first 25% of resected bowel was exteriorized as a Thiry-Vella fistula. Seven control rats underwent jejunal transection. Twelve days postoperatively the fasted animals were killed, and circulating and tissue concentrations of enteroglucagon and CCK were estimated by radioimmunoassay. Crypt-cell production rate was used as an index of cellular proliferation in the Thiry-Vella fistulae. Proximal small bowel defunctioned in the Thirty-Vella fistulae had a significantly lower crypt-cell production rate and enteroglucagon and CCK content than the equivalent segment in transected rats. Further small bowel resection produced a subsequent increase in circulating enteroglucagon and CCK concentrations, an increase in the Thiry-Vella fistula content of these hormones, and a doubling of the crypt-cell production rate in the Thiry-Vella fistulae. These results show that circulating enteroglucagon and CCK concentrations match closely with enterocyte production even when luminal influences are excluded. It is suggested that circulating factors may play a major role in postresectional ileal hyperplasia. This hyperplasia apparently affects endocrine cells as well as enterocytes.

Red Blood Cell Volume, Plasma Volume and Total Blood Volume in Healthy Elderly Men and Women Aged 64 to 100

DTIC Science & Technology

1992-05-06

Robert Valeri, Linda E. Pivacek, Hiliary Siebens, and Mark D. Altschule ». PERFORMING ORGANIZATION NAME AND AOORESS Naval Blood Research Laboratory...Gibson JG, Peacock WC, Seligman AM, Sack T: Circulating red cell volume measured simultaneously by the radioactive iron and dye methods. J Clin

The role of the habenula-interpeduncular pathway in modulating levels of circulating adrenal hormones.

PubMed

Murray, M; Murphy, C A; Ross, L L; Haun, F

1994-01-01

The fasciculus retroflexus (FR) is the major pathway by which the medial and lateral habenular nuclei project to the interpeduncular nucleus (IPN) and ventral tegmentum. Recent work has suggested that the habenula-interpeduncular system may be involved in the regulation of states of arousal. Bilateral FR lesions have been shown to disrupt chronically, and habenula transplants have been shown to restore normal sleep patterns in rats [J. NeuroscL, 12 (1992) 3282-3290]. In this study, we examined whether FR lesions and habenula cell transplants would also modify chronically the circulating plasma levels of the stress-related hormones, norepinephrine (NE), epinephrine (EPI) and corticosterone. When plasma samples were obtained via retro-orbital eye-bleed during anesthesia, animals with FR lesions had significantly increased levels of plasma NE, EPI and corticosterone 2-3 months postoperatively compared to unoperated controls. Transplants of embryonic habenula cells placed near the denervated IPN in FR-lesioned animals restored levels of NE and EPI to normal, but did not attenuate elevated corticosterone levels. When plasma samples were obtained in conscious animals via indwelling arterial cannulae, FR-lesioned rats likewise exhibited increased basal levels of corticosterone but plasma levels of catecholamines were similar to those of unoperated controls. Differences in our results obtained using the two methods of blood sampling may be explained by the effects of anesthesia and stress associated with the eye-bleed method. Thus, the effect of FR lesions in increasing plasma levels of catecholamines may not reflect a difference in basal hormone levels, but a heightened sympathetic adrenomedullary response to stress. While these results indicate that the integrity of the habenular efferent pathway is important in modulating circulating levels of hormones associated with the stress response, two separate mechanisms appear to control its interactions with sympathetic-adrenal medullary and adrenocortical pathways.

The Acid-Secreting Parietal Cell as an Endocrine Source of Sonic Hedgehog During Gastric Repair

PubMed Central

Engevik, Amy C.; Feng, Rui; Yang, Li

2013-01-01

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shhflx/flx) and mice expressing a Cre transgene under the control of the H+,K+-ATPase β-subunit promoter. Shhflx/flx, the H+,K+-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair. PMID:24092639

Resistance to Fluid Shear Stress Is a Conserved Biophysical Property of Malignant Cells

PubMed Central

Henry, Michael D.

2012-01-01

During metastasis, cancer cells enter the circulation in order to gain access to distant tissues, but how this fluid microenvironment influences cancer cell biology is poorly understood. A longstanding view is that circulating cancer cells derived from solid tissues may be susceptible to damage from hemodynamic shear forces, contributing to metastatic inefficiency. Here we report that compared to non-transformed epithelial cells, transformed cells are remarkably resistant to fluid shear stress (FSS) in a microfluidic protocol, exhibiting a biphasic decrease in viability when subjected to a series of millisecond pulses of high FSS. We show that magnitude of FSS resistance is influenced by several oncogenes, is an adaptive and transient response triggered by plasma membrane damage and requires extracellular calcium and actin cytoskeletal dynamics. This novel property of malignant cancer cells may facilitate hematogenous metastasis and indicates, contrary to expectations, that cancer cells are quite resistant to destruction by hemodynamic shear forces. PMID:23226552

Impact of Hepatitis C Virus on the Circulating Levels of IL-7 in HIV-1 Coinfected Women.

PubMed

Kerzerho, Jerome; McIlvaine, Elizabeth J; Anthony, Patricia; Mack, Wendy J; Wang, Chia-Hao; Frederick, Toni; Operskalski, Eva; Chen, Zhi; Al-Harthi, Lena; Landay, Alan; Young, Mary A; Tien, Phyllis C; Augenbraun, Michael; Strickler, Howard D; Akbari, Omid; Golub, Elizabeth T; Sharp, Gerald B; Kovacs, Andrea

2016-02-01

Hepatitis C virus (HCV) infection causes an alteration in T-cell maturation and activation in patients coinfected with human immunodeficiency virus (HIV). Because interleukin 7 (IL-7) is a major cytokine controlling T-cell homeostasis, we analyzed the potential influence of HCV coinfection on circulating IL-7 levels in HIV-infected women before and after highly active antiretroviral therapy (HAART). This prospective study included 56 HIV monoinfected, 55 HIV/HCV coinfected without HCV viremia, 132 HIV/HCV coinfected with HCV viremia, and 61 HIV/HCV-uninfected women for whom plasma levels of IL-7 were determined by enzyme-linked immunosorbent assay at 1 or more follow-up visits before and after HAART. Cross-sectional analyses of the associations between plasma IL-7 levels and HCV infection, demographic, clinical, and immunologic characteristics were evaluated using univariate and multivariate linear regression models before and after HAART. In multivariate models, IL-7 levels were significantly higher in coinfected HCV viremic women than in HIV monoinfected women (multiplicative effect = 1.48; 95% confidence interval: 1.01 to 2.16; P = 0.04) before HAART, but were similar between these two groups among women after HAART. In addition to HCV viremia, higher IL-7 levels were associated with older age (P = 0.02), lower CD4(+) T-cell count (P = 0.0007), and higher natural killer T-cell count (P = 0.02) in women before HAART. Among HAART-treated women, only lower CD4(+) T-cell count was significantly associated with IL-7 level (P = 0.006). Our data demonstrate that in HIV-infected women, circulating levels of IL-7 are strongly associated with CD4 T-cell depletion both before and after HAART. Our data also demonstrate that HCV viremia increases circulating IL-7 levels before HAART but not after HAART in coinfected women. This suggests that the effect of HCV on lymphopenia is abrogated by HAART.

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

PubMed

de Barros, Cintia M; Andrade, Leonardo R; Allodi, Silvana; Viskov, Christian; Mourier, Pierre A; Cavalcante, Moisés C M; Straus, Anita H; Takahashi, Helio K; Pomin, Vitor H; Carvalho, Vinicius F; Martins, Marco A; Pavão, Mauro S G

2007-01-19

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

The Clinical Development of Thalildomide as an Angiogenesis Inhibitor Therapy for Prostate Cancer

DTIC Science & Technology

2005-10-01

regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti- TNF - alpha agent thalidomide . Settles B, Stevenson A...Plasma levels of circulating TNF -α and VEGF measured in 16 thalidomide treated patients seemed to increase after treatment (Table 2). Serum levels of...Logothetis, Christopher J. Page 6 Table 2. Levels of circulating VEGF, bFGF, TNF -α and IL-6 before and after thalidomide treatment. Comparison of

Angptl4 does not control hyperglucagonemia or α-cell hyperplasia following glucagon receptor inhibition

PubMed Central

Okamoto, Haruka; Cavino, Katie; Na, Erqian; Krumm, Elizabeth; Kim, Steven; Stevis, Panayiotis E.; Harp, Joyce; Murphy, Andrew J.; Yancopoulos, George D.; Gromada, Jesper

2017-01-01

Genetic disruption or pharmacologic inhibition of glucagon signaling effectively lowers blood glucose but results in compensatory glucagon hypersecretion involving expansion of pancreatic α-cell mass. Ben-Zvi et al. recently reported that angiopoietin-like protein 4 (Angptl4) links glucagon receptor inhibition to hyperglucagonemia and α-cell proliferation [Ben-Zvi et al. (2015) Proc Natl Acad Sci USA 112:15498–15503]. Angptl4 is a secreted protein and inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance. We report that Angptl4−/− mice treated with an anti-glucagon receptor monoclonal antibody undergo elevation of plasma glucagon levels and α-cell expansion similar to wild-type mice. Overexpression of Angptl4 in liver of mice caused a 8.6-fold elevation in plasma triglyceride levels, but did not alter plasma glucagon levels or α-cell mass. Furthermore, administration of glucagon receptor-blocking antibody to healthy individuals increased plasma glucagon and amino acid levels, but did not change circulating Angptl4 concentration. These data show that Angptl4 does not link glucagon receptor inhibition to compensatory hyperglucagonemia or expansion of α-cell mass, and that it cannot be given to induce such secretion and growth. The reduction of plasma triglyceride levels in Angptl4−/− mice and increase following Angptl4 overexpression suggest that changes in plasma triglyceride metabolism do not regulate α-cells in the pancreas. Our findings corroborate recent data showing that increased plasma amino acids and their transport into α-cells link glucagon receptor blockage to α-cell hyperplasia. PMID:28143927

Plasma levels of lysine, tyrosine, and valine during pregnancy are independent risk factors of insulin resistance and gestational diabetes.

PubMed

Park, Sunmin; Park, Jin Young; Lee, Ju Hong; Kim, Sung-Hoon

2015-03-01

This study compared plasma concentrations of amino acids in pregnant women with and without gestational diabetes mellitus (GDM) and identified the association between plasma amino acid levels and GDM, insulin resistance, and insulin secretion at 24-28 weeks of pregnancy. Circulating amino acid levels were evaluated using high-performance liquid chromatography at 24-28 weeks of pregnancy in 25 non-GDM and 64 GDM women after adjusting for covariates such as maternal age, body mass index (BMI) before pregnancy, BMI and gestational age at screening GDM, and daily caloric intake. Backward stepwise logistic regression analysis was used to identify the predictors of developing GDM, and homeostatic model assessments for insulin resistance (HOMA-IR) and β-cell function (HOMA-B). Circulating levels of amino acids except threonine and tyrosine were significantly higher in GDM women than non-GDM women. Along with the intakes of energy, protein, and fat from animal sources, the intakes of each amino acid were significantly higher in the GDM group without a direct correlation to plasma amino acid levels. The variation in GDM development was explained by maternal age, diastolic blood pressure, and plasma lysine levels (R(2)=0.691). Height, BMI before pregnancy, systolic blood pressure, and plasma tyrosine and valine levels accounted for the variation in HOMA-IR (R(2)=0.589). The 53.3% variation of HOMA-B was explained by maternal age, BMI at GDM screening, plasma insulin level at 1 h during the oral glucose tolerance test (OGTT), and plasma valine level. Circulating concentrations of lysine, tyrosine, and valine were independently and positively associated with GDM through modifying insulin resistance and secretion.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Circulating Cell-Free Tumour DNA in the Management of Cancer

PubMed Central

Francis, Glenn; Stein, Sandra

2015-01-01

With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease. PMID:26101870

STUDIES ON TUBERCULIN FEVER. 3. MECHANISMS INVOLVED IN THE RELEASE OF ENDOGENOUS PYROGEN IN VITRO.

PubMed

ATKINS, E; HEIJN, C

1965-08-01

In a search for the source of the circulating endogenous pyrogen (EP) that mediates tuberculin-induced fever, tuberculin was incubated in vitro with various tissues of rabbits sensitized by intravenous infection with BCG. Evidence was obtained that tuberculin specifically stimulates cells in the blood of sensitized rabbits to generate pyrogen in vitro, whereas both lymph node and spleen cells from the same donors were inactive. Since normal blood cells, incubated in plasma of sensitized donors, were similarly activated, it is postulated that circulating antibodies play a role in sensitizing cells (presumably granulocytes) to release pyrogen on contact with tuberculin) both in vitro and in vivo. Release of endogenous pyrogen in vitro may be a sensitive means of detecting immunologic reactions between antigen and specifically sensitized blood cells-in other allergic states accompanied by fever.

Distribution, persistence and interchange of Epstein-Barr virus strains among PBMC, plasma and saliva of primary infection subjects.

PubMed

Kwok, Hin; Chan, Koon Wing; Chan, Kwok Hung; Chiang, Alan Kwok Shing

2015-01-01

Our study aimed at investigating the distribution, persistence and interchange of viral strains among peripheral blood mononuclear cells (PBMC), plasma and saliva of primary Epstein-Barr virus (EBV) infection subjects. Twelve infectious mononucleosis (IM) patients and eight asymptomatic individuals (AS) with primary EBV infection were followed longitudinally at several time points for one year from the time of diagnosis, when blood and saliva samples were collected and separated into PBMC, plasma and saliva, representing circulating B cell, plasma and epithelial cell compartments, respectively. To survey the viral strains, genotyping assays for the natural polymorphisms in two latent EBV genes, EBNA2 and LMP1, were performed and consisted of real-time PCR on EBNA2 to distinguish type 1 and 2 viruses, fluorescent-based 30-bp typing assay on LMP1 to distinguish deletion and wild type LMP1, and fluorescent-based heteroduplex tracking assays on both EBNA2 and LMP1 to distinguish defined polymorphic variants. No discernible differences were observed between IM patients and AS. Multiple viral strains were acquired early at the start of infection. Stable persistence of dominant EBV strains in the same tissue compartment was observed throughout the longitudinal samples. LMP1-defined strains, China 1, China 2 and Mediterranean+, were the most common strains observed. EBNA2-defined groups 1 and 3e predominated the PBMC and saliva compartments. Concordance of EBNA2 and LMP1 strains between PBMC and saliva suggested ready interchange of viruses between circulating B cell and epithelial cell pools, whilst discordance of viral strains observed between plasma and PBMC/saliva indicated presence of viral pools in other undetermined tissue compartments. Taken together, the results indicated that the distribution, persistence and interchange of viral strains among the tissue compartments are more complex than those proposed by the current model of EBV life cycle.

Distribution, Persistence and Interchange of Epstein-Barr Virus Strains among PBMC, Plasma and Saliva of Primary Infection Subjects

PubMed Central

Kwok, Hin; Chan, Koon Wing; Chan, Kwok Hung; Chiang, Alan Kwok Shing

2015-01-01

Our study aimed at investigating the distribution, persistence and interchange of viral strains among peripheral blood mononuclear cells (PBMC), plasma and saliva of primary Epstein-Barr virus (EBV) infection subjects. Twelve infectious mononucleosis (IM) patients and eight asymptomatic individuals (AS) with primary EBV infection were followed longitudinally at several time points for one year from the time of diagnosis, when blood and saliva samples were collected and separated into PBMC, plasma and saliva, representing circulating B cell, plasma and epithelial cell compartments, respectively. To survey the viral strains, genotyping assays for the natural polymorphisms in two latent EBV genes, EBNA2 and LMP1, were performed and consisted of real-time PCR on EBNA2 to distinguish type 1 and 2 viruses, fluorescent-based 30-bp typing assay on LMP1 to distinguish deletion and wild type LMP1, and fluorescent-based heteroduplex tracking assays on both EBNA2 and LMP1 to distinguish defined polymorphic variants. No discernible differences were observed between IM patients and AS. Multiple viral strains were acquired early at the start of infection. Stable persistence of dominant EBV strains in the same tissue compartment was observed throughout the longitudinal samples. LMP1-defined strains, China 1, China 2 and Mediterranean+, were the most common strains observed. EBNA2-defined groups 1 and 3e predominated the PBMC and saliva compartments. Concordance of EBNA2 and LMP1 strains between PBMC and saliva suggested ready interchange of viruses between circulating B cell and epithelial cell pools, whilst discordance of viral strains observed between plasma and PBMC/saliva indicated presence of viral pools in other undetermined tissue compartments. Taken together, the results indicated that the distribution, persistence and interchange of viral strains among the tissue compartments are more complex than those proposed by the current model of EBV life cycle. PMID:25807555

PLASMA CELL LEUKEMIA

PubMed Central

de Larrea, Carlos Fernandez; Kyle, Robert A.; Durie, Brian GM; Ludwig, Heinz; Usmani, Saad; Vesole, David H.; Hajek, Roman; Miguel, Jésus San; Sezer, Orhan; Sonneveld, Pieter; Kumar, Shaji K.; Mahindra, Anuj; Comenzo, Ray; Palumbo, Antonio; Mazumber, Amitabha; Anderson, Kenneth C.; Richardson, Paul G.; Badros, Ashraf Z.; Caers, Jo; Cavo, Michele; LeLeu, Xavier; Dimopoulos, Meletios A.; Chim, CS; Schots, Rik; Noeul, Amara; Fantl, Dorotea; Mellqvist, Ulf-Henrik; Landgren, Ola; Chanan-Khan, Asher; Moreau, Philippe; Fonseca, Rafael; Merlini, Giampaolo; Lahuerta, JJ; Bladé, Joan; Orlowski, Robert Z.; Shah, Jatin J.

2014-01-01

Plasma cell leukemia (PCL) is a rare and aggressive variant of myeloma characterized by the presence of circulating plasma cells. It is classified as either primary PCL occurring at diagnosis or as secondary PCL in patients with relapsed/refractory myeloma. Primary PCL is a distinct clinic-pathologic entity with different cytogenetic and molecular findings. The clinical course is aggressive with short remissions and survival duration. The diagnosis is based upon the percentage (≥ 20%) and absolute number (≥ 2 × 10 9/L) of plasma cells in the peripheral blood. It is proposed that the thresholds for diagnosis be reexamined and consensus recommendations are made for diagnosis, as well as, response and progression criteria. Induction therapy needs to begin promptly and have high clinical activity leading to rapid disease control in an effort to minimize the risk of early death. Intensive chemotherapy regimens and bortezomib-based regimens are recommended followed by high-dose therapy with autologous stem-cell transplantation (HDT/ASCT) if feasible. Allogeneic transplantation can be considered in younger patients. Prospective multicenter studies are required to provide revised definitions and better understanding of the pathogenesis of PCL. PMID:23288300

Influence of electromagnetic radiation produced by mobile phone on some biophysical blood properties in rats.

PubMed

El-Bediwi, Abu Bakr; Saad, Mohamed; El-kott, Attall F; Eid, Eman

2013-04-01

Effects of electromagnetic radiation produced by mobile phone on blood viscosity, plasma viscosity, hemolysis, Osmotic fragility, and blood components of rats have been investigated. Experimental results show that there are significant change on blood components and its viscosity which affects on a blood circulation due to many body problems. Red blood cells, White blood cells, and Platelets are broken after exposure to electromagnetic radiation produced by mobile phone. Also blood viscosity and plasma viscosity values are increased but Osmotic fragility value decreased after exposure to electromagnetic radiation produced by mobile phone.

Combination of plasma HA and circulating M2-like monocytes may serve as a diagnostic marker for breast cancer.

PubMed

Zhang, Boke; Cao, Manlin; He, Yiqing; Liu, Yiwen; Zhang, Guoliang; Yang, Cuixia; Du, Yan; Xu, Jing; Hu, Jiajie; Gao, Feng

2017-01-01

Background: Breast cancer (BC)-derived hyaluronan (HA) can induce the formation of M2-like tumor-associated macrophages (TAMs) in tumor context. However, little is known about the correlation between circulating M2-like monocytes and plasma HA in BC patients. This study focused on evaluating the relationship between circulating M2-like monocytes and plasma HA, and further appraised the diagnostic value of them in BC. Methods: The expression of M2-like TAMs and HA was determined in pathological tissues by immunohistochemistry. Flow cytometry was used to detect the levels of circulating CD14 + CD204 + M2-like monocytes in 81 BC patients, 45 patients with breast benign diseases, and 46 healthy subjects. The levels of HA, CEA, and CA15-3 were measured in plasma samples using chemiluminescence method. Results: M2-like TAMs and HA expressions were elevated in BC tissues compared with benign tissues. In correspondence, the frequency of circulating CD14 + CD204 + M2-like monocytes and the plasma HA levels were significantly higher in patients with BC than those in control groups. Importantly, there was a positive correlation between circulating M2-like monocytes and the plasma HA (Spearman r = 0.404, p < 0.001). Area under receiver operating characteristic curve (ROC) for the combination of circulating M2-like monocytes and HA was 0.899 (95% CI: 0.853-0.946), which was higher than the panel of CEA and CA15-3. Conclusions: The frequency of circulating CD14 + CD204 + M2-like monocytes was positively correlated to plasma HA levels. The combination of circulating CD14 + CD204 + M2-like monocytes and plasma HA could provide considerable diagnostic value in BC.

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

PubMed Central

Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

2015-01-01

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

Cortisol-induced immune suppression by a blockade of lymphocyte egress in traumatic brain injury.

PubMed

Dong, Tingting; Zhi, Liang; Bhayana, Brijesh; Wu, Mei X

2016-08-25

Acute traumatic brain injury (TBI) represents one of major causes of mortality and disability in the USA. Neuroinflammation has been regarded both beneficial and detrimental, probably in a time-dependent fashion. To address a role for neuroinflammation in brain injury, C57BL/6 mice were subjected to a closed head mild TBI (mTBI) by a standard controlled cortical impact, along with or without treatment of sphingosine 1-phosphate (S1P) or rolipram, after which the brain tissue of the impact site was evaluated for cell morphology via histology, inflammation by qRT-PCR and T cell staining, and cell death with Caspase-3 and TUNEL staining. Circulating lymphocytes were quantified by flow cytometry, and plasma hydrocortisone was analyzed by LC-MS/MS. To investigate the mechanism whereby cortisol lowered the number of peripheral T cells, T cell egress was tracked in lymph nodes by intravital confocal microscopy after hydrocortisone administration. We detected a decreased number of circulating lymphocytes, in particular, T cells soon after mTBI, which was inversely correlated with a transient and robust increase of plasma cortisol. The transient lymphocytopenia might be caused by cortisol in part via a blockade of lymphocyte egress as demonstrated by the ability of cortisol to inhibit T cell egress from the secondary lymphoid tissues. Moreover, exogenous hydrocortisone severely suppressed periphery lymphocytes in uninjured mice, whereas administering an egress-promoting agent S1P normalized circulating T cells in mTBI mice and increased T cells in the injured brain. Likewise, rolipram, a cAMP phosphodiesterase inhibitor, was also able to elevate cAMP levels in T cells in the presence of hydrocortisone in vitro and abrogate the action of cortisol in mTBI mice. The investigation demonstrated that the number of circulating T cells in the early phase of TBI was positively correlated with T cell infiltration and inflammatory responses as well as cell death at the cerebral cortex and hippocampus beneath the impact site. Decreases in intracellular cAMP might be part of the mechanism behind cortisol-mediated blockade of T cell egress. The study argues strongly for a protective role of cortisol-induced immune suppression in the early stage of TBI.

The effects of conventional extracorporeal circulation versus miniaturized extracorporeal circulation on microcirculation during cardiopulmonary bypass-assisted coronary artery bypass graft surgery.

PubMed

Yuruk, Koray; Bezemer, Rick; Euser, Mariska; Milstein, Dan M J; de Geus, Hilde H R; Scholten, Evert W; de Mol, Bas A J M; Ince, Can

2012-09-01

OBJECTIVES To reduce the complications associated with cardiopulmonary bypass (CPB) during cardiac surgery, many modifications have been made to conventional extracorporeal circulation systems. This trend has led to the development of miniaturized extracorporeal circulation systems. Cardiac surgery using conventional extracorporeal circulation systems has been associated with significantly reduced microcirculatory perfusion, but it remains unknown whether this could be prevented by an mECC system. Here, we aimed to test the hypothesis that microcirculatory perfusion decreases with the use of a conventional extracorporeal circulation system and would be preserved with the use of an miniaturized extracorporeal circulation system. METHODS Microcirculatory density and perfusion were assessed using sublingual side stream dark-field imaging in patients undergoing on-pump coronary artery bypass graft (CABG) surgery before, during and after the use of either a conventional extracorporeal circulation system (n = 10) or a miniaturized extracorporeal circulation system (n = 10). In addition, plasma neutrophil gelatinase-associated lipocalin and creatinine levels and creatinine clearance were assessed up to 5 days post-surgery to monitor renal function. RESULTS At the end of the CPB, one patient in the miniaturized extracorporeal circulation-treated group and five patients in the conventional extracorporeal circulation-treated group received one bag of packed red blood cells (300 ml). During the CPB, the haematocrit and haemoglobin levels were slightly higher in the miniaturized extracorporeal circulation-treated patients compared with the conventional extracorporeal circulation-treated patients (27.7 ± 3.3 vs 24.7 ± 2.0%; P = 0.03; and 6.42 ± 0.75 vs 5.41 ± 0.64 mmol/l; P < 0.01). The density of perfused vessels with a diameter <25 µm (i.e. perfused vessel density) decreased slightly in the conventional extracorporeal circulation-treated group from 16.4 ± 3.8 to 12.8 ± 3.3 mm/mm(2) (P < 0.01) and remained stable in the miniaturized extracorporeal circulation-treated group (16.3 ± 2.7 and 15.2 ± 2.9 mm/mm(2) before and during the pump, respectively). Plasma neutrophil gelatinase-associated lipocalin levels were increased following the use of extracorporeal circulation in both groups, and no differences were observed between the groups. Plasma creatinine levels and creatinine clearance were not affected by CABG surgery or CPB. CONCLUSIONS The results from this relatively small study suggest that the use of the miniaturized extracorporeal circulation system is associated with a statistically significant (but clinically insignificant) reduction in haemodilution and microcirculatory hypoperfusion compared with the use of the conventional extracorporeal circulation system.

Plasma levels of endothelial and B-cell-derived microparticles are restored by fingolimod treatment in multiple sclerosis patients.

PubMed

Zinger, Anna; Latham, Sharissa L; Combes, Valery; Byrne, Scott; Barnett, Michael H; Hawke, Simon; Grau, Georges E

2016-12-01

No molecular marker can monitor disease progression and treatment efficacy in multiple sclerosis (MS). Circulating microparticles represent a potential snapshot of disease activity at the blood brain barrier. To profile plasma microparticles by flow cytometry in MS and determine how fingolimod could impact endothelial microparticles production. In non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticle release in vitro. Fingolimod restored dysregulated endothelial and B-cell-microparticle numbers, which could serve as a biomarker in MS. © The Author(s), 2016.

Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer

PubMed Central

Lococo, Filippo; Paci, Massimiliano; Rapicetta, Cristian; Rossi, Teresa; Sancisi, Valentina; Braglia, Luca; Cavuto, Silvio; Bisagni, Alessandra; Bongarzone, Italia; Noonan, Douglas M.; Albini, Adriana; Maramotti, Sally

2015-01-01

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies. PMID:26295387

The role of ctDNA detection and the potential of the liquid biopsy for breast cancer monitoring.

PubMed

Openshaw, Mark Robert; Page, Karen; Fernandez-Garcia, Daniel; Guttery, David; Shaw, Jacqueline Amanda

2016-07-01

Recent advances in deep amplicon sequencing have enabled rapid assessment of somatic mutations and structural changes in multiple cancer genes in DNA isolated from tumour tissues and circulating cell-free DNA (cfDNA). This cfDNA is under investigation as a 'liquid biopsy' for the real time monitoring of patients with cancer in a growing number of research studies and clinical trials. Here we will provide a brief overview of the potential clinical utility of cfDNA profiling for detection and monitoring of patients with breast cancer. The review was conducted in English using PubMed and search terms including 'breast cancer', 'plasma DNA', 'circulating cell free DNA' and 'circulating tumour DNA'. Expert commentary: Liquid biopsies through circulating tumor DNA (ctDNA) enable monitoring of patients with breast cancer. The challenge ahead will be to incorporate cfDNA mutation profiling into routine clinical practice to provide patients with the most appropriate and timely treatment.

Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies.

PubMed

Lambert-Messerlian, Geralyn; Kloza, Edward M; Williams, John; Loucky, Jaroslav; O'Brien, Barbara; Wilkins-Haug, Louise; Mahoney, Maurice J; De Biasio, Pierangela; Borrell, Antoni; Ehrich, Mathias; van den Boom, Dirk; Bombard, Allan T; Deciu, Cosmin; Palomaki, Glenn E

2014-05-01

We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies. Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis. Analyses were restricted to 632 euploid (5.2% assisted reproductive technologies) and 73 Down syndrome (13.7% assisted reproductive technologies), including 16 twin pregnancies. No differences were found for fetal or total circulating cell-free DNA, or for the fetal fraction in euploid (P = 0.70) or Down syndrome (P = 0.58) pregnancies by method of conception. There appeared to be systematic z-score reductions for chromosomes 21, 18, and 13 in assisted reproductive technologies versus natural euploid pregnancies (P = 0.048, 0.0032, and 0.36, respectively). Assisted reproductive technologies and naturally conceived pregnancies contribute similar levels of circulating cell-free DNA into maternal circulation. Small differences in the z-scores of pregnancies achieved by assisted reproductive technologies were observed and do not appear to be test-related artifacts. However, the findings need confirmation before any consideration of changes to testing and reporting protocols.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation.

PubMed

Banz, Yara; Beldi, Guido; Wu, Yan; Atkinson, Ben; Usheva, Anny; Robson, Simon C

2008-08-01

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Clinical Significance of PD-L1+ Exosomes in Plasma of Head and Neck Cancer Patients.

PubMed

Theodoraki, Marie-Nicole; Yerneni, Saigopalakrishna S; Hoffmann, Thomas K; Gooding, William E; Whiteside, Theresa L

2018-02-15

Purpose: The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1 + exosomes to immune suppression and disease activity are evaluated. Experimental Design: Exosomes isolated from plasma of 40 HNSCC patients by size exclusion chromatography were captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. The percentages and mean fluorescence intensities (MFI) of PD-L1 + and PD-1 + exosome/bead complexes were correlated with the patients' clinicopathologic data. PD-L1 high or PD-L1 low exosomes were incubated with activated CD69 + human CD8 + T cells ± PD-1 inhibitor. Changes in CD69 expression levels on T cells were measured. Patients' plasma was tested for soluble PD-L1 (sPD-L1) by ELISA. Results: Levels of PD-L1 carried by exosomes correlated with patients' disease activity, the UICC stage and the lymph node status ( P = 0.0008-0.013). In contrast, plasma levels of sPD-L1 or exosome PD-1 levels did not correlate with any clinicopathologic parameters. CD69 expression levels were inhibited ( P < 0.03) by coincubation with PD-L1 high but not by PD-L1 low exosomes. Blocking of PD-L1 + exosome signaling to PD-1 + T cells attenuated immune suppression. Conclusions: PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1 + exosomes emerge as useful metrics of disease and immune activity in HNSCC patients. Circulating PD-L1 high exosomes in HNC patients' plasma but not soluble PD-L1 levels associate with disease progression. Clin Cancer Res; 24(4); 896-905. ©2017 AACR . ©2017 American Association for Cancer Research.

Immune and endocrine responses of adult spring Chinook salmon during freshwater migration and sexual maturation

USGS Publications Warehouse

Maule, A.G.; Schrock, R.M.; Slater, C.; Fitzpatrick, M.S.; Schreck, C. B.

1996-01-01

The immune –endocrine responses in spring chinook salmon (Oncorhynchus tshawytscha) were examined during their freshwater migration and final maturation. In 1990, migrating fish had high plasma cortisol titres (means 200 ng ml−1) and generated relatively few antibody-producing cells (APC) from peripheral blood leukocytes (PBL) (100 –200 per culture). After three weeks acclimation in constant environmental conditions, plasma cortisol was reduced and APC increased. There were no changes in number or affinity of glucocorticoid receptors. Concentrations of several sex steroids correlated with APC in females, but there were no such correlations in males. In 1993, fish in a hatchery had significantly greater cortisol concentrations in primary circulation than in secondary circulation, but sex steroid concentrations did not differ between circulations. Mean lysozyme activity in the primary and secondary circulation did not differ in June. In August, activity in the primary circulation was significantly less than that of the secondary, perhaps the result of acute stress associated with sampling. While some sex steroids correlated with lysozyme activity, the fact that in both years all endocrine and immune variables that correlated with each other also correlated with the date of sample, raises the question as to whether or not these are cause-and-effect relations.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schock, Sarah C.; Edrissi, Hamidreza; Burger, Dylan

Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPsmore » were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.« less

Absence of cell surface expression of human ACE leads to perinatal death

PubMed Central

Michaud, Annie; Acharya, K. Ravi; Masuyer, Geoffrey; Quenech'du, Nicole; Gribouval, Olivier; Morinière, Vincent; Gubler, Marie-Claire; Corvol, Pierre

2014-01-01

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges). PMID:24163131

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume

PubMed Central

Proskurnin, Mikhail A.; Zhidkova, Tatyana V.; Volkov, Dmitry S.; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I.; Mock, Donald; Zharov, Vladimir P.

2011-01-01

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (ICG, MB, and TB) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including CV and BG were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is, safe for human, and its applications for studying the liver function are further highlighted. PMID:21905207

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

PubMed

van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

2017-10-01

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

PubMed

Laget, Sophie; Broncy, Lucile; Hormigos, Katia; Dhingra, Dalia M; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Renal ammonia metabolism and transport.

PubMed

Weiner, I David; Verlander, Jill W

2013-01-01

Renal ammonia metabolism and transport mediates a central role in acid-base homeostasis. In contrast to most renal solutes, the majority of renal ammonia excretion derives from intrarenal production, not from glomerular filtration. Renal ammoniagenesis predominantly results from glutamine metabolism, which produces 2 NH4(+) and 2 HCO3(-) for each glutamine metabolized. The proximal tubule is the primary site for ammoniagenesis, but there is evidence for ammoniagenesis by most renal epithelial cells. Ammonia produced in the kidney is either excreted into the urine or returned to the systemic circulation through the renal veins. Ammonia excreted in the urine promotes acid excretion; ammonia returned to the systemic circulation is metabolized in the liver in a HCO3(-)-consuming process, resulting in no net benefit to acid-base homeostasis. Highly regulated ammonia transport by renal epithelial cells determines the proportion of ammonia excreted in the urine versus returned to the systemic circulation. The traditional paradigm of ammonia transport involving passive NH3 diffusion, protonation in the lumen and NH4(+) trapping due to an inability to cross plasma membranes is being replaced by the recognition of limited plasma membrane NH3 permeability in combination with the presence of specific NH3-transporting and NH4(+)-transporting proteins in specific renal epithelial cells. Ammonia production and transport are regulated by a variety of factors, including extracellular pH and K(+), and by several hormones, such as mineralocorticoids, glucocorticoids and angiotensin II. This coordinated process of regulated ammonia production and transport is critical for the effective maintenance of acid-base homeostasis.

Circulating endothelial progenitor cells, Th1/Th2/Th17-related cytokines, and endothelial dysfunction in resistant hypertension.

PubMed

Magen, Eli; Feldman, Arie; Cohen, Ziona; Alon, Dora Ben; Minz, Evegeny; Chernyavsky, Alexey; Linov, Lina; Mishal, Joseph; Schlezinger, Menacham; Sthoeger, Zev

2010-02-01

A possible link between chronic vascular inflammation and arterial hypertension is now an object of intensive studies. To compare Th1/Th2/Th17 cells-related cytokines, circulating endothelial progenitor cells (EPC), and endothelial function in subjects with resistant arterial hypertension (RAH) and controlled arterial hypertension (CAH). Blood pressure was measured by electronic sphygmomanometer. EPC were identified as CD34+/CD133+/kinase insert domain receptor (KDR)+ cells by flow cytometry. Th1/Th2/Th17 cells-related cytokines were identified using the Human Th1/Th2/Th17 Cytokines MultiAnalyte ELISArray Kit. Endothelium-dependent (FMD) vasodilatation of brachial artery was measured by Doppler ultrasound scanning. RAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, lower blood levels of EPC number (42.4 +/- 16.7 cells/mL) and EPC% (0.19 +/- 0.08%) were observed than in the CAH group (93.1 +/- 88.7 cells/mL; P = 0.017; 0.27 +/- 0.17; P = 0.036) and control group (68.5 +/- 63.6 cells/mL; P < 0.001; 0.28 +/- 0.17%; P = 0.003), respectively. Plasma transforming growth factor-beta1 levels were significantly higher in the RAH group (1767 +/- 364 pg/mL) than in the CAH group (1292 +/- 349; P < 0.001) and in control group (1203 +/- 419 pg/mL; P < 0.001). In the RAH group, statistically significant negative correlation was observed between systolic blood pressure and EPC% (r = -0.72, P < 0.01). FMD in the RAH group was significantly lower (5.5 +/- 0.8%) than in the CAH group (9.2 +/- 1.4; P < 0.001) and in healthy controls (10.1 +/- 1.1%; P < 0.001). RAH is characterized by reduced circulating EPC, substantial endothelial dysfunction, and increased plasma transforming growth factor-beta1 levels.

Estradiol selectively enhances auditory function in avian forebrain neurons

PubMed Central

Caras, Melissa L.; O’Brien, Matthew; Brenowitz, Eliot A.; Rubel, Edwin W

2012-01-01

Sex steroids modulate vertebrate sensory processing, but the impact of circulating hormone levels on forebrain function remains unclear. We tested the hypothesis that circulating sex steroids modulate single-unit responses in the avian telencephalic auditory nucleus, field L. We mimicked breeding or non-breeding conditions by manipulating plasma 17β-estradiol levels in wild-caught female Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii). Extracellular responses of single neurons to tones and conspecific songs presented over a range of intensities revealed that estradiol selectively enhanced auditory function in cells that exhibited monotonic rate-level functions to pure tones. In these cells, estradiol treatment increased spontaneous and maximum evoked firing rates, increased pure tone response strengths and sensitivity, and expanded the range of intensities over which conspecific song stimuli elicited significant responses. Estradiol did not significantly alter the sensitivity or dynamic ranges of cells that exhibited non-monotonic rate-level functions. Notably, there was a robust correlation between plasma estradiol concentrations in individual birds and physiological response properties in monotonic, but not non-monotonic neurons. These findings demonstrate that functionally distinct classes of anatomically overlapping forebrain neurons are differentially regulated by sex steroid hormones in a dose-dependent manner. PMID:23223283

Associations of aldosterone and renin concentrations with inflammation-the Study of Health in Pomerania and the German Conn's Registry.

PubMed

Grotevendt, A; Wallaschofski, H; Reincke, M; Adolf, C; Quinkler, M; Nauck, M; Hoffmann, W; Rettig, R; Hannemann, A

2017-08-01

Chronic inflammation is an age-independent and body mass index-independent contributor to the development of multi-morbidity. Alterations of the renin-angiotensin-aldosterone system are observed within the context of proinflammatory states. We assessed circulating aldosterone, renin, and inflammatory biomarker concentrations in healthy, normotensive subjects and patients with primary aldosteronism. We included 1177 normotensive individuals from the population-based Study of Health in Pomerania (first follow-up, Study of Health in Pomerania-1) and 103 primary aldosteronism patients from the German Conn's Registry. A 1:1 matching for sex, age, body mass index, smoking status, diabetes mellitus, and the estimated glomerular filtration rate was performed to determine whether primary aldosteronism patients exhibit higher inflammatory biomarker concentrations than normotensive controls. The associations of plasma aldosterone concentration or plasma renin concentration with circulating fibrinogen concentrations, white blood cell count, and high sensitive C-reactive protein concentrations in the normotensive sample were determined with multivariable linear and logistic regression analyses. 1:1 matched primary aldosteronism patients demonstrated significantly (p < 0.01) higher plasma aldosterone concentration (198 vs. 47 ng/l), lower plasma renin concentration (3.1 vs. 7.7 ng/l) and higher high sensitive C-reactive protein concentrations (1.5 vs. 1.0 mg/l) than normotensive controls. Within the normotensive cohort, plasma renin concentration but not plasma aldosterone concentration was positively associated with fibrinogen concentrations and white blood cell count. Further, a J-shaped association between plasma renin concentration and high sensitive C-reactive protein concentrations was detected. High plasma aldosterone concentration in a primary aldosteronism cohort and high plasma renin concentration in normotensive subjects are associated with increased concentrations of inflammatory biomarkers. This suggests a link between the renin-angiotensin-aldosterone system and inflammatory processes in patients with primary aldosteronism and even in normotensive subjects.

Characterization of CD4+ T cell-mediated cytotoxicity in patients with multiple myeloma.

PubMed

Zhang, Xiaole; Gao, Lei; Meng, Kai; Han, Chunting; Li, Qiang; Feng, Zhenjun; Chen, Lei

2018-05-01

Multiple myeloma (MM) is an incurable cancer characterized by the development of malignant plasma cells. The CD8 T cell-mediated cytotoxicity is considered a major player in antitumor immunity, but in MM patients, the CD8 T cells displayed senescence markers and were functionally impaired. To investigate whether cytotoxic CD4 T cells could act as a treatment alternative in MM, we examined the frequency and function of naturally occurring cytotoxic CD4 T cells in MM patients. The cytotoxic CD4 T cells were identified as granzyme-A, granzyme B-, and perforin-expressing CD4 T cells, and their frequencies were significantly upregulated in MM patients when compared with healthy controls. The frequencies of cytotoxic CD4 T cells in MM patients were not associated with the frequencies of cytotoxic CD8 T cells, but were negatively associated with disease severity. Interestingly, the expression levels of inhibitory molecules, including PD-1 and CTLA-4, were significantly lower in cytotoxic CD4 T cells than in cytotoxic CD8 T cells. When co-incubated with autologous CD38 + CD138 + plasma cells, CD4 T cells were capable of eliminating plasma cells with varying degrees of efficacy. In MM patients, the frequency of circulating plasma cells was negatively correlated with the frequency of cytotoxic CD4 T cells. Therefore, CD4 T cell-mediated cytotoxicity existed naturally in MM patients and could potentially act as an option in antitumor therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

The effects of conventional extracorporeal circulation versus miniaturized extracorporeal circulation on microcirculation during cardiopulmonary bypass-assisted coronary artery bypass graft surgery

PubMed Central

Yuruk, Koray; Bezemer, Rick; Euser, Mariska; Milstein, Dan M.J.; de Geus, Hilde H.R.; Scholten, Evert W.; de Mol, Bas A.J.M.; Ince, Can

2012-01-01

OBJECTIVES To reduce the complications associated with cardiopulmonary bypass (CPB) during cardiac surgery, many modifications have been made to conventional extracorporeal circulation systems. This trend has led to the development of miniaturized extracorporeal circulation systems. Cardiac surgery using conventional extracorporeal circulation systems has been associated with significantly reduced microcirculatory perfusion, but it remains unknown whether this could be prevented by an mECC system. Here, we aimed to test the hypothesis that microcirculatory perfusion decreases with the use of a conventional extracorporeal circulation system and would be preserved with the use of an miniaturized extracorporeal circulation system. METHODS Microcirculatory density and perfusion were assessed using sublingual side stream dark-field imaging in patients undergoing on-pump coronary artery bypass graft (CABG) surgery before, during and after the use of either a conventional extracorporeal circulation system (n = 10) or a miniaturized extracorporeal circulation system (n = 10). In addition, plasma neutrophil gelatinase-associated lipocalin and creatinine levels and creatinine clearance were assessed up to 5 days post-surgery to monitor renal function. RESULTS At the end of the CPB, one patient in the miniaturized extracorporeal circulation-treated group and five patients in the conventional extracorporeal circulation-treated group received one bag of packed red blood cells (300 ml). During the CPB, the haematocrit and haemoglobin levels were slightly higher in the miniaturized extracorporeal circulation-treated patients compared with the conventional extracorporeal circulation-treated patients (27.7 ± 3.3 vs 24.7 ± 2.0%; P = 0.03; and 6.42 ± 0.75 vs 5.41 ± 0.64 mmol/l; P < 0.01). The density of perfused vessels with a diameter <25 µm (i.e. perfused vessel density) decreased slightly in the conventional extracorporeal circulation-treated group from 16.4 ± 3.8 to 12.8 ± 3.3 mm/mm2 (P < 0.01) and remained stable in the miniaturized extracorporeal circulation-treated group (16.3 ± 2.7 and 15.2 ± 2.9 mm/mm2 before and during the pump, respectively). Plasma neutrophil gelatinase-associated lipocalin levels were increased following the use of extracorporeal circulation in both groups, and no differences were observed between the groups. Plasma creatinine levels and creatinine clearance were not affected by CABG surgery or CPB. CONCLUSIONS The results from this relatively small study suggest that the use of the miniaturized extracorporeal circulation system is associated with a statistically significant (but clinically insignificant) reduction in haemodilution and microcirculatory hypoperfusion compared with the use of the conventional extracorporeal circulation system. PMID:22700685

ESR1 Methylation: A Liquid Biopsy-Based Epigenetic Assay for the Follow-up of Patients with Metastatic Breast Cancer Receiving Endocrine Treatment.

PubMed

Mastoraki, Sophia; Strati, Areti; Tzanikou, Eleni; Chimonidou, Maria; Politaki, Eleni; Voutsina, Alexandra; Psyrri, Amanda; Georgoulias, Vassilis; Lianidou, Evi

2018-03-15

Purpose: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Experimental Design: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in (i) 65 primary breast tumors formalin-fixed paraffin-embedded (FFPE), (ii) EpCAM + CTC fractions (122 patients and 30 healthy donors; HD), (iii) plasma ctDNA (108 patients and 30HD), and (iv) in CTCs (CellSearch) and in paired plasma ctDNA for 58 patients with breast cancer. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER + /HER2 - advanced breast cancer receiving everolimus/exemestane. Results: ESR1 methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) EpCAM + CTC fractions : 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) plasma ctDNA: 8/108 (7.4%) patients and 1/30 (3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment ( P = 0.023, Fisher exact test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. Clin Cancer Res; 24(6); 1500-10. ©2017 AACR . ©2017 American Association for Cancer Research.

Identification and analysis of circulating exosomal microRNA in human body fluids.

PubMed

Lässer, Cecilia

2013-01-01

Exosomes are 40-100 nm sized vesicles released from cells when multivesicular bodies fuse with the plasma membrane. These vesicles take part in cell-to-cell communication by binding and signalling through membrane receptors on cells or by transferring proteins, RNA, and lipids into the cells. Exosomal RNA in body fluids, such as plasma and urine, has been associated with malignancies, making the exosomal RNA a potential biomarker for early detection of these diseases. This has increased the interest in the field of extracellular RNA and in particular, the interest in exosomal RNA.In this chapter, a well-established exosome isolation method is described, as well as how to characterize the isolated vesicles by electron microscopy. Furthermore, two types of RNA isolation methods are described with a focus on isolating RNA from body fluids, which can be more viscous than cell culture media.

Public speaking stress-induced neuroendocrine responses and circulating immune cell redistribution in irritable bowel syndrome.

PubMed

Elsenbruch, Sigrid; Lucas, Ayscha; Holtmann, Gerald; Haag, Sebastian; Gerken, Guido; Riemenschneider, Natalie; Langhorst, Jost; Kavelaars, Annemieke; Heijnen, Cobi J; Schedlowski, Manfred

2006-10-01

Augmented neuroendocrine stress responses and altered immune functions may play a role in the manifestation of functional gastrointestinal (GI) disorders. We tested the hypothesis that IBS patients would demonstrate enhanced psychological and endocrine responses, as well as altered stress-induced redistribution of circulating leukocytes and lymphocytes, in response to an acute psychosocial stressor when compared with healthy controls. Responses to public speaking stress were analyzed in N = 17 IBS patients without concurrent psychiatric conditions and N = 12 healthy controls. At baseline, immediately following public speaking, and after a recovery period, state anxiety, acute GI symptoms, cardiovascular responses, serum cortisol and plasma adrenocorticotropic hormone (ACTH) were measured, and numbers of circulating leukocytes and lymphocyte subpopulations were analyzed by flow cytometry. Public speaking led to significant cardiovascular activation, a significant increase in ACTH, and a redistribution of circulating leukocytes and lymphocyte subpopulations, including significant increases in natural killer cells and cytotoxic/suppressor T cells. IBS patients demonstrated significantly greater state anxiety both at baseline and following public speaking. However, cardiovascular and endocrine responses, as well as the redistribution of circulating leukocytes and lymphocyte subpopulations after public speaking stress, did not differ for IBS patients compared with controls. In IBS patients without psychiatric comorbidity, the endocrine response as well as the circulation pattern of leukocyte subpopulations to acute psychosocial stress do not differ from healthy controls in spite of enhanced emotional responses. Future studies should discern the role of psychopathology in psychological and biological stress responses in IBS.

Circulating vascular cell adhesion molecule-1 in pre-eclampsia, gestational hypertension, and normal pregnancy: evidence of selective dysregulation of vascular cell adhesion molecule-1 homeostasis in pre-eclampsia.

PubMed

Higgins, J R; Papayianni, A; Brady, H R; Darling, M R; Walshe, J J

1998-08-01

Our purpose was to investigate circulating levels of vascular cell adhesion molecule-1 in the peripheral and uteroplacental circulations during normotensive and hypertensive pregnancies. This prospective observational study involved 2 patient groups. Group 1 consisted of 22 women with pre-eclampsia and 30 normotensive women followed up longitudinally through pregnancy and post partum. There were an additional 13 women with established gestational hypertension. Group 2 consisted of 20 women with established pre-eclampsia and 19 normotensive control subjects undergoing cesarean delivery. Plasma levels of vascular cell adhesion molecule-1 were measured in blood drawn from the antecubital vein (group 1) and from both the antecubital and uterine veins (group 2). Data were analyzed by analysis of variance. In group 1 vascular cell adhesion molecule-1 levels did not change significantly throughout normal pregnancy and post partum. Women with established pre-eclampsia had increased vascular cell adhesion molecule-1 levels compared with the normotensive pregnancy group (P = .01). Vascular cell adhesion molecule-1 levels were not elevated in women with established gestational hypertension. In group 2 significantly higher levels of vascular cell adhesion molecule-1 were detected in the uteroplacental (P < .0001) and peripheral (P < .0001) circulations of pre-eclamptic women by comparison with normotensive women. In the pre-eclamptic group there was a tendency toward higher vascular cell adhesion molecule-1 levels in the peripheral circulation than in the uteroplacental circulation (P = .06). In contrast to vascular cell adhesion molecule-1, circulating levels of E-selectin and intercellular adhesion molecule-1, other major leukocyte adhesion molecules expressed by the endothelium, were not different in pre-eclamptic and normotensive pregnancies. Established pre-eclampsia is characterized by selective dysregulation of vascular cell adhesion molecule-1 homeostasis. This event is not an early preclinical feature of pre-eclampsia, does not persist post partum, is not a feature of nonproteinuric gestational hypertension, and is not observed with other major leukocyte adhesion molecules. Induction of vascular cell adhesion molecule-1 expression in pre-eclampsia may contribute to leukocyte-mediated tissue injury in this condition or may reflect perturbation of other, previously unrecognized, functions of this molecule in pregnancy.

Origin and Function of Circulating Plasmablasts during Acute Viral Infections.

PubMed

Fink, Katja

2012-01-01

Activated B cells proliferate and differentiate into antibody-producing cells, long-lived plasma cells, and memory B cells after immunization or infection. Repeated encounter of the same antigen triggers the rapid re-activation of pre-existing specific memory B cells, which then potentially enter new germinal center reactions and differentiate into short-lived plasmablasts or remain in the system as memory B cells. Short-lived class-switched IgG and IgA plasmablasts appear in the circulation transiently and the frequency of these cells can be remarkably high. The specificities and affinities of single plasmablasts in humans have been reported for several viral infections, so far most extensively for influenza and HIV. In general, the immunoglobulin variable regions of plasmablasts are highly mutated and diverse, suggesting that plasmablasts are derived from memory B cells, yet it is unclear which memory B cell subsets are activated and whether activated memory B cells adapt or mature before differentiation. This review summarizes what is known about the phenotype and the origin of human plasmablasts in the context of viral infections and whether these cells can be predictors of long-lived immunity.

Insights Into Circulating Cytokine Dynamics during Pregnancy in HIV-Infected Beninese Exposed to Plasmodium falciparum Malaria

PubMed Central

Ibitokou, Samad A.; Denoeud-Ndam, Lise; Ezinmegnon, Sèm; Ladékpo, Rodolphe; Zannou, Djimon-Marcel; Massougbodji, Achille; Girard, Pierre-Marie; Cot, Michel; Luty, Adrian J. F.; Ndam, Nicaise Tuikue

2015-01-01

We investigated the circulating plasma levels of Th1- (Interleukin-2 [IL-2], tumor necrosis factor-α [TNF-α], interferon-gamma [IFN-γ]) and Th2-type (IL-4, IL-5, IL-10) cytokines in human immunodeficiency virus (HIV)-infected pregnant women living in a malaria-endemic area. We analyzed samples from 200 pregnant women included in the prevention of pregnancy-associated malaria in HIV-infected women: cotrimoxazole prophylaxis versus mefloquine (PACOME) clinical trial who were followed until delivery. Cytokine concentrations were measured by flow cytometry-based multiplex bead array. Significantly elevated levels of IL-10 and lower levels of TNF-α were observed at delivery compared with inclusion (P = 0.005). At inclusion, the presence of circulating IFN-γ, a higher CD4+ T cell count and having initiated intermittent preventive treatment of malaria with sulfadoxine pyrimethamine (SP-IPTp) were all associated with a lower likelihood of Plasmodium falciparum infection. At delivery, the inverse relationship between the presence of infection and circulating IFN-γ persisted, although there was a positive association between the likelihood of infection and the presence of circulating TNF-α. Initiation of antiretroviral therapy was associated with elevated IL-5 production. Consistent with our own and others' observations in HIV seronegative subjects, this study shows circulating IL-10 to be a marker of infection with P. falciparum during pregnancy even in HIV-infected women, although plasma IFN-γ may be a marker of anti-malarial protection in such women. PMID:26101276

Detection of melanoma cells suspended in mononuclear cells and blood plasma using photoacoustic generation

NASA Astrophysics Data System (ADS)

Spradling, Emily M.; Viator, John A.

2009-02-01

Melanoma is the deadliest form of skin cancer. Although the initial malignant cells are removed, it is impossible to determine whether or not the cancer has metastasized until a secondary tumor forms that is large enough to detect with conventional imaging. Photoacoustic detection of circulating melanoma cells in the bloodstream has shown promise for early detection of metastasis that may aid in treatment of this aggressive cancer. When blood is irradiated with energy from an Nd:YAG laser at 532 nm, photoacoustic signals are created and melanoma cells can be differentiated from the surrounding cells based on waveforms produced by an oscilloscope. Before this can be used as a diagnostic technique, however, we needed to investigate several parameters. Specifically, the current technique involves the in vitro separation of blood through centrifugation to isolate and test only the white blood cell layer. Using this method, we have detected a single cultured melanoma cell among a suspension of white blood cells. However, the process could be made simpler if the plasma layer were used for detection instead of the white blood cell layer. This layer is easier to obtain after blood separation, the optical difference between plasma and melanoma cells is more pronounced in this layer than in the white blood cell layer, and the possibility that any stray red blood cells could distort the results is eliminated. Using the photoacoustic apparatus, we detected no melanoma cells within the plasma of whole blood samples spiked with cultured melanoma cells.

Diagnostic Value of Circulating CXC Chemokines in Non-small Cell Lung Cancer.

PubMed

Spaks, Artjoms; Jaunalksne, Inta; Spaka, Irina; Chudasama, Dimple; Pirtnieks, Ainis; Krievins, Dainis

2015-12-01

To evaluate the diagnostic value of circulating CXC chemokines as biomarkers for non-small cell lung cancer and compare them against a standard panel of already existing cancer biomarkers. A total of 90 individuals were enrolled in the study. We analyzed 30 patients with stage IA-IIB carcinoma of the lung who underwent pulmonary resection, 30 patients with metastatic NSCLC, and 30 healthy volunteers. The biomarkers levels were measured in plasma blood samples, by ELISA and immunoassays. The levels of circulating CXCL4, CXCL8, CXCL9, CXCL10 and CXCL11 were higher and those of circulating CXCL1 were lower in patients with early-stage NSCLC compared to metastatic NSCLC patients and controls (p<0.05). CXCL4, CXCL9 and CXCL11 were included in the panel that showed a sensitivity of 100% versus 60% for CEA, CA125 and CYFRA21-1 (p<0.001). Combination of CXCL4, CXCL9 and CXCL11 has a high diagnostic value. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing.

PubMed

Wong, David; Moturi, Sharmili; Angkachatchai, Vach; Mueller, Reinhold; DeSantis, Grace; van den Boom, Dirk; Ehrich, Mathias

2013-08-01

Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE). These BCTs were designed to minimize cellular degradation, and thus effectively prevent dilution of fetal ccf DNA by maternal genomic DNA, was evaluated. Peripheral venous maternal blood was collected into Streck BCTs to investigate four aspects of handling and processing conditions: (1) time from blood draw to plasma processing; (2) storage temperature; (3) mechanical stress; and (4) lot-to-lot tube variations. Maternal blood stored in Streck BCTs for up to 7 days at ambient temperature provides stable concentrations of ccffDNA. The amount of fetal DNA did not change over a broad range of storage temperatures (4°C, 23°C, 37°C, 40°C), but the amount of total (largely maternal) DNA increased in samples stored at 23°C and above, indicating maternal cell degradation and genomic DNA release at elevated temperatures. Shipping maternal blood in Streck BCTs, did not affect sample quality. Maternal plasma DNA stabilized for 0 to 7 days in Streck BCTs can be used for non-invasive prenatal molecular applications, when temperatures are maintained within the broad parameters assessed in this study. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Immunoglobulins against Tyrosine Nitrated Epitopes in Coronary Artery Disease

PubMed Central

Thomson, Leonor; Tenopoulou, Margarita; Lightfoot, Richard; Tsika, Epida; Parastatidis, Ioannis; Martinez, Marissa; Greco, Todd M.; Doulias, Paschalis-Thomas; Wu, Yuping; Tang, W. H. Wilson; Hazen, Stanley L.; Ischiropoulos, Harry

2012-01-01

Background Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine nitrated proteins, a footprint of oxygen and nitrogen derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of coronary artery disease (CAD) subjects. However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of CAD subjects. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in CAD patients (n=374) and subjects without CAD (non CAD controls, n=313). A ten-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in the CAD subjects (3.75 ± 1.8 μg antibody Eq/mL plasma vs. 0.36 ± 0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions The results of this cross sectional study suggest that post-translational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neoepitopes for elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD. PMID:23081989

Detection of circulating Bmi-1 mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancer.

PubMed

Zhang, Xin; Wang, Chuanxin; Wang, Lili; Du, Lutao; Wang, Shun; Zheng, Guixi; Li, Wei; Zhuang, Xuewei; Zhang, Xuhua; Dong, Zhaogang

2012-07-01

Bmi-1 is overexpressed in uterine cervical cancer (UCC) and is found to be associated with adverse clinical characteristics and poor prognosis. However, little information is available on the status of circulating Bmi-1 mRNA in UCC. Because circulating cell-free nucleic acids have emerged as a novel class of markers for cancer detection, our research aims to address this question by detecting the circulating Bmi-1 mRNA and to assess its diagnostic and prognostic potential in UCC. Reverse transcription quantitative real-time PCR method was established to detect the circulating Bmi-1 mRNA in plasma of 109 patients with UCC, 138 patients with cervical intraepithelial neoplasia (CIN) and 80 healthy volunteers, and found that it was significantly increased in UCC compared with CINs and healthy controls (all at p < 0.001). Moreover, its high level was significantly correlated with advanced clinical stage (p < 0.001) and positive lymph nodes metastasis (p = 0.002). The area under the receiver operating characteristic curve (AUC) was 0.881, and the optimal cut-off value was 0.057, providing a sensitivity of 69.7% and a specificity of 95.9%. The AUC for circulating Bmi-1 mRNA showed higher diagnosis capability than that for SCC-Ag (p = 0.035) or CA125 (p < 0.001) currently utilized. Kaplan-Meier analysis demonstrated a correlation between increased circulating Bmi-1 mRNA level and reduced disease-free survival (DFS) (p = 0.001) and overall survival (OS) (p = 0.015). And, Cox analysis indicated that it was an independent prognostic factor for DFS and OS. We conclude that circulating Bmi-1 mRNA may be a potential noninvasive molecular marker for diagnosis and prognosis of UCC. Copyright © 2011 UICC.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

PubMed

Proskurnin, Mikhail A; Zhidkova, Tatyana V; Volkov, Dmitry S; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Mock, Donald; Nedosekin, Dmitry A; Zharov, Vladimir P

2011-10-01

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

PubMed

Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

2004-12-01

In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

The Effect of Pulsatile Versus Nonpulsatile Blood Flow on Viscoelasticity and Red Blood Cell Aggregation in Extracorporeal Circulation

PubMed Central

Ahn, Chi Bum; Kang, Yang Jun; Kim, Myoung Gon; Yang, Sung; Lim, Choon Hak; Son, Ho Sung; Kim, Ji Sung; Lee, So Young; Son, Kuk Hui; Sun, Kyung

2016-01-01

Background Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Methods Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was performed before and at 1, 2, and 3 hours after ECC commencement. To eliminate bias induced by hematocrit and plasma, all blood samples were adjusted to a hematocrit of 45% using baseline plasma. Blood viscoelasticity, plasma viscosity, hematocrit, arterial blood gas analysis, central venous O2 saturation, and lactate were measured. Results The blood viscosity and aggregation index decreased abruptly 1 hour after ECC and then remained low during ECC in both groups, but blood elasticity did not change during ECC. Blood viscosity, blood elasticity, plasma viscosity, and the aggregation index were not significantly different in the groups at any time. Hematocrit decreased abruptly 1 hour after ECC in both groups due to dilution by the priming solution used. Conclusion After ECC, blood viscoelasticity and RBC aggregation were not different in the pulsatile and nonpulsatile groups in the adult dog model. Furthermore, pulsatile flow did not have a more harmful effect on blood viscoelasticity or RBC aggregation than nonpulsatile flow. PMID:27298790

The Effect of Pulsatile Versus Nonpulsatile Blood Flow on Viscoelasticity and Red Blood Cell Aggregation in Extracorporeal Circulation.

PubMed

Ahn, Chi Bum; Kang, Yang Jun; Kim, Myoung Gon; Yang, Sung; Lim, Choon Hak; Son, Ho Sung; Kim, Ji Sung; Lee, So Young; Son, Kuk Hui; Sun, Kyung

2016-06-01

Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was performed before and at 1, 2, and 3 hours after ECC commencement. To eliminate bias induced by hematocrit and plasma, all blood samples were adjusted to a hematocrit of 45% using baseline plasma. Blood viscoelasticity, plasma viscosity, hematocrit, arterial blood gas analysis, central venous O2 saturation, and lactate were measured. The blood viscosity and aggregation index decreased abruptly 1 hour after ECC and then remained low during ECC in both groups, but blood elasticity did not change during ECC. Blood viscosity, blood elasticity, plasma viscosity, and the aggregation index were not significantly different in the groups at any time. Hematocrit decreased abruptly 1 hour after ECC in both groups due to dilution by the priming solution used. After ECC, blood viscoelasticity and RBC aggregation were not different in the pulsatile and nonpulsatile groups in the adult dog model. Furthermore, pulsatile flow did not have a more harmful effect on blood viscoelasticity or RBC aggregation than nonpulsatile flow.

Circulation Plasma Centrifuge with Product Flow

NASA Astrophysics Data System (ADS)

Borisevich, V. D.; Potanin, E. P.

2018-05-01

We have analyzed the isotope separation in a high-frequency plasma circulating centrifuge operating with a product flow. The rotation of a weakly ionized plasma is ensured by a rotating magnetic field, while the countercurrent flow (circulation) is produced by a traveling magnetic field. We have calculated the dependences of the enrichment factor and the separative power of the centrifuge on a product flow. The optimal characteristics of the separation unit have been determined.

Some potential blood flow experiments for space

NASA Technical Reports Server (NTRS)

Cokelet, G. R.; Meiselman, H. J.; Goldsmith, H. L.

1979-01-01

Blood is a colloidal suspension of cells, predominantly erythrocytes, (red cells) in an aqueous solution called plasma. Because the red cells are more dense than the plasma, and because they tend to aggregate, erythrocyte sedimentation can be significant when the shear stresses in flowing blood are small. This behavior, coupled with equipment restrictions, has prevented certain definitive fluid mechanical studies from being performed with blood in ground-based experiments. Among such experiments, which could be satisfactorily performed in a microgravity environment, are the following: (1) studies of blood flow in small tubes, to obtain pressure-flow rate relationships, to determine if increased red cell aggregation can be an aid to blood circulation, and to determine vessel entrance lengths, and (2) studies of blood flow through vessel junctions (bifurcations), to obtain information on cell distribution in downstream vessels of (arterial) bifurcations, and to test flow models of stratified convergent blood flows downstream from (venous) bifurcations.

Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24h after venipuncture.

PubMed

Buysse, Karen; Beulen, Lean; Gomes, Ingrid; Gilissen, Christian; Keesmaat, Chantal; Janssen, Irene M; Derks-Willemen, Judith J H T; de Ligt, Joep; Feenstra, Ilse; Bekker, Mireille N; van Vugt, John M G; Geurts van Kessel, Ad; Vissers, Lisenka E L M; Faas, Brigitte H W

2013-12-01

Circulating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS). Whole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads. Both T21 samples were correctly identified as such at all time-points. However, after 48h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h. The quality and quantity of ccffDNA extracted from plasma samples processed up to 24h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction. © 2013.

Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.

PubMed

Tang, Xiaoyun; Wang, Xianyan; Zhao, Yuan Y; Curtis, Jonathan M; Brindley, David N

2017-02-08

We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces. In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting. Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1β, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80 + macrophages and CD31 + blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate. These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Kato, Mamoru; Shibata, Tatsuhiro; Yachida, Shinichi

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.

A Gestational Profile of Placental Exosomes in Maternal Plasma and Their Effects on Endothelial Cell Migration

PubMed Central

Salomon, Carlos; Torres, Maria Jose; Kobayashi, Miharu; Scholz-Romero, Katherin; Sobrevia, Luis; Dobierzewska, Aneta; Illanes, Sebastian E.; Mitchell, Murray D.; Rice, Gregory E.

2014-01-01

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6–12 weeks), second (ST, 22–24 weeks) and third (TT, 32–38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction. PMID:24905832

Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing

PubMed Central

Peng, Xianlu Laura; Jiang, Peiyong

2017-01-01

The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons. PMID:28230760

Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing.

PubMed

Peng, Xianlu Laura; Jiang, Peiyong

2017-02-20

The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons.

Effect of heterogeneity of carcinoembryonic antigen on liver cell membrane binding and its kinetics of removal from circulation.

PubMed

Byrn, R A; Medrek, P; Thomas, P; Jeanloz, R W; Zamcheck, N

1985-07-01

Carcinoembryonic antigen (CEA) is a glycoprotein metabolized primarily by the liver. Subcellular fractions of rat liver were examined for CEA binding activity. Hepatocyte plasma membrane and microsome fractions bound CEA, and this binding shared the calcium requirement, neuraminidase sensitivity, and carbohydrate specificity of the hepatocyte asialoglycoprotein receptor. CEA had previously been shown to react with this galactose-specific receptor, in vivo, only following neuraminidase treatment. Galactose receptor binding of CEA was measured in three different purified CEA preparations. The fraction of CEA capable of binding to excess levels of galactose receptor on membranes varied (46.5%, 40.2%, and 4.7% for CEA-1, -2, and -3, respectively). These CEAs were shown to be 2.3%, 7.9%, and 0.7% as effective, respectively, as asialo-alpha 1-acid glycoprotein in inhibiting the binding of radiolabeled asialo-alpha 1-acid glycoprotein to liver cell membranes. Each of the three CEA preparations showed different clearance kinetics from the circulation of mice. Coinjection of asialo-alpha 1-acid glycoprotein with the CEAs revealed differing inhibition of the clearances. These results show that differences in the carbohydrate components of purified CEA preparations affect their rate of removal from circulation and thus possibly the relationship between CEA production and observed plasma levels in patients. The possible origin of these CEA differences is discussed with their clinical implications.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

PubMed

Marlicz, Wojciech; Sielatycka, Katarzyna; Serwin, Karol; Kubis, Ewa; Tkacz, Marta; Głuszko, Rafał; Białek, Andrzej; Starzyńska, Teresa; Ratajczak, Mariusz Z

2016-12-01

Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF‑1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

Circulating platelet aggregates damage endothelial cells in culture.

PubMed

Aluganti Narasimhulu, Chandrakala; Nandave, Mukesh; Bonilla, Diana; Singaravelu, Janani; Sai-Sudhakar, Chittoor B; Parthasarathy, Sampath

2017-06-01

Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). Endothelial cells were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Endogenous circulating sympatholytic factor in orthostatic intolerance

NASA Technical Reports Server (NTRS)

Shapiro, R. E.; Winters, B.; Hales, M.; Barnett, T.; Schwinn, D. A.; Flavahan, N.; Berkowitz, D. E.

2000-01-01

Sympathotonic orthostatic hypotension (SOH) is an idiopathic syndrome characterized by tachycardia, hypotension, elevated plasma norepinephrine, and symptoms of orthostatic intolerance provoked by assumption of an upright posture. We studied a woman with severe progressive SOH with blood pressure unresponsive to the pressor effects of alpha(1)-adrenergic receptor (AR) agonists. We tested the hypothesis that a circulating factor in this patient interferes with vascular adrenergic neurotransmission. Preincubation of porcine pulmonary artery vessel rings with patient plasma produced a dose-dependent inhibition of vasoconstriction to phenylephrine in vitro, abolished vasoconstriction to direct electrical stimulation, and had no effect on nonadrenergic vasoconstrictive stimuli (endothelin-1), PGF-2alpha (or KCl). Preincubation of vessels with control plasma was devoid of these effects. SOH plasma inhibited the binding of an alpha(1)-selective antagonist radioligand ([(125)I]HEAT) to membrane fractions derived from porcine pulmonary artery vessel rings, rat liver, and cell lines selectively overexpressing human ARs of the alpha(1B) subtype but not other AR subtypes (alpha(1A) and alpha(1D)). We conclude that a factor in SOH plasma can selectively and irreversibly inhibit adrenergic ligand binding to alpha(1B) ARs. We propose that this factor contributes to a novel pathogenesis for SOH in this patient. This patient's syndrome represents a new disease entity, and her plasma may provide a unique tool for probing the selective functions of alpha(1)-ARs.

Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR.

PubMed

Conte, Davide; Verri, Carla; Borzi, Cristina; Suatoni, Paola; Pastorino, Ugo; Sozzi, Gabriella; Fortunato, Orazio

2015-10-23

Research efforts for the management of cancer, in particular for lung cancer, are directed to identify new strategies for its early detection. MicroRNAs (miRNAs) are a new promising class of circulating biomarkers for cancer detection, but lack of consensus on data normalization methods has affected the diagnostic potential of circulating miRNAs. There is a growing interest in techniques that allow an absolute quantification of miRNAs which could be useful for early diagnosis. Recently, digital PCR, mainly based on droplets generation, emerged as an affordable technology for precise and absolute quantification of nucleic acids. In this work, we described a new interesting approach for profiling circulating miRNAs in plasma samples using a chip-based platform, the QuantStudio 3D digital PCR. The proposed method was validated using synthethic oligonucleotide at serial dilutions in plasma samples of lung cancer patients and in lung tissues and cell lines. Given its reproducibility and reliability, our approach could be potentially applied for the identification and quantification of miRNAs in other biological samples such as circulating exosomes or protein complexes. As chip-digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number for diagnosis of lung cancer and other diseases.

Renal Ammonia Metabolism and Transport

PubMed Central

Weiner, I. David; Verlander, Jill W.

2015-01-01

Renal ammonia metabolism and transport mediates a central role in acid-base homeostasis. In contrast to most renal solutes, the majority of renal ammonia excretion derives from intrarenal production, not from glomerular filtration. Renal ammoniagenesis predominantly results from glutamine metabolism, which produces 2 NH4+ and 2 HCO3− for each glutamine metabolized. The proximal tubule is the primary site for ammoniagenesis, but there is evidence for ammoniagenesis by most renal epithelial cells. Ammonia produced in the kidney is either excreted into the urine or returned to the systemic circulation through the renal veins. Ammonia excreted in the urine promotes acid excretion; ammonia returned to the systemic circulation is metabolized in the liver in a HCO3−-consuming process, resulting in no net benefit to acid-base homeostasis. Highly regulated ammonia transport by renal epithelial cells determines the proportion of ammonia excreted in the urine versus returned to the systemic circulation. The traditional paradigm of ammonia transport involving passive NH3 diffusion, protonation in the lumen and NH4+ trapping due to an inability to cross plasma membranes is being replaced by the recognition of limited plasma membrane NH3 permeability in combination with the presence of specific NH3-transporting and NH4+-transporting proteins in specific renal epithelial cells. Ammonia production and transport are regulated by a variety of factors, including extracellular pH and K+, and by several hormones, such as mineralocorticoids, glucocorticoids and angiotensin II. This coordinated process of regulated ammonia production and transport is critical for the effective maintenance of acid-base homeostasis. PMID:23720285

Benzene exposure is associated with cardiovascular disease risk.

PubMed

Abplanalp, Wesley; DeJarnett, Natasha; Riggs, Daniel W; Conklin, Daniel J; McCracken, James P; Srivastava, Sanjay; Xie, Zhengzhi; Rai, Shesh; Bhatnagar, Aruni; O'Toole, Timothy E

2017-01-01

Benzene is a ubiquitous, volatile pollutant present at high concentrations in toxins (e.g. tobacco smoke) known to increase cardiovascular disease (CVD) risk. Despite its prevalence, the cardiovascular effects of benzene have rarely been studied. Hence, we examined whether exposure to benzene is associated with increased CVD risk. The effects of benzene exposure in mice were assessed by direct inhalation, while the effects of benzene exposure in humans was assessed in 210 individuals with mild to high CVD risk by measuring urinary levels of the benzene metabolite trans,trans-muconic acid (t,t-MA). Generalized linear models were used to assess the association between benzene exposure and CVD risk. Mice inhaling volatile benzene had significantly reduced levels of circulating angiogenic cells (Flk-1+/Sca-1+) as well as an increased levels of plasma low-density lipoprotein (LDL) compared with control mice breathing filtered air. In the human cohort, urinary levels of t,t-MA were inversely associated several populations of circulating angiogenic cells (CD31+/34+/45+, CD31+/34+/45+/AC133-, CD34+/45+/AC133+). Although t,t-MA was not associated with plasma markers of inflammation or thrombosis, t,t-MA levels were higher in smokers and in individuals with dyslipidemia. In smokers, t,t-MA levels were positively associated with urinary metabolites of nicotine (cotinine) and acrolein (3-hydroxymercapturic acid). Levels of t,t-MA were also associated with CVD risk as assessed using the Framingham Risk Score and this association was independent of smoking. Thus, benzene exposure is associated with increased CVD risk and deficits in circulating angiogenic cells in both smokers and non-smokers.

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients

PubMed Central

Chang, Ting-Yu; Tsai, Wei-Chi; Huang, Tse-Shun; Su, Shu-Han; Chang, Chih-Young; Ma, Hsiu-Yen; Wu, Chun-Hsien; Yang, Chih-Yung; Lin, Chi-Hung; Huang, Po-Hsun; Cheng, Cheng-Chung; Wang, Hsei-Wei

2017-01-01

Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases. PMID:28727754

Vaspin plasma concentrations and mRNA expressions in patients with stable and unstable angina pectoris.

PubMed

Li, Hai Ling; Peng, Wen Hui; Cui, Shi Tao; Lei, Hou; Wei, Yi Dong; Li, Wei Ming; Xu, Ya Wei

2011-09-01

Vaspin was a recently identified adipokine, playing a protective role in many metabolic diseases. The present study aimed to investigate the association between vaspin plasma level and stable angina pectoris (SAP) and unstable angina pectoris (UAP). A total of 88 patients with angiographically-proved coronary artery disease (CAD) (SAP 47, UAP 41) and 103 control subjects without cardiovascular diseases were enrolled in this study. Circulating vaspin, mRNA expression of vaspin in peripheral blood mononuclear cells (PBMC), clinical parameters, lipid profile and high-sensitivity C-reactive protein (hsCRP) were assayed. The severity of CAD was also assessed according to the number of vessels diseased. There are significant differences in circulating vaspin levels and mRNA levels of PBMC between SAP and UAP groups (SAP 0.91±0.95 ng/mL and UAP 0.43±0.38 ng/mL, p<0.01 in circulating vaspin level; SAP 1.19±0.85 and UAP 0.82±0.56, p<0.05 in mRNA level of PBMC). An inverse correlation between the number of diseased vessels and plasma vaspin concentration was observed (r=-0.350, p<0.01) in the CAD group. Construction of receiver operating characteristic curves confirmed that vaspin plasma concentrations significantly differentiated CAD patients (area under the curve=0.684, p<0.001), as well as UAP (area under the curve=0.640, p<0.05). Decreased vaspin plasma levels and mRNA levels in PBMC were observed in patients with UAP. Low vaspin concentrations correlate with CAD severity. The findings suggested that vaspin could serve as a novel biomarker of CAD as well as UAP.

Circulating tumor DNA profiling reveals clonal evolution and real-time disease progression in advanced hepatocellular carcinoma.

PubMed

Cai, Zhi-Xiong; Chen, Geng; Zeng, Yong-Yi; Dong, Xiu-Qing; Lin, Min-Jie; Huang, Xin-Hui; Zhang, Da; Liu, Xiao-Long; Liu, Jing-Feng

2017-09-01

Circulating tumor DNA (ctDNA) provides a potential non-invasive biomarker for cancer diagnosis and prognosis, but whether it could reflect tumor heterogeneity and monitor therapeutic responses in hepatocellular carcinoma (HCC) is unclear. Focusing on 574 cancer genes known to harbor actionable mutations, we identified the mutation repertoire of HCC tissues, and monitored the corresponding ctDNA features in blood samples to evaluate its clinical significance. Analysis of 3 HCC patients' mutation profiles revealed that ctDNA could overcome tumor heterogeneity and provide information of tumor burden and prognosis. Further analysis was conducted on the 4th HCC case with multiple lesion samples and sequential plasma samples. We identified 160 subclonal SNVs in tumor tissues as well as matched peritumor tissues with PBMC as control. 96.9% of this patient's tissue mutations could be also detected in plasma samples. These subclonal SNVs were grouped into 9 clusters according to their trends of cellular prevalence shift in tumor tissues. Two clusters constituted of tumor stem somatic mutations showed circulating levels relating with cancer progression. Analysis of tumor somatic mutations revealed that circulating level of such tumor stem somatic mutations could reflect tumor burden and even predict prognosis earlier than traditional strategies. Furthermore, HCK (p.V174M), identified as a recurrent/metastatic related mutation site, could promote migration and invasion of HCC cells. Taken together, study of mutation profiles in biopsy and plasma samples in HCC patients showed that ctDNA could overcome tumor heterogeneity and real-time track the therapeutic responses in the longitudinal monitoring. © 2017 UICC.

Plasma Shh levels reduced in pancreatic cancer patients

PubMed Central

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A.; Cantu, Esperanza F.; Lee, Jamie; Plonka, Caitlyn M.; Simeone, Diane M.; Anderson, Michelle A.; Merchant, Juanita L.

2012-01-01

Objectives Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an ELISA to detect human Shh in blood, and determine the levels in subjects with and without pancreatic cancer. Methods A human Shh ELISA assay was developed, and plasma Shh levels were measured in blood samples from normal volunteers and subjects with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. Results The average levels of Shh in human blood were lower in pancreatitis and pancreatic cancer patients than in normal individuals. Hematopoietic cells did not express Shh suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched for Shh did not induce Gli-1 mRNA suggesting that the protein was not biologically active. Conclusions Shh is secreted from tissues and organs into the circulation but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer. PMID:22513293

Plasma Shh levels reduced in pancreatic cancer patients.

PubMed

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A; Cantu, Esperanza F; Lee, Jamie; Plonka, Caitlyn M; Simeone, Diane M; Anderson, Michelle A; Merchant, Juanita L

2012-10-01

Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an enzyme-linked immunosorbent assay to detect human Shh in blood and determine its levels in subjects with and without pancreatic cancer. A human Shh enzyme-linked immunosorbent assay was developed, and plasma Shh levels were measured in blood samples from healthy subjects and patients with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. The mean levels of Shh in human blood were lower in patients with pancreatitis and pancreatic cancer than in healthy subjects. Hematopoietic cells did not express Shh, suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched with Shh did not induce Gli-1 messenger RNA, suggesting that the protein was not biologically active. Shh is secreted from tissues and organs into the circulation, but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer.

Circulating metastasis associated in colon cancer 1 transcripts in gastric cancer patient plasma as diagnostic and prognostic biomarker

PubMed Central

Burock, Susen; Herrmann, Pia; Wendler, Ina; Niederstrasser, Markus; Wernecke, Klaus-Dieter; Stein, Ulrike

2015-01-01

AIM: To evaluate the diagnostic and prognostic value of circulating Metastasis Associated in Colon Cancer 1 (MACC1) transcripts in plasma of gastric cancer patients. METHODS: We provide for the first time a blood-based assay for transcript quantification of the metastasis inducer MACC1 in a prospective study of gastric cancer patient plasma. MACC1 is a strong prognostic biomarker for tumor progression and metastasis in a variety of solid cancers. We conducted a study to define the diagnostic and prognostic power of MACC1 transcripts using 76 plasma samples from gastric cancer patients, either newly diagnosed with gastric cancer, newly diagnosed with metachronous metastasis of gastric cancer, as well as follow-up patients. Findings were controlled by using plasma samples from 54 tumor-free volunteers. Plasma was separated, RNA was isolated, and levels of MACC1 as well as S100A4 transcripts were determined by quantitative RT-PCR. RESULTS: Based on the levels of circulating MACC1 transcripts in plasma we significantly discriminated tumor-free volunteers and gastric cancer patients (P < 0.001). Levels of circulating MACC1 transcripts were increased in gastric cancer patients of each disease stage, compared to tumor-free volunteers: patients with tumors without metastasis (P = 0.005), with synchronous metastasis (P = 0.002), with metachronous metastasis (P = 0.005), and patients during follow-up (P = 0.021). Sensitivity was 0.68 (95%CI: 0.45-0.85) and specificity was 0.89 (95%CI: 0.77-0.95), respectively. Importantly, gastric cancer patients with high circulating MACC1 transcript levels in plasma demonstrated significantly shorter survival when compared with patients demonstrating low MACC1 levels (P = 0.0015). Furthermore, gastric cancer patients with high circulating transcript levels of MACC1 as well as of S100A4 in plasma demonstrated significantly shorter survival when compared with patients demonstrating low levels of both biomarkers or with only one biomarker elevated (P = 0.001). CONCLUSION: Levels of circulating MACC1 transcripts in plasma of gastric cancer patients are of diagnostic value and are prognostic for patient survival in a prospective study. PMID:25574109

Nonclinical evaluation of the serum pharmacodynamic biomarkers HGF and shed MET following dosing with the anti-MET monovalent monoclonal antibody onartuzumab.

PubMed

Mai, Elaine; Zheng, Zhong; Chen, Youjun; Peng, Jing; Severin, Christophe; Filvaroff, Ellen; Romero, Mally; Mallet, William; Kaur, Surinder; Gelzleichter, Thomas; Nijem, Ihsan; Merchant, Mark; Young, Judy C

2014-02-01

Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab.

A Bioartificial Liver-State of the Art

NASA Astrophysics Data System (ADS)

Strain, Alastair J.; Neuberger, James M.

2002-02-01

End-stage liver disease is treated by liver transplantation, but donor organ shortages remain a serious problem. This has prompted the design of bioartificial liver devices to ``bridge'' patients until they either recover or receive a liver transplant. In these devices, patient plasma is circulated extracorporeally through a bioreactor that houses liver cells (hepatocytes) sandwiched between artificial plates or capillaries.

The characterization of exosome from blood plasma of patients with colorectal cancer

NASA Astrophysics Data System (ADS)

Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Afanas'ev, S. G.; Frolova, A. Y.; Kondakova, I. V.

2016-08-01

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of "International Society for Extracellular Vesicles". The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

HPV circulating tumor DNA to monitor the efficacy of anti-PD-1 therapy in metastatic squamous cell carcinoma of the anal canal: A case report.

PubMed

Cabel, Luc; Bidard, François-Clément; Servois, Vincent; Cacheux, Wulfran; Mariani, Pascale; Romano, Emanuela; Minsat, Mathieu; Bieche, Ivan; Farkhondeh, Fereshteh; Jeannot, Emmanuelle; Buecher, Bruno

2017-10-15

Squamous cell carcinoma of the anal canal (SCCA) is a rare HPV-associated cancer with limited sensitivity to standard chemotherapy. In a phase 2 study, nivolumab, an anti PD-1 immune checkpoint inhibitor, demonstrated significant efficacy as single-agent therapy in metastatic SCCA patients. Nevertheless, imaging assessment by standard RECIST criteria of the efficacy of immune therapy can be difficult in some patients due to tumor immune cell infiltration, and biomarkers of treatment efficacy are needed. We have previously developed a quantitative droplet digital PCR (ddPCR) technique to detect HPV circulating tumor DNA (HPV ctDNA), with excellent sensitivity and specificity. Here, we report, for the first time, the kinetics of HPV ctDNA during therapy in a patient with metastatic SCCA, who obtained sustained partial response to single-agent nivolumab. We observed an early and very significant decrease of HPV ctDNA during therapy from the baseline level of 3713 copies/ml plasma to 564 copies/ml plasma at 4 weeks, and 156 copies/ml at 6 weeks, followed by a plateau. This observation provides proof-of-concept that HPV ctDNA can be used as a noninvasive early dynamic biomarker to monitor the efficacy of new immunotherapy agents. © 2017 UICC.

PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.

PubMed

Kooijmans, S A A; Fliervoet, L A L; van der Meel, R; Fens, M H A M; Heijnen, H F G; van Bergen En Henegouwen, P M P; Vader, P; Schiffelers, R M

2016-02-28

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery. Copyright © 2015. Published by Elsevier B.V.

Acute high-intensity interval exercise induces comparable levels of circulating cell-free DNA and Interleukin-6 in obese and normal-weight individuals.

PubMed

Ferrandi, Peter J; Fico, Brandon G; Whitehurst, Michael; Zourdos, Michael C; Bao, Fanchen; Dodge, Katelyn M; Rodriguez, Alexandra L; Pena, Gabriel; Huang, Chun-Jung

2018-06-01

Obesity is associated with lipid aggregation in adipocytes and macrophage infiltration, leading to increased oxidative stress and inflammation. Increased cell-free DNA (cfDNA) concentrations have been observed in clinical conditions of systemic inflammation. While the beneficial effects of regular physical activity on the release of circulating cfDNA still remain unknown, acute intense exercise has been shown to increase inflammatory cytokines and cfDNA concentrations in normal-weight individuals. Therefore, the primary purpose of this study was to examine the effect of acute high-intensity interval Exercise (HIIE) on plasma cfDNA and interleukin-6 (IL-6) responses in obese and normal-weight subjects. Fourteen male subjects (7 obese and 7 normal-weight) participated in an acute HIIE protocol (30 min, 4x4min @ 80% - 90% of VO 2max ) on a treadmill. Between HIIE intervals, subjects performed 3 min of active recovery at 50-60% VO 2max . Blood samples were collected prior to, immediately following exercise, and one hour into recovery for measurements of plasma cfDNA and IL-6. Our results demonstrated a significant elevation in plasma cfDNA immediately following acute HIIE in both obese and normal-weight subjects. A comparable elevation in the concentration of plasma IL-6 was also found between two groups in response to acute HIIE. Furthermore, the level of plasma cfDNA was not correlated with IL-6 either at baseline or in response to acute HIIE. These findings may support the utilization of HIIE as a time-efficient exercise protocol to understand the obesity-associated cfDNA and inflammatory responses. Published by Elsevier Inc.

Cell-Free Circulating Methylated SEPT9 for Noninvasive Diagnosis and Monitoring of Colorectal Cancer

PubMed Central

Fu, Bo; Yan, Peng; Zhang, Shan; Lu, Yan; Pan, Li; Tang, Wenqiang; Chen, Shen; Chen, Shuangfeng

2018-01-01

Identification of early-stage tumor and monitoring therapeutic efficacy and recurrence or metastasis of colorectal cancer (CRC) are urgently warranted for improving the outcome of CRC patients and reducing the disease-related mortality. In this study, we evaluated the diagnostic value of cell-free circulating methylated SEPT9 (mSEPT9) for CRC and beyond CRC and examined the potentiality of mSEPT9 in assessing therapeutic efficacy and monitoring recurrence of CRC. Our results confirmed the favorable diagnostic value of plasma mSEPT9 for CRC, with a sensitivity of 61.22% (95% confidence interval (CI): 51.33%–70.27%) and specificity of 93.7% (95% CI: 91.09%–95.57%) using 2/3 algorithm. The positive rate of mSEPT9 in CRC was correlated with tumor size, histological grade, and general histological type (P < 0.05). Beyond CRC, gastric cancer patients also presented a high positive rate of plasma mSEPT9 (70%). The conversions between preoperative and postoperative plasma mSEPT9 reflected the therapeutic efficacy of curatively intended surgery for CRC patients. The persistent positivity of plasma mSEPT9 after surgery (within 7–14 days) was highly associated with impending recurrences or metastases (within one year), with a sensitivity of 100%. Postoperative mSEPT9 status during follow-up also provided valuable indication for CRC recurrence or metastases, with a good consistency (kappa = 0.818, P = 0.001). Our results verified the reliability of plasma mSEPT9 as a biomarker for noninvasive diagnosis of CRC. More significantly, we revealed its valuable role in appraising CRC therapeutic efficacy and monitoring CRC recurrences or metastases. Further studies with larger sample sizes are needed to verify and elucidate the clinical utility of the promising findings. PMID:29849824

Circulating microRNAs as Biomarkers for Detection of Autologous Blood Transfusion

PubMed Central

Leuenberger, Nicolas; Schumacher, Yorck Olaf; Pradervand, Sylvain; Sander, Thomas; Saugy, Martial; Pottgiesser, Torben

2013-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs measured in blood plasma have emerged as specific and sensitive markers of physiological processes and disease. In this study, we investigated whether circulating miRNAs can serve as biomarkers for the detection of autologous blood transfusion, a major doping technique that is still undetectable. Plasma miRNA levels were analyzed using high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after autologous blood transfusion (blood bag storage time 42 days) in 10 healthy subjects and 10 controls without transfusion. Other serum markers of erythropoiesis were determined in the same samples. Our results revealed a distinct change in the pattern of circulating miRNAs. Ten miRNAs were upregulated in transfusion samples compared with control samples. Among these, miR-30b, miR-30c, and miR-26b increased significantly and showed a 3.9-, 4.0-, and 3.0-fold change, respectively. The origin of these miRNAs was related to pulmonary and liver tissues. Erythropoietin (EPO) concentration decreased after blood reinfusion. A combination of miRNAs and EPO measurement in a mathematical model enhanced the efficiency of autologous transfusion detection through miRNA analysis. Therefore, our results lay the foundation for the development of miRNAs as novel blood-based biomarkers to detect autologous transfusion. PMID:23840438

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

PubMed Central

Laget, Sophie; Dhingra, Dalia M.; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion. PMID:28060956

Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; Rassenti, Laura; Ghia, Emanuela M.; Skowronski, Elaine A.; Manouchehri, Sareh; McCanna, James; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

2014-01-01

BACKGROUND Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a “liquid biopsy” may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification,PCR,and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15–20 mL blood. CONCLUSIONS Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring. PMID:24270796

Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood.

PubMed

Sonnenberg, Avery; Marciniak, Jennifer Y; Rassenti, Laura; Ghia, Emanuela M; Skowronski, Elaine A; Manouchehri, Sareh; McCanna, James; Widhopf, George F; Kipps, Thomas J; Heller, Michael J

2014-03-01

Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Circulating Plasma and Exosomal microRNAs as Indicators of Drug-Induced Organ Injury in Rodent Models

PubMed Central

Cho, Young-Eun; Kim, Sang-Hyun; Lee, Byung-Heon; Baek, Moon-Chang

2017-01-01

This study was performed to evaluate whether microRNAs (miRNAs) in circulating exosomes may serve as biomarkers of drug-induced liver, kidney, or muscle-injury. Quantitative PCR analyses were performed to measure the amounts of liver-specific miRNAs (miR-122, miR-192, and miR-155), kidney-specific miR-146a, or muscle-specific miR-206 in plasma and exosomes from mice treated with liver, kidney or muscle toxicants. The levels of liver-specific miRNAs in circulating plasma and exosomes were elevated in acetaminophen-induced liver injury and returned to basal levels by treatment with antioxidant N-acetyl-cysteine. Circulating miR-146a and miR-206 were increased in cisplatin-induced nephrotoxicity and bupivacaine-induced myotoxicity, respectively. Taken together, these results indicate that circulating plasma and exosomal miRNAs can be used as potential biomarkers specific for drug-induced liver, kidney or muscle injury. PMID:28208010

Immune dysregulation in severe influenza.

PubMed

Heltzer, Meredith L; Coffin, Susan E; Maurer, Kelly; Bagashev, Asen; Zhang, Zhe; Orange, Jordan S; Sullivan, Kathleen E

2009-06-01

Among previously healthy children with severe influenza, the mechanisms leading to increased pathology are not understood. We hypothesized that children with severe influenza would have high levels of circulating cytokines. To examine this, we recruited patients with severe influenza and examined plasma cytokine levels as well as the ability of peripheral blood cells to respond to stimuli. Ten patients with severe influenza were enrolled during the 2005-2007 influenza seasons. We evaluated plasma cytokine levels, circulating NK cells, and responses to TLR ligands during the illness. We compared these patients with five patients with moderate influenza, six patients with respiratory syncytial virus (RSV), and 24 noninfected controls. Patients with influenza showed depressed responses to TLR ligands when compared with RSV patients and healthy controls (P<0.05). These normalized when retested during a convalescent phase. Plasma levels of IL-6, IL-12, and IFN- were elevated in influenza patients compared with controls (P<0.05). A compromised ability to produce TNF- was reproduced by in vitro infection, and the magnitude of the effect correlated with the multiplicity of infection and induction of IFN regulatory factor 4 expression. Aberrant, systemic, innate responses to TLR ligands during influenza infection may be a consequence of specific viral attributes such as a high inoculum or rapid replication and may underlie the known susceptibility of influenza-infected patients to secondary bacterial infections.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

PubMed

Mazloom, Amin R; Džakula, Željko; Oeth, Paul; Wang, Huiquan; Jensen, Taylor; Tynan, John; McCullough, Ron; Saldivar, Juan-Sebastian; Ehrich, Mathias; van den Boom, Dirk; Bombard, Allan T; Maeder, Margo; McLennan, Graham; Meschino, Wendy; Palomaki, Glenn E; Canick, Jacob A; Deciu, Cosmin

2013-06-01

Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR. © 2013 John Wiley & Sons, Ltd.

Fibronectin on circulating extracellular vesicles as a liquid biopsy to detect breast cancer.

PubMed

Moon, Pyong-Gon; Lee, Jeong-Eun; Cho, Young-Eun; Lee, Soo Jung; Chae, Yee Soo; Jung, Jin Hyang; Kim, In-San; Park, Ho Yong; Baek, Moon-Chang

2016-06-28

Extracellular vesicles (EVs) secreted from cancer cells have potential for generating cancer biomarker signatures. Fibronectin (FN) was selected as a biomarker candidate, due to the presence in surface on EVs secreted from human breast cancer cell lines. A subsequent study used two types of enzyme-linked immunosorbent assays (ELISA) to determine the presence of these proteins in plasma samples from disease-free individuals (n=70), patients with BC (n=240), BC patients after surgical resection (n=40), patients with benign breast tumor (n=55), and patients with non-cancerous diseases (thyroiditis, gastritis, hepatitis B, and rheumatoid arthritis; n=80). FN levels were significantly elevated (p< .0001) at all stages of BC, and returned to normal after tumor removal. The diagnostic accuracy for FN detection in extracellular vesicles (ELISA method 1) (area under the curve, 0.81; 95% CI, 0.76 to 0.86; sensitivity of 65.1% and specificity of 83.2%) were also better than those for FN detection in the plasma (ELISA method 2) (area under the curve, 0.77; 95% CI, 0.72 to 0.83; sensitivity of 69.2% and specificity of 73.3%) in BC. The diagnostic accuracy of plasma FN was similar in both the early-stage BC and all BC patients, as well as in the two sets. This liquid biopsy to detect FN on circulating EVs could be a promising method to detect early breast cancer.

One-year enzyme-linked immunosorbent assay follow-up of human interleukin for Da cells/leukemia inhibitory factor in blood and urine of 22 kidney transplant recipients.

PubMed

Morel, D; Taupin, J L; Combe, C; Potaux, L; Gualde, N; Moreau, J F

1994-12-15

The cytokine human interleukin for Da cells/leukemia inhibitory factor (HILDA/LIF) exerts multiple biological effects in vitro. In mice, high circulating levels of HILDA/LIF induce a wide range of pathophysiological events, some of them closely involved with immunological and inflammatory responses. Using a sandwich ELISA recognizing the natural human HILDA/LIF molecule with a threshold of 50 pg/ml in urine and 150 pg/ml in plasma, we monitored the urine and plasma HILDA/LIF levels of 22 patients in their first year after a kidney transplant. HILDA/LIF urine excretion is increased during acute rejection, and infections also trigger heavy HILDA/LIF plasma concentrations or urine excretion. In addition, this study raises the question of HILDA/LIF involvement in post-kidney-transplant phenomena such as hypercalcemia, osteoporosis, or the reversal of anemia.

Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.

PubMed

Khoo, Bee Luan; Warkiani, Majid Ebrahimi; Tan, Daniel Shao-Weng; Bhagat, Ali Asgar S; Irwin, Darryl; Lau, Dawn Pingxi; Lim, Alvin S T; Lim, Kiat Hon; Krisna, Sai Sakktee; Lim, Wan-Teck; Yap, Yoon Sim; Lee, Soo Chin; Soo, Ross A; Han, Jongyoon; Lim, Chwee Teck

2014-01-01

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.

Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

NASA Astrophysics Data System (ADS)

Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

2013-06-01

Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

Reduction of circulating and selective limbic brain levels of (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) following forced swim stress in C57BL/6J mice

PubMed Central

Maldonado-Devincci, Antoniette M.; Beattie, Matthew C.; Morrow, Danielle H.; McKinley, Raechel E.; Cook, Jason B.; O’Buckley, Todd K.

2014-01-01

Rationale Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, and GABAergic neuroactive steroids contribute to homeostatic regulation of this circuitry. Acute forced swim stress (FSS) increases plasma, cortical, and hypothalamic (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) levels in rats. However, there have not been systemic investigations of acute stress on changes in plasma and brain levels of 3α,5α-THP in mouse models. Objectives The present experiments aimed to assess circulating and local brain levels of 3α,5α-THP following acute FSS in C57BL/6J mice. Methods Mice were exposed to FSS (10 min), and 50 min later, blood and brains were collected. Circulating pregnenolone and 3α,5α-THP levels were assessed in serum. Free-floating brain sections (40 µm, four to five sections/region) were immunostained and analyzed in cortical and limbic brain structures. Results FSS decreased circulating 3α,5α-THP (−41.6± 10.4 %) and reduced 3α,5α-THP immunolabeling in the paraventricular nucleus of the hypothalamus (−15.2±5.7 %), lateral amygdala (LA, −31.1±13.4 %), and nucleus accumbens (NAcc) shell (−31.9±14.6). Within the LA, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter were localized in 3α,5α-THP-positively stained cells, while in the NAcc shell, only VGLUT1 was localized in 3α,5α-THP-positively stained cells, suggesting that both glutamatergic and GABAergic cells within the LA are 3α,5α-THP-positive, while in the NAcc shell, 3α,5α-THP only localizes to glutamatergic cells. Conclusions The decrease in circulating and brain levels of 3α,5α-THP may be due to alterations in the biosynthesis/ metabolism or changes in the regulation of the HPA axis following FSS. Changes in GABAergic neuroactive steroids in response to stress likely mediate functional adaptations in neuronal activity. This may provide a potential targeted therapeutic avenue to address maladaptive stress responsivity. PMID:24744202

Circulating endothelial cells in acute ischaemic stroke.

PubMed

Nadar, Sunil K; Lip, Gregory Y H; Lee, Kaeng W; Blann, Andrew D

2005-10-01

Increased numbers of CD146-bearing circulating endothelial cells (CECs) in the peripheral blood probably represent the most direct evidence of endothelial cell damage. As acute ischaemic strokes are associated with endothelial abnormalities, we hypothesised that these CECs are raised in acute stroke, and that they would correlate with the other indices of endothelial perturbation, i.e. plasma von Willebrand factor (vWf) and soluble E-selectin. We studied 29 hypertensive patients (19 male; mean age 63 years) who presented with an acute stroke and compared them with 30 high risk hypertensive patients (21 male; mean age 62 years) and 30 normotensive controls (16 male; mean age 58 years). CECs were estimated by CD146 immunobead capture, vWf and soluble E-selectin by ELISA. Patients with an acute ischaemic stroke had significantly higher numbers of CECs/ml of blood (p<0.001) plasma vWf (p=0.008) soluble E-selectin (p=0.002) and higher systolic blood pressure (SBP) as compared to the other groups. The number of CECs significantly correlated with soluble E-selectin (r=0.432, p<0.001) and vWf (r=0.349, p=0.001) but not with SBP (r=0.198, p=0.069). However, in multivariate analysis, only disease group (i.e. health, hypertension or stroke) was associated with increased CECs. Acute ischaemic stroke is associated with increased numbers of CECs. The latter correlate well with established plasma markers of endothelial dysfunction or damage, thus unequivocally confirming severe vasculopathy in this condition. However, the greatest influence on CECs numbers was clinical group.

KRAS mutations in blood circulating cell-free DNA: a pancreatic cancer case-control

PubMed Central

Le Calvez-Kelm, Florence; Foll, Matthieu; Wozniak, Magdalena B.; Delhomme, Tiffany M.; Durand, Geoffroy; Chopard, Priscilia; Pertesi, Maroulio; Fabianova, Eleonora; Adamcakova, Zora; Holcatova, Ivana; Foretova, Lenka; Janout, Vladimir; Vallee, Maxime P.; Rinaldi, Sabina; Brennan, Paul; McKay, James D.; Byrnes, Graham B.; Scelo, Ghislaine

2016-01-01

The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series. PMID:27705932

Circulating inhibitor of ouabain-insensitive cation transport in malignantrenal hypertension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simon, G.

1986-03-01

The role of circulating humoral agents in the pathogenesis of vascular wall Na depletion in malignant hypertension (MHT) was investigated. Plasma was collected from 33 male F344 rats with malignant one-kidney, one clip HT and 22 normotensive control rats. MHT developed spontaneously and was characterized by inactivity, weight loss, edema, anemia or hemoconcentration, hyperkalemia, and renal insufficiency. For bioassay, monolayers of quiescent vascular smooth muscle cells from F344 rats were incubated in deproteinized or whole plasma for measurement of /sup 86/Rb uptake with or without 2 mM ouabain or 1 mM furosemide. Compared to controls, ouabain-insensitive /sup 86/Rb uptake wasmore » reduced from 8.2 +- 2.0 nmol/mg protein min/sup -1/ (mean +- SD) to 5.2 +- 1.4 in deproteinized plasma (p < 0.01, N = 12) and from 6.6 +- 1.9 to 4.0 +- 0.3 in whole plasma (p < 0.05, N=5) of rats with MHT, due in part to a reduction in furosemide-sensitive uptake (p < 0.01, N = 6). There were no differences in ouabain-sensitive /sup 86/Rb uptake of cells between groups. In rats with MHT the increased Na content of the aorta that characterizes benign one-kidney, one clip HT was reversed, and bladder wall Na content was reduced (p < 0.001, N = 9). In MHT, a furosemide-like, ouabain-insensitive cation transport inhibitor in blood and urine may be the cause of vascular wall Na loss and of natriuresis that triggers the syndrome.« less

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

PubMed Central

Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

2008-01-01

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. PMID:18974299

Fragment Length of Circulating Tumor DNA.

PubMed

Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

2016-07-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Circulating microRNAs and treatment response in the Phase II SWOG S0925 study for patients with new metastatic hormone-sensitive prostate cancer.

PubMed

Cheng, Heather H; Plets, Melissa; Li, Hongli; Higano, Celestia S; Tangen, Catherine M; Agarwal, Neeraj; Vogelzang, Nicholas J; Hussain, Maha; Thompson, Ian M; Tewari, Muneesh; Yu, Evan Y

2018-02-01

Previous studies suggest circulating, blood-based microRNAs (miRNAs) may serve as minimally invasive prostate cancer biomarkers, however there is limited data from prospective clinical trials. Here, we explore the role of candidate plasma miRNAs as potential biomarkers in the SWOG 0925 randomized phase II study of androgen deprivation combined with cixutumumab versus androgen deprivation alone in patients with new metastatic hormone-sensitive prostate cancer. Correlative biospecimens, including circulating tumor cells (CTCs) and plasma for miRNA analysis, were collected at baseline and after 12 weeks on treatment from 50 patients enrolled on SWOG 0925. Circulating microRNAs were quantified using real-time RT-PCR microRNA array that allowed specific analysis of previously identified candidate miRNAs (miR-141, miR-200a, miR-200b, miR-210, and miR-375) as well as discovery analysis to identify new candidate miRNAs. MiRNA levels were correlated to previously reported CTC counts using CellSearch® (Veridex) and with the primary study outcome of 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL), previously shown to correlate with overall survival. We observed a correlation between baseline circulating miR-141, miR-200a, and miR-375 levels with baseline CTCs. Baseline miR-375 levels were associated with 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL, P = 0.007). Using ROC curve analysis, there was no significant difference between baseline miR-375 and baseline CTC in predicting 28-week PSA response (≤0.2 vs >0.2 ng/mL). To discover novel candidate miRNAs, we analyzed 365 miRNAs for association with the 28-week PSA response endpoint and identified new candidate miRNAs along with the existing candidates miR-375 and miR-200b (P = 0.0012, P = 0.0046, respectively. Baseline plasma miR-141, miR-200a, and miR-375 levels are associated with baseline CTC count. Baseline miR-375 was also associated with the trial endpoint of 28-week PSA response. Our results provide evidence that circulating miRNA biomarkers may have value as prognostic biomarkers and warrant further study in larger prospective clinical trials. © 2017 Wiley Periodicals, Inc.

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

PubMed

van Ginkel, Joost H; Huibers, Manon M H; van Es, Robert J J; de Bree, Remco; Willems, Stefan M

2017-06-19

During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data. In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%. Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

Systemic influences contribute to prolonged microvascular rarefaction after brain irradiation: a role for endothelial progenitor cells

PubMed Central

Ashpole, Nicole M.; Warrington, Junie P.; Mitschelen, Matthew C.; Yan, Han; Sosnowska, Danuta; Gautam, Tripti; Farley, Julie A.; Csiszar, Anna; Ungvari, Zoltan

2014-01-01

Whole brain radiation therapy (WBRT) induces profound cerebral microvascular rarefaction throughout the hippocampus. Despite the vascular loss and localized cerebral hypoxia, angiogenesis fails to occur, which subsequently induces long-term deficits in learning and memory. The mechanisms underlying the absence of vessel recovery after WBRT are unknown. We tested the hypotheses that vascular recovery fails to occur under control conditions as a result of loss of angiogenic drive in the circulation, chronic tissue inflammation, and/or impaired endothelial cell production/recruitment. We also tested whether systemic hypoxia, which is known to promote vascular recovery, reverses these chronic changes in inflammation and endothelial cell production/recruitment. Ten-week-old C57BL/6 mice were subjected to a clinical series of fractionated WBRT: 4.5-Gy fractions 2 times/wk for 4 wk. Plasma from radiated mice increased in vitro endothelial cell proliferation and adhesion compared with plasma from control mice, indicating that WBRT did not suppress the proangiogenic drive. Analysis of cytokine levels within the hippocampus revealed that IL-10 and IL-12(p40) were significantly increased 1 mo after WBRT; however, systemic hypoxia did not reduce these inflammatory markers. Enumeration of endothelial progenitor cells (EPCs) in the bone marrow and circulation indicated that WBRT reduced EPC production, which was restored with systemic hypoxia. Furthermore, using a bone marrow transplantation model, we determined that bone marrow-derived endothelial-like cells home to the hippocampus after systemic hypoxia. Thus, the loss of production and homing of EPCs have an important role in the prolonged vascular rarefaction after WBRT. PMID:25038144

Developmental changes in circulating IL-8/CXCL8 isoforms in neonates.

PubMed

Maheshwari, Akhil; Voitenok, Nikolai N; Akalovich, Svetlana; Shaik, Sadiq S; Randolph, David A; Sims, Brian; Patel, Rakesh P; Killingsworth, Cheryl R; Fallon, Michael B; Ohls, Robin K

2009-04-01

Interleukin-8 (IL-8/CXCL8) is widely expressed in fetal tissues although inflammatory changes are not seen. Circulating IL-8 is comprised of an endothelial-derived [ala-IL-8](77) isoform and another, more potent [ser-IL-8](72) secreted by most other cells; [ala-IL-8](77) can be converted into [ser-IL-8](72) by proteolytic removal of an N-terminal pentapeptide from [ala-IL-8](77). In this study, we show [ala-IL-8](77) is the predominant circulating isoform of IL-8 in premature neonates but not in term neonates/adults, who have [ser-IL-8](72) as the major isoform. This isoform switch from the less potent [ala-IL-8](77) to [ser-IL-8](72) correlates with a maturational increase in the neutrophil chemotactic potency of plasma IL-8. The emergence of [ser-IL-8](72) as the major isoform is likely due to increased plasma [ala-IL-8](77)-convertase activity and/or changes in the cellular sources of IL-8. Developmental changes in IL-8 isoforms may serve to minimize its inflammatory effects in the fetus and also provide a mechanism to restore its full activity after birth.

Unloading oxygen in a capillary vessel under a pathological condition.

PubMed

Escobar, C; Méndez, F

2008-10-01

In this work, we study theoretically the unloading of oxygen from a hemoglobin molecule to the wall of a typical capillary vessel, considering that the hemoglobin under pathological conditions, obeys the rheological Maxwell model. Based on recent experimental evidences in hypertension, we consider that the red blood cells (RBCs) are composed by a single continuous medium in contrast with the classical particulate or discrete RBC models, which are only valid under normal physiological conditions. The analysis considers the hemodynamic interactions between the plasma and the hemoglobin, both circulating in a long horizontal capillary. We apply numerical and analytical methods to obtain the main fluid-dynamic characteristics for both fluids in the limit of low Reynolds and Womersley numbers. A diffusion boundary layer formulation for the oxygen transport in the combined plasma-hemoglobin core region is presented. The main aspects derived are the time and spatial evolution of the membrane. The hemoglobin and plasma velocities and the pressure distributions are shown. For the oxygen unloading the results are the oxy-hemoglobin saturation, the oxygen flux and the oxygen concentration in the cell-free plasma layer. The volume fraction of red blood cells and the Strouhal number have a great influence on the hemodynamic interactions.

Apoptosis and dysfunction of blood dendritic cells in patients with falciparum and vivax malaria

PubMed Central

Woodberry, Tonia; Kienzle, Vivian; McPhun, Virginia; Minigo, Gabriela; Lampah, Daniel A.; Kenangalem, Enny; Engwerda, Christian; López, J. Alejandro; Anstey, Nicholas M.

2013-01-01

Malaria causes significant morbidity worldwide and a vaccine is urgently required. Plasmodium infection causes considerable immune dysregulation, and elicitation of vaccine immunity remains challenging. Given the central role of dendritic cells (DCs) in initiating immunity, understanding their biology during malaria will improve vaccination outcomes. Circulating DCs are particularly important, as they shape immune responses in vivo and reflect the functional status of other subpopulations. We performed cross-sectional and longitudinal assessments of the frequency, phenotype, and function of circulating DC in 67 Papuan adults during acute uncomplicated P. falciparum, P. vivax, and convalescent P. falciparum infections. We demonstrate that malaria patients display a significant reduction in circulating DC numbers and the concurrent accumulation of immature cells. Such alteration is associated with marked levels of spontaneous apoptosis and impairment in the ability of DC to mature, capture, and present antigens to T cells. Interestingly, sustained levels of plasma IL-10 were observed in patients with acute infection and were implicated in the induction of DC apoptosis. DC apoptosis was reversed upon IL-10 blockade, and DC function recovered when IL-10 levels returned to baseline by convalescence. Our data provide key information on the mechanisms behind DC suppression during malaria and will assist in developing strategies to better harness DC’s immunotherapeutic potential. PMID:23835848

Dietary glutamine supplementation enhances endothelial progenitor cell mobilization in streptozotocin-induced diabetic mice subjected to limb ischemia.

PubMed

Su, Shiau-Tsz; Yeh, Chiu-Li; Hou, Yu-Chen; Pai, Man-Hui; Yeh, Sung-Ling

2017-02-01

Diabetes is a metabolic disorder with increased risk of vascular diseases. Tissue ischemia may occur with diabetic vascular complications. Bone marrow-derived endothelial progenitor cells (EPCs) constitute a reparative response to ischemic injury. This study investigated the effects of oral glutamine (GLN) supplementation on circulating EPC mobilization and expression of tissue EPC-releasing markers in diabetic mice subjected to limb ischemia. Diabetes was induced by a daily intraperitoneal injection of streptozotocin for 5 days. Diabetic mice were divided into 2 nonischemic groups and 6 ischemic groups. One of the nonischemic and 3 ischemic groups were fed the control diet, while the remaining 4 groups received diets with identical components except that part of the casein was replaced by GLN. The respective diets were fed to the mice for 3 weeks, and then the nonischemic mice were sacrificed. Unilateral hindlimb ischemia was created in the ischemic groups, and mice were sacrificed at 1, 7 or 21 days after ischemia. Their blood and ischemic muscle tissues were collected for further analyses. Results showed that plasma matrix metallopeptidase (MMP)-9 and the circulating EPC percentage increased after limb ischemia in a diabetic condition. Compared to groups without GLN, GLN supplementation up-regulated plasma stromal cell-derived factor (SDF)-1 and muscle MMP-9, SDF-1, hypoxia-inducible factor-1 and vascular endothelial growth factor gene expression. The CD31-immunoreactive intensities were also higher in the ischemic limb. These findings suggest that GLN supplementation enhanced circulating EPC mobilization that may promote endothelium repair at ischemic tissue in diabetic mice subjected to limb ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

Benzene exposure is associated with cardiovascular disease risk

PubMed Central

Riggs, Daniel W.; Conklin, Daniel J.; McCracken, James P.; Srivastava, Sanjay; Xie, Zhengzhi; Rai, Shesh; Bhatnagar, Aruni; O’Toole, Timothy E.

2017-01-01

Benzene is a ubiquitous, volatile pollutant present at high concentrations in toxins (e.g. tobacco smoke) known to increase cardiovascular disease (CVD) risk. Despite its prevalence, the cardiovascular effects of benzene have rarely been studied. Hence, we examined whether exposure to benzene is associated with increased CVD risk. The effects of benzene exposure in mice were assessed by direct inhalation, while the effects of benzene exposure in humans was assessed in 210 individuals with mild to high CVD risk by measuring urinary levels of the benzene metabolite trans,trans-muconic acid (t,t-MA). Generalized linear models were used to assess the association between benzene exposure and CVD risk. Mice inhaling volatile benzene had significantly reduced levels of circulating angiogenic cells (Flk-1+/Sca-1+) as well as an increased levels of plasma low-density lipoprotein (LDL) compared with control mice breathing filtered air. In the human cohort, urinary levels of t,t-MA were inversely associated several populations of circulating angiogenic cells (CD31+/34+/45+, CD31+/34+/45+/AC133–, CD34+/45+/AC133+). Although t,t-MA was not associated with plasma markers of inflammation or thrombosis, t,t-MA levels were higher in smokers and in individuals with dyslipidemia. In smokers, t,t-MA levels were positively associated with urinary metabolites of nicotine (cotinine) and acrolein (3-hydroxymercapturic acid). Levels of t,t-MA were also associated with CVD risk as assessed using the Framingham Risk Score and this association was independent of smoking. Thus, benzene exposure is associated with increased CVD risk and deficits in circulating angiogenic cells in both smokers and non-smokers. PMID:28886060

Low-calorie cranberry juice supplementation reduces plasma oxidized LDL and cell adhesion molecule concentrations in men.

PubMed

Ruel, Guillaume; Pomerleau, Sonia; Couture, Patrick; Lemieux, Simone; Lamarche, Benoît; Couillard, Charles

2008-02-01

Elevated circulating concentrations of oxidized LDL (OxLDL) and cell adhesion molecules are considered to be relevant markers of oxidative stress and endothelial activation which are implicated in the development of CVD. On the other hand, it has been suggested that dietary flavonoid consumption may be cardioprotective through possible favourable impacts on LDL particle oxidation and endothelial activation. The present study was undertaken to determine the effect of the daily consumption of low-calorie cranberry juice cocktail on plasma OxLDL, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin concentrations in men. Thirty men (mean age 51 (sd 10) years) were recruited and asked to consume increasing daily doses of cranberry juice cocktail (125, 250 and 500 ml/d) over three successive periods of 4 weeks. Plasma OxLDL and adhesion molecule concentrations were measured by ELISA before and after each phase. We noted a significant decrease in plasma OxLDL concentrations following the intervention (P < 0.0001). We also found that plasma ICAM-1 (P < 0.0001) and VCAM-1 (P < 0.05) concentrations decreased significantly during the course of the study. In summary, the present results show that daily cranberry juice cocktail consumption is associated with decreases in plasma OxLDL, ICAM-1 and VCAM-1 concentrations in men.

Potential Function of Granulysin, Other Related Effector Molecules and Lymphocyte Subsets in Patients with TB and HIV/TB Coinfection

PubMed Central

Pitabut, Nada; Sakurada, Shinsaku; Tanaka, Takahiro; Ridruechai, Chutharut; Tanuma, Junko; Aoki, Takahiro; Kantipong, Pacharee; Piyaworawong, Surachai; Kobayashi, Nobuyuki; Dhepakson, Panadda; Yanai, Hideki; Yamada, Norio; Oka, Shinichi; Okada, Masaji; Khusmith, Srisin; Keicho, Naoto

2013-01-01

Background: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. Objective: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9+Vδ2+ T cells, CD4+ T cells and CD8+ T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. Methods: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. Results: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8+ T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. Conclusion: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection. PMID:23801887

Plasma Membrane Integrity and Survival of Melanoma Cells After Nanosecond Laser Pulses

PubMed Central

Pérez-Gutiérrez, Francisco G.; Camacho-López, Santiago; Evans, Rodger; Guillén, Gabriel; Goldschmidt, Benjamin S.; Viator, John A.

2010-01-01

Circulating tumor cells (CTCs) photoacoustic detection systems can aid clinical decision-making in the treatment of cancer. Interaction of melanin within melanoma cells with nanosecond laser pulses generates photoacoustic waves that make its detection possible. This study aims at: (1) determining melanoma cell survival after laser pulses of 6 ns at λ = 355 and 532 nm; (2) comparing the potential enhancement in the photoacoustic signal using λ = 355 nm in contrast with λ = 532 nm; (3) determining the critical laser fluence at which melanin begins to leak out from melanoma cells; and (4) developing a time-resolved imaging (TRI) system to study the intracellular interactions and their effect on the plasma membrane integrity. Monolayers of melanoma cells were grown on tissue culture-treated clusters and irradiated with up to 1.0 J/cm2. Surviving cells were stained with trypan blue and counted using a hemacytometer. The phosphate buffered saline absorbance was measured with a nanodrop spectrophotometer to detect melanin leakage from the melanoma cells post-laser irradiation. Photoacoustic signal magnitude was studied at both wavelengths using piezoelectric sensors. TRI with 6 ns resolution was used to image plasma membrane damage. Cell survival decreased proportionally with increasing laser fluence for both wavelengths, although the decrease is more pronounced for 355 nm radiation than for 532 nm. It was found that melanin leaks from cells equally for both wavelengths. No significant difference in photoacoustic signal was found between wavelengths. TRI showed clear damage to plasma membrane due to laser-induced bubble formation. PMID:20589533

Prediagnostic plasma vitamin C and risk of gastric adenocarcinoma and esophageal squamous cell carcinoma in a Chinese population123

PubMed Central

Freedman, Neal D; Fan, Jin-Hu; Qiao, You-Lin; Dawsey, Sanford M; Taylor, Philip R; Abnet, Christian C

2013-01-01

Background: China has some of the highest incidence rates for gastric adenocarcinoma (GA) and esophageal squamous cell carcinoma (ESCC) in the world. Prospective studies suggested that vitamin C may reduce risks; however, associations are unclear because of limited sample size. Objective: The objective was to examine the relation between prediagnostic plasma vitamin C and the risk of GA and ESCC. Design: A case-cohort study was used to assess the association between prediagnostic plasma vitamin C and incidence of GA (n = 467) and ESCC (n = 618) in the General Population Nutrition Intervention Trial. With the use of multivariate Cox proportional hazards models, we estimated the HRs and 95% CIs. We also conducted a meta-analysis of the literature up to 1 October 2012 on the relation between circulating vitamin C and gastric cancer incidence. Two cohort studies and the current study were included to assess the body of evidence. Results: For GA, each 20-μmol/L increase in plasma vitamin C was associated with a 14% decrease in risk (HR: 0.86; 95% CI: 0.76, 0.96). Compared with individuals with low plasma vitamin C concentrations (≤28 μmol/L), those with normal concentrations (>28 μmol/L) had a 27% reduced risk of GA (HR: 0.73; 95% CI: 0.56, 0.94). No association between vitamin C concentrations and ESCC was seen. Meta-analysis showed that the risk of incident GA among those with the highest concentration of plasma vitamin C was 31% lower (random-effects-pooled-odds ratio 0.69; 95% CI: 0.54, 0.89) than those in the lowest category. Conclusion: Our data provide evidence that higher circulating vitamin C was associated with a reduced risk of incident GA, but no association was seen for ESCC. PMID:24025629

Circulating levels of cell-derived microparticles are reduced by mild hypobaric hypoxia: data from a randomised controlled trial.

PubMed

Ayers, Lisa; Stoewhas, Anne-Christin; Ferry, Berne; Latshang, Tsogyal D; Lo Cascio, Christian M; Sadler, Ross; Stadelmann, Katrin; Tesler, Noemi; Huber, Reto; Achermann, Peter; Bloch, Konrad E; Kohler, Malcolm

2014-05-01

Hypoxia is known to induce the release of microparticles in vitro. However, few publications have addressed the role of hypoxia in vivo on circulating levels of microparticles. This randomised, controlled, crossover trial aimed to determine the effect of mild hypoxia on in vivo levels of circulating microparticles in healthy individuals. Blood was obtained from 51 healthy male volunteers (mean age of 26.9 years) at baseline altitude (490 m) and after 24 and 48 h at moderate altitude (2,590 m). The order of altitude exposure was randomised. Flow cytometry was used to assess platelet-poor plasma for levels of circulating microparticles derived from platelets, endothelial cells, leucocytes, granulocytes, monocytes, red blood cells and procoagulant microparticles. Mean (standard deviation) oxygen saturation was significantly lower on the first and second day after arrival at 2,590 m, 91.0 (2.0) and 92.0 (2.0) %, respectively, compared to 490 m, 96 (1.0) %, p < 0.001 for both comparisons. A significant decrease in the levels of procoagulant microparticles (annexin V+ -221/μl 95 % CI -370.8/-119.0, lactadherin+ -202/μl 95 % CI -372.2/-93.1), platelet-derived microparticles (-114/μl 95 % CI -189.9/-51.0) and red blood cell-derived microparticles (-81.4 μl 95 % CI -109.9/-57.7) after 48 h at moderate altitude was found. Microparticles derived from endothelial cells, granulocytes, monocytes and leucocytes were not significantly altered by exposure to moderate altitude. In healthy male individuals, mild hypobaric hypoxia, induced by a short-term stay at moderate altitude, is associated with lower levels of procoagulant microparticles, platelet-derived microparticles and red blood cell-derived microparticles, suggesting a reduction in thrombotic potential.

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease

PubMed Central

van Beers, Eduard J.; Schaap, Marianne C.L.; Berckmans, René J.; Nieuwland, Rienk; Sturk, Augueste; van Doormaal, Frederiek F.; Meijers, Joost C.M.; Biemond, Bart J.

2009-01-01

Background Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease. Design and Methods In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in 25 consecutive patients with sickle cell disease. Results The majority of microparticles originated from platelets (GPIIIa,CD61) and erythrocytes (glycophorin A,CD235), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=−0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.023). Conclusions We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles. PMID:19815831

Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18

PubMed Central

Harms, Robert Z.; Creer, Austin J.; Lorenzo-Arteaga, Kristina M.; Ostlund, Katie R.; Sarvetnick, Nora E.

2017-01-01

The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor. PMID:28900426

Hypoxic exercise training improves cardiac/muscular hemodynamics and is associated with modulated circulating progenitor cells in sedentary men.

PubMed

Wang, Jong-Shyan; Lee, Mei-Yi; Lien, Hen-Yu; Weng, Tzu-Pin

2014-01-01

Circulating progenitor cells (CPCs) improve cardiovascular function and organ perfusion by enhancing the capacities of endothelial repair and neovasculogenesis. This study investigates whether exercise regimens with/without hypoxia affect cardiac and muscular hemodynamics by modulating CPCs and angiogenic factors. Forty sedentary males were randomly divided into hypoxic (HT, n=20) and normoxic (NT, n=20) training groups. The subjects were trained on a bicycle ergometer at 60%VO(2max) under 15% (HT) or 21% (NT) O2 conditions for 30 min daily, five days weekly for five weeks. After the five-week interventions, the HT group exhibited a larger improvement in aerobic capacity than the NT group. Furthermore, the HT regimen (i) enhanced cardiac output (Q(H)) and perfusion (Q(M))/oxygenation of vastus lateralis during exercise; (ii) increased levels of CD34(+)/KDR(+)/CD117(+), CD34(+)/KDR(+)/CD133(+), and CD34(+)/KDR(+)/CD31(+) cells in blood; (iii) promoted the proliferative capacity of these CPC subsets, and (iv) elevated plasma nitrite/nitrate, stromal cell-derived factor-1 (SDF-1), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor-A (VEGF-A) concentrations. Despite the lack of changes in Q(H) and the number or proliferative capacity of CD34(+)/KDR(+)/CD117(+) or CD34(+)/KDR(+)/CD31(+) cells, the NT regimen elevated both Q(M) and plasma nitrite/nitrate levels and suppressed the shedding of endothelial cells (CD34(-)/KDR(+)/phosphatidylserine(+) cells). The HT regimen improves cardiac and muscular hemodynamic adaptations, possibly by promoting the mobilization/function of CPCs and the production of angiogenic factors. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

[The physiology of glucagon-like peptide-1 and its role in the pathophysiology of type 2 diabetes mellitus].

PubMed

Escalada, Francisco Javier

2014-01-01

The hormone glucagon-like peptide-1 (GLP-1) is synthesized and secreted by L cells in the small intestine in response to food ingestion. After reaching the general circulation it has a half-life of 2-3 minutes due to degradation by the enzyme dipeptidyl peptidase-4. Its physiological role is directed to control plasma glucose concentration, though GLP-1 also plays other different metabolic functions following nutrient absorption. Biological activities of GLP-1 include stimulation of insulin biosynthesis and glucose-dependent insulin secretion by pancreatic beta cell, inhibition of glucagon secretion, delay of gastric emptying and inhibition of food intake. GLP-1 is able to reduce plasma glucose levels in patients with type 2 diabetes and also can restore beta cell sensitivity to exogenous secretagogues, suggesting that the increasing GLP-1 concentration may be an useful therapeutic strategy for the treatment of patients with type 2 diabetes.

Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells

PubMed Central

Mairbäurl, Heimo

2013-01-01

During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called “sports anemia.” This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise. PMID:24273518

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

PubMed Central

Shi, Guixin; Cui, Wenjin; Benchimol, Michael; Liu, Yu-Tsueng; Mattrey, Robert F.; Mukthavaram, Rajesh; Kesari, Santosh; Esener, Sadik C.; Simberg, Dmitri

2013-01-01

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells. PMID:23516425

EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: analysis of LUX-Lung 3 and 6

PubMed Central

Wu, Yi-Long; Sequist, Lecia V; Hu, Cheng-Ping; Feng, Jifeng; Lu, Shun; Huang, Yunchao; Li, Wei; Hou, Mei; Schuler, Martin; Mok, Tony; Yamamoto, Nobuyuki; O'Byrne, Kenneth; Hirsh, Vera; Gibson, Neil; Massey, Dan; Kim, Miyoung; Yang, James Chih-Hsin

2017-01-01

Background: In the Phase III LUX-Lung 3/6 (LL3/LL6) trials in epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma patients, we evaluated feasibility of EGFR mutation detection using circulating cell-free DNA (cfDNA) and prognostic and predictive utility of cfDNA positivity (cfDNA+). Methods: Paired tumour and blood samples were prospectively collected from randomised patients. Mutations were detected using cfDNA from serum (LL3) or plasma (LL6) by a validated allele-specific quantitative real-time PCR kit. Results: EGFR mutation detection rates in cfDNA were 28.6% (serum) and 60.5% (plasma). Mutation detection in blood was associated with advanced disease characteristics, including higher performance score, number of metastatic sites and bone/liver metastases, and poorer prognosis. In patients with common EGFR mutations, afatinib improved progression-free survival vs chemotherapy in cfDNA+ (LL3: HR, 0.35; P=0.0009; LL6: HR, 0.25; P<0.0001) and cfDNA− (LL3: HR, 0.46; P<0.0001; LL6: HR, 0.12; P<0.0001) cohorts. A trend towards overall survival benefit with afatinib was observed in cfDNA+ patients. Conclusions: Plasma cfDNA is a promising alternative to biopsy for EGFR testing. Detectable mutation in blood was associated with more advanced disease and poorer prognosis. Afatinib improved outcomes in EGFR mutation-positive patients regardless of blood mutation status. PMID:28006816

CONVERSION OF PLASMA PROTEIN TO TISSUE PROTEIN WITHOUT EVIDENCE OF PROTEIN BREAKDOWN

PubMed Central

Yuile, C. L.; Lamson, B. G.; Miller, L. L.; Whipple, G. H.

1951-01-01

Labeled plasma proteins obtained from donor dogs, previously fed ε-C14-dl-lysine, have been given intravenously to recipient dogs. The disappearance of labeled globulin from the plasma at a rate considerably faster than albumin has been confirmed. Evidence suggesting that the mass of protein in solution in the extravascular, extracellular fluid is approximately equal to the plasma proteins in circulation has been derived from a study of the dilution of labeled plasma protein by repeated injections of non-labeled plasma protein. In a period of 7 days the transfer of C14 from plasma to tissue proteins amounted to between 30 and 40 per cent of the activity in the labeled plasma protein injected intravenously. The conversion was accompanied by a very small loss of activity in the urine and expired air and the activity remained in the lysine residue of the liver and probably of other tissues. The data presented favor the view that plasma proteins are utilized in the body economy after partial catabolism within the cell area and provide no evidence of complete breakdown to the amino acid level. PMID:14832401

A novel CD4-conjugated ultraviolet light-activated photocatalyst inactivates HIV-1 and SIV efficiently.

PubMed

Yamaguchi, Koushi; Sugiyama, Takahiro; Kato, Shinji; Kondo, Yoichi; Ageyama, Naohide; Kanekiyo, Masaru; Iwata, Misao; Koyanagi, Yoshio; Yamamoto, Naoki; Honda, Mitsuo

2008-08-01

In this study, we found that the electric potential derived from the redox reaction of ultraviolet (UV)-illuminated CD4-conjugated titanium dioxide (TiO2) inactivated a wide range of high-titered primary HIV-1 isolates, regardless of virus co-receptor usage or genetic clade. In vitro incubation of HIV-1 isolates with CD4-conjugated TiO2 (CD4-TiO2) followed by UV illumination led to inhibition of viral infectivity in both H9 cells and peripheral blood mononuclear cells as well as to the complete inactivation of plasma virions from HIV-1-infected individuals. Treatment with a newly established extra-corporeal circulation system with the photocatalyst in rhesus macaques completely inactivated plasma virus in the system and effectively reduced the infectious plasma viral load. Furthermore, plasma viremia and infectious viral loads were controlled following a second therapeutic photocatalyst treatment during primary SIV(mac239) infection of macaques. Our findings suggest that this therapeutic immunophysical strategy may help control human immunodeficiency viral infection in vivo.

The characterization of exosome from blood plasma of patients with colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yunusova, N. V., E-mail: Bochkarevanv@oncology.tomsk.ru; Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050; Tamkovich, S. N., E-mail: s.tamk@niboch.nsc.ru

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of “International Society for Extracellular Vesicles”. The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. Themore » results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.« less

The concept of a plasma centrifuge with a high frequency rotating magnetic field and axial circulation

NASA Astrophysics Data System (ADS)

Borisevich, V. D.; Potanin, E. P.

2017-07-01

The possibility of using a rotating magnetic field (RMF) in a plasma centrifuge (PC), with axial circulation to multiply the radial separation effect in an axial direction, is considered. For the first time, a traveling magnetic field (TMF) is proposed to drive an axial circulation flow in a PC. The longitudinal separation effect is calculated for a notional model, using specified operational parameters and the properties of a plasma, comprising an isotopic mixture of 20Ne-22Ne and generated by a high frequency discharge. The optimal intensity of a circulation flow, in which the longitudinal separation effect reaches its maximum value, is studied. The optimal parameters of the RMF and TMF for effective separation, as well as the centrifuge performance, are calculated.

Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing.

PubMed

Lo, Y M Dennis

2013-12-01

The discovery of cell-free fetal DNA in maternal plasma in 1997 has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. Fetal trisomies 21, 18 and 13 are now robustly detectable in maternal plasma and such analyses have been available clinically since 2011. Fetal genome-wide molecular karyotyping and whole-genome sequencing have now been demonstrated in a number of proof-of-concept studies. Genome-wide and targeted sequencing of maternal plasma has been shown to allow the non-invasive prenatal testing of β-thalassaemia and can potentially be generalized to other monogenic diseases. It is thus expected that plasma DNA-based non-invasive prenatal testing will play an increasingly important role in future obstetric care. It is thus timely and important that the ethical, social and legal issues of non-invasive prenatal testing be discussed actively by all parties involved in prenatal care. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-05-31

The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients

PubMed Central

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-01-01

Background The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). Methods A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information. PMID:27102299

Fragment Length of Circulating Tumor DNA

PubMed Central

Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

2016-01-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

Assessment of the ``cross-bridge''-induced interaction of red blood cells by optical trapping combined with microfluidics

NASA Astrophysics Data System (ADS)

Lee, Kisung; Wagner, Christian; Priezzhev, Alexander V.

2017-09-01

Red blood cell (RBC) aggregation is an intrinsic property of the blood that has a direct effect on the blood viscosity and circulation. Nevertheless, the mechanism behind the RBC aggregation has not been confirmed and is still under investigation with two major hypotheses, known as "depletion layer" and "cross-bridging." We aim to ultimately understand the mechanism of the RBC aggregation and clarify both models. To measure the cell interaction in vitro in different suspensions (including plasma, isotonic solution of fibrinogen, isotonic solution of fibrinogen with albumin, and phosphate buffer saline) while moving the aggregate from one solution to another, an approach combining optical trapping and microfluidics has been applied. The study reveals evidence that RBC aggregation in plasma is at least partly due to the cross-bridging mechanism. The cell interaction strength measured in the final solution was found to be significantly changed depending on the initial solution where the aggregate was formed.

Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

PubMed Central

Jison, Maria L.; Munson, Peter J.; Barb, Jennifer J.; Suffredini, Anthony F.; Talwar, Shefali; Logun, Carolea; Raghavachari, Nalini; Beigel, John H.; Shelhamer, James H.; Danner, Robert L.; Gladwin, Mark T.

2016-01-01

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury. PMID:15031206

Exercise intensity modulates the appearance of circulating microvesicles with proangiogenic potential upon endothelial cells.

PubMed

Wilhelm, Eurico N; González-Alonso, José; Parris, Christopher; Rakobowchuk, Mark

2016-11-01

The effect of endurance exercise on circulating microvesicle dynamics and their impact on surrounding endothelial cells is unclear. Here we tested the hypothesis that exercise intensity modulates the time course of platelet (PMV) and endothelial-derived (EMV) microvesicle appearance in the circulation through hemodynamic and biochemical-related mechanisms, and that microvesicles formed during exercise would stimulate endothelial angiogenesis in vitro. Nine healthy young men had venous blood samples taken before, during, and throughout the recovery period after 1 h of moderate [46 ± 2% maximal oxygen uptake (V̇o 2max )] or heavy (67 ± 2% V̇o 2max ) intensity semirecumbent cycling and a time-matched resting control trial. In vitro experiments were performed by incubating endothelial cells with rest and exercise-derived microvesicles to examine their effects on cell angiogenic capacities. PMVs (CD41 + ) increased from baseline only during heavy exercise (from 21 ± 1 × 10 3 to 55 ± 8 × 10 3 and 48 ± 6 × 10 3 PMV/μl at 30 and 60 min, respectively; P < 0.05), returning to baseline early in postexercise recovery (P > 0.05), whereas EMVs (CD62E + ) were unchanged (P > 0.05). PMVs were related to brachial artery shear rate (r 2 = 0.43) and plasma norepinephrine concentrations (r 2 = 0.21) during exercise (P < 0.05). Exercise-derived microvesicles enhanced endothelial proliferation, migration, and tubule formation compared with rest microvesicles (P < 0.05). These results demonstrate substantial increases in circulating PMVs during heavy exercise and that exercise-derived microvesicles stimulate human endothelial cells by enhancing angiogenesis and proliferation. This involvement of microvesicles may be considered a novel mechanism through which exercise mediates vascular healing and adaptation. Copyright © 2016 the American Physiological Society.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

PubMed Central

Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2016-01-01

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles].

PubMed

de la Cuesta, F; Baldan-Martin, M; Mourino-Alvarez, L; Sastre-Oliva, T; Alvarez-Llamas, G; Gonzalez-Calero, L; Ruiz-Hurtado, G; Segura, J; Vivanco, F; Ruilope, L M; Barderas, M G

2016-01-01

Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma.

PubMed

Treon, S P; Maimonis, P; Bua, D; Young, G; Raje, N; Mollick, J; Chauhan, D; Tai, Y T; Hideshima, T; Shima, Y; Hilgers, J; von Mensdorff-Pouilly, S; Belch, A R; Pilarski, L M; Anderson, K C

2000-11-01

Soluble MUC1 (sMUC1) levels are elevated in many MUC1(+) cancers. We and others have shown that MUC1 is expressed on multiple myeloma (MM) plasma cells and B cells. In this study, we measured sMUC1 levels in bone marrow (BM) plasma from 71 MM patients and 21 healthy donors (HDs), and in peripheral blood (PB) plasma from 42 MM patients and 13 HDs using an immunoassay that detects the CA27.29 epitope of MUC1. sMUC1 levels were found to be significantly greater (mean 31.76 U/mL, range 5.69 to 142.48 U/mL) in MM patient BM plasma versus HD BM plasma (mean 9.68 U/mL, range 0.65 to 39.83 U/mL) (P <. 001). Importantly, BM plasma sMUC1 levels were related to tumor burden because sMUC1 levels were significantly higher for MM patients with active disease (34.62 U/mL, range 5.69 to 142.48 U/mL) versus MM patients with minimal residual disease (16.16 U/mL, range 5.7 to 56.68 U/mL) (P =.0026). sMUC1 levels were also elevated in the PB plasma of MM patients (32.79 U/mL, range 4.15 to 148.84 U/mL) versus HDs (18.47 U/mL, range 8.84 to 42.49) (P =.0052). Lastly, circulating immunglobulin M (IgM) and IgG antibodies to MUC1 were measured in 114 MM patients and 31 HDs, because natural antibodies to MUC1 have been detected in patients with other MUC1-bearing malignancies. These studies demonstrated lower levels of circulating IgM (P <.001) and IgG (P =.078) antibodies to MUC1 in MM patients compared with HDs. Our data therefore show that in MM patients, sMUC1 levels are elevated and correlate with disease burden, whereas anti-MUC1 antibody levels are decreased.

Seminal Plasma Enhances Cervical Adenocarcinoma Cell Proliferation and Tumour Growth In Vivo

PubMed Central

Sutherland, Jason R.; Sales, Kurt J.; Jabbour, Henry N.; Katz, Arieh A.

2012-01-01

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A(VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway. PMID:22442729

Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo.

PubMed

Sutherland, Jason R; Sales, Kurt J; Jabbour, Henry N; Katz, Arieh A

2012-01-01

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

Circulating Immunoglobulins Are Not Associated with Intraplaque Mast Cell Number and Other Vulnerable Plaque Characteristics in Patients with Carotid Artery Stenosis

PubMed Central

Quax, Paul H. A.; de Borst, Gert Jan; de Vries, Jean-Paul P. M.; Moll, Frans L.; Kuiper, Johan; Toes, René E. M.; de Jager, Saskia C. A.; de Kleijn, Dominique P. V.; Hoefer, Imo E.; Pasterkamp, Gerard; Bot, Ilze

2014-01-01

Background Recently, we have shown that intraplaque mast cell numbers are associated with atherosclerotic plaque vulnerability and with future cardiovascular events, which renders inhibition of mast cell activation of interest for future therapeutic interventions. However, the endogenous triggers that activate mast cells during the progression and destabilization of atherosclerotic lesions remain unidentified. Mast cells can be activated by immunoglobulins and in the present study, we aimed to establish whether specific immunoglobulins in plasma of patients scheduled for carotid endarterectomy were related to (activated) intraplaque mast cell numbers and plasma tryptase levels. In addition, the levels were related to other vulnerable plaque characteristics and baseline clinical data. Methods and Results OxLDL-IgG, total IgG and total IgE levels were measured in 135 patients who underwent carotid endarterectomy. No associations were observed between the tested plasma immunoglobulin levels and total mast cell numbers in atherosclerotic plaques. Furthermore, no associations were found between IgG levels and the following plaque characteristics: lipid core size, degree of calcification, number of macrophages or smooth muscle cells, amount of collagen and number of microvessels. Interestingly, statin use was negatively associated with plasma IgE and oxLDL-IgG levels. Conclusions In patients suffering from carotid artery disease, total IgE, total IgG and oxLDL-IgG levels do not associate with plaque mast cell numbers or other vulnerable plaque histopathological characteristics. This study thus does not provide evidence that the immunoglobulins tested in our cohort play a role in intraplaque mast cell activation or grade of atherosclerosis. PMID:24586471

Novel Technology for Enrichment of Biomolecules from Cell-Free Body Fluids and Subsequent DNA Sizing.

PubMed

Patel, Vipulkumar; Celec, Peter; Grunt, Magdalena; Schwarzenbach, Heidi; Jenneckens, Ingo; Hillebrand, Timo

2016-01-01

Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length. This method allows isolation of low-abundant DNA as well as separation of long and short DNA molecules. This procedure may be important e.g., in prenatal diagnostics and cancer research that have been already confirmed by our primary experiments. Here, we report the results of selective separation of 200- and 500-bp long synthetic DNA fragments spiked in plasma samples. Furthermore, we size-fractionated ccfDNA from the plasma of pregnant women and verified the prevalence of fetal ccfDNA in all fractions.

Hypercholesterolemia induces T cell expansion in humanized immune mice.

PubMed

Proto, Jonathan D; Doran, Amanda C; Subramanian, Manikandan; Wang, Hui; Zhang, Mingyou; Sozen, Erdi; Rymond, Christina C; Kuriakose, George; D'Agati, Vivette; Winchester, Robert; Sykes, Megan; Yang, Yong-Guang; Tabas, Ira

2018-06-01

Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting.

Non-invasive detection of human cardiomyocyte death using methylation patterns of circulating DNA.

PubMed

Zemmour, Hai; Planer, David; Magenheim, Judith; Moss, Joshua; Neiman, Daniel; Gilon, Dan; Korach, Amit; Glaser, Benjamin; Shemer, Ruth; Landesberg, Giora; Dor, Yuval

2018-04-24

Detection of cardiomyocyte death is crucial for the diagnosis and treatment of heart disease. Here we use comparative methylome analysis to identify genomic loci that are unmethylated specifically in cardiomyocytes, and develop these as biomarkers to quantify cardiomyocyte DNA in circulating cell-free DNA (cfDNA) derived from dying cells. Plasma of healthy individuals contains essentially no cardiomyocyte cfDNA, consistent with minimal cardiac turnover. Patients with acute ST-elevation myocardial infarction show a robust cardiac cfDNA signal that correlates with levels of troponin and creatine phosphokinase (CPK), including the expected elevation-decay dynamics following coronary angioplasty. Patients with sepsis have high cardiac cfDNA concentrations that strongly predict mortality, suggesting a major role of cardiomyocyte death in mortality from sepsis. A cfDNA biomarker for cardiomyocyte death may find utility in diagnosis and monitoring of cardiac pathologies and in the study of normal human cardiac physiology and development.

Circulating Levels of Human salusin-β,a Potent Hemodynamic and Atherogenesis Regulator

PubMed Central

Fujimoto, Kazumi; Hayashi, Akinori; Kamata, Yuji; Ogawa, Akifumi; Watanabe, Takuya; Ichikawa, Raishi; Iso, Yoshitaka; Koba, Shinji; Kobayashi, Youichi; Koyama, Takatoshi; Shichiri, Masayoshi

2013-01-01

Using bioinformatics analysis, we previously identified salusin-β, an endogenous bioactive peptide with diverse physiological activities. Salusin-β is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-β acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-β concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-β was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-β peak coincided with synthetic authentic salusin-β. Plasma salusin-β concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-β concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-β levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-β levels and for confirming the presence of authentic salusin-β in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-β concentrations and a role for plasma total salusin-β concentrations as an indicator of systemic vascular diseases. PMID:24098553

Detection of EGFR Variants in Plasma: A Multilaboratory Comparison of the cobas EGFR Mutation Test v2 in Europe.

PubMed

Keppens, Cleo; Palma, John F; Das, Partha M; Scudder, Sidney; Wen, Wei; Normanno, Nicola; Van Krieken, J Han; Sacco, Alessandra; Fenizia, Francesca; de Castro, David Gonzalez; Hönigschnabl, Selma; Kern, Izidor; Lopez-Rios, Fernando; Lozano, Maria D; Marchetti, Antonio; Halfon, Philippe; Schuuring, Ed; Setinek, Ulrike; Sorensen, Boe; Taniere, Phillipe; Tiemann, Markus; Vosmikova, Hana; Dequeker, Elisabeth M C

2018-04-25

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small-cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of the cobas EGFR Mutation Test v2 to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild-type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per mL. The circulating tumor DNA was extracted by the cobas cfDNA Sample Preparation kit, followed by duplicate analysis with the EGFRv2 kit (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. Specificities were all 98.8% to 100.0%. Coefficients of variation indicated good intra and interlaboratory repeatability and reproducibility, but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the pre-defined copy number and the observed semiquantitative index values which reflects the samples' plasma mutation load. This study demonstrates an overall robust performance of the EGFRv2 kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Plasma functionalization of powdery nanomaterials using porous filter electrode and sample circulation

NASA Astrophysics Data System (ADS)

Lee, Deuk Yeon; Choi, Jae Hong; Shin, Jung Chul; Jung, Man Ki; Song, Seok Kyun; Suh, Jung Ki; Lee, Chang Young

2018-06-01

Compared with wet processes, dry functionalization using plasma is fast, scalable, solvent-free, and thus presents a promising approach for grafting functional groups to powdery nanomaterials. Previous approaches, however, had difficulties in maintaining an intimate sample-plasma contact and achieving uniform functionalization. Here, we demonstrate a plasma reactor equipped with a porous filter electrode that increases both homogeneity and degree of functionalization by capturing and circulating powdery carbon nanotubes (CNTs) via vacuum and gas blowing. Spectroscopic measurements verify that treatment with O2/air plasma generates oxygen-containing groups on the surface of CNTs, with the degree of functionalization readily controlled by varying the circulation number. Gas sensors fabricated using the plasma-treated CNTs confirm alteration of molecular adsorption on the surface of CNTs. A sequential treatment with NH3 plasma following the oxidation pre-treatment results in the functionalization with nitrogen species of up to 3.2 wt%. Our approach requiring no organic solvents not only is cost-effective and environmentally friendly, but also serves as a versatile tool that applies to other powdery micro or nanoscale materials for controlled modification of their surfaces.

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube.

PubMed

György, Bence; Pálóczi, Krisztina; Kovács, Alexandra; Barabás, Eszter; Bekő, Gabriella; Várnai, Katalin; Pállinger, Éva; Szabó-Taylor, Katalin; Szabó, Tamás G; Kiss, Attila A; Falus, András; Buzás, Edit I

2014-02-01

Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples. We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes. Agitation and storage of blood samples at 37 °C for 1 hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes. We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impact of Delayed Whole Blood Processing Time on Plasma Levels of miR-1 and miR-423-5p up to 24 Hours.

PubMed

Borges, Danielle Pazzotti; Cunha-Neto, Edecio; Bocchi, Edimar A; Rigaud, Vagner Oliveira Carvalho

2018-03-21

Circulating cell-free miRNAs hold great promise as a new class of biomarkers due to their high stability in body fluids and association with disease stages. However, even using sensitive and specific methods, technical challenges are associated with miRNA analysis in body fluids. A major source of variation in plasma and serum is the potential cell-derived miRNA contamination from hemolysis. To evaluate the effect of the delayed whole blood processing time on the concentrations of miR-1 and -423-5p. Ten blood samples were incubated for 0, 3 and 24 hours at room temperature prior processing into plasma. For each time point, hemolysis was assessed in plasma by UV spectrophotometry at 414nm wavelength (λ414). Circulating levels of miR-1 and -423-5p were measured by RT-qPCR; miR-23a and -451 were also analyzed as controls. A significant hemolysis was observed only after 24h (λ414 0.3±0.02, p<0.001). However, only small changes in miR-1 and -423-5p levels were observed up to 24h of storage at room temperature (Ct 31.5±0.5 to 31.8±0.6for miR-1, p=0.989; and 29.01±0.3 to 29.04±0.3, p=0.614 for -423-5p). No correlation was observed between hemolysis and levels of miR-1 and -423-5p. Our data indicate the storage of whole blood samples at room temperature for up to 24h prior their processing into plasma does not appear to have a significant impact on miR-1 and -423-5p concentrations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quantification of Circulating Clonal Plasma Cells via Multiparametric Flow Cytometry Identifies Patients with Smoldering Multiple Myeloma at High Risk of Progression

PubMed Central

Gonsalves, Wilson I.; Rajkumar, S. Vincent; Dispenzieri, Angela; Dingli, David; Timm, Michael M.; Morice, William G.; Lacy, Martha Q.; Buadi, Francis K.; Go, Ronald S.; Leung, Nelson; Kapoor, Prashant; Hayman, Suzanne R.; Lust, John A.; Russell, Stephen J.; Zeldenrust, Steven R.; Hwa, Lisa; Kourelis, Taxiarchis V.; Kyle, Robert A.; Gertz, Morie A.; Kumar, Shaji K.

2017-01-01

The presence of high numbers of circulating clonal plasma cells (cPCs) in patients with smoldering multiple myeloma (SMM), detected by a slide-based immunofluorescence assay, has been associated with a shorter time to progression (TTP) to multiple myeloma (MM). The significance of quantifying cPCs via multiparameter flow cytometry, a much more readily available diagnostic modality, in patients with SMM has not been evaluated. This study evaluated 100 patients with a known or new diagnosis of SMM who were seen at the Mayo Clinic, Rochester from January 2008 until December 2013. Patients with ≥ 150 cPCs (N = 9) were considered to have high number of cPCs based on the 97% specificity and 78% PPV of progression to MM within 2 years of cPC assessment. The median TTP of patients with ≥ 150 cPCs was 9 months compared to not reached for patients with < 150 cPCs (P < 0.001). Thus, quantification of cPCs via multiparametric flow cytometry identifies patients with SMM at very high risk of progression to MM within 2 years and warrants confirmation in larger studies. In the future, this may allow reclassification of such patients as having MM requiring therapy prior to them enduring end-organ damage. PMID:27457702

Formation and plasma circulation of solar prominences and coronal rains

NASA Astrophysics Data System (ADS)

Xia, C.

2016-12-01

Solar prominences are long-lived cool and dense plasma curtains in the hot and rarefied corona. The physical mechanism responsible for their formation and especially for their internal plasma circulation has been uncertain for decades. The observed ubiquitous down flows in quiescent prominences are difficult to interpret as plasma with high conductivity seems to move across horizontal magnetic field lines. Here we present three-dimensional (3D) numerical simulations of prominence formation and evolution in an elongated magnetic flux rope as a result of in-situ plasma condensations fueled by continuous plasma evaporation from the solar chromosphere. The prominence is born and maintained in a fragmented, highly dynamic state with continuous reappearance of multiple blobs and thread structures that move mainly downward dragging along mass-loaded field lines. The prominence plasma circulation is characterized by the dynamic balance between the drainage of prominence plasma back to the chromosphere and the formation of prominence plasma via continuous condensation. Plasma evaporates from the chromosphere, condenses into the prominence in the corona, and drains back to the chromosphere, establishing a stable chromosphere-corona plasma cycle. Another form of cool and dense plasma in the corona is coronal rain, which forms in-situ and drain down arched pathways along loops near active regions. We present 3D simulations of coronal rain in a bipolar arcade and compare it with observational results.

Vitamin A levels and human immunodeficiency virus load in injection drug users.

PubMed Central

Semba, R D; Farzadegan, H; Vlahov, D

1997-01-01

Although low plasma vitamin A levels are associated with increased mortality and higher vertical transmission during human immunodeficiency virus (HIV) infection, it is unknown whether plasma low vitamin A levels are a marker for circulating HIV load. We conducted a cross-sectional study within a prospective cohort study of injection drug users in order to evaluate the relationship between plasma vitamin A levels and HIV viral load. Plasma vitamin A level was measured by high-performance liquid chromatography. Infectious viral load was measured by quantitative microculture of serial fivefold dilutions of 10(6) peripheral blood mononuclear cells. A total of 284 HIV-infected adults (79 women, 205 men) were studied. Plasma vitamin A levels consistent with deficiency were found in 28.9% of adults. A total of 38.0% of women and 25.3% of men had vitamin A deficiency (P < 0.04). The median infectious viral load for the entire study population was 8 infectious units per million cells. No significant relationship between plasma vitamin A levels and infectious viral load was observed in these injection drug users. This study suggests that there is no correlation between HIV viral load and plasma vitamin A levels in injection drug users, and these variables may represent independent risk factors during HIV infection. HIV-infected adult women appear to be at higher risk of developing vitamin A deficiency. PMID:9008289

Correlations between the Dielectric Properties and Exterior Morphology of Cells Revealed by Dielectrophoretic Field-Flow Fractionation

PubMed Central

Gascoyne, Peter R. C.; Shim, Sangjo; Noshari, Jamileh; Becker, Frederick F.; Stemke-Hale, Katherine

2013-01-01

Although dielectrophoresis (DEP) has great potential for addressing clinical cell isolation problems based on cell dielectric differences, a biological basis for predicting the DEP behavior of cells has been lacking. Here, the dielectric properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic (DEP) field-flow fractionation, correlated with the exterior morphologies of the cells during growth, and compared with the dielectric and morphological characteristics of the subpopulations of peripheral blood. In agreement with earlier findings, cell total capacitance varied with both cell size and plasma membrane folding and the dielectric properties of the NCI-60 cell types in suspension reflected the plasma membrane area and volume of the cells at their growth sites. Therefore, the behavior of cells in DEP-based manipulations is largely determined by their exterior morphological characteristics prior to release into suspension. As a consequence, DEP is able to discriminate between cells of similar size having different morphological origins, offering a significant advantage over size-based filtering for isolating circulating tumor cells, for example. The findings provide a framework for anticipating cell dielectric behavior on the basis of structure-function relationships and suggest that DEP should be widely applicable as a surface marker-independent method for sorting cells. PMID:23172680

Concentrations of amino acids in plasma from 45- to 47-week gestation mares and foetuses (Equus caballus).

PubMed

Zicker, S C; Vivrette, S; Rogers, Q R

1994-06-01

Concentrations of 16 of 24 amino acids in plasma of foetuses were significantly higher, while four of 24 were lower, than their concentration in maternal plasma. The higher foetal concentrations of amino acids in plasma are similar to other species, with some exceptions, and suggest that equine placenta actively transports and concentrates amino acids into the umbilical circulation. Concentrations of nine of 24 amino acids were significantly lower in plasma from the umbilical artery compared to plasma from the umbilical vein, while no significant differences were present between maternal artery and vein plasma. The umbilical venous-arterial difference in concentrations of amino acids in plasma suggests the foetus extracts amino acids from the umbilical circulation for catabolism or protein synthesis, as in other species.

The Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

NASA Astrophysics Data System (ADS)

Liberda, Eric N.

Introduction. Particulate air pollution, specifically nickel found on or in particulate matter, has been associated with an increased risk of mortality in human population studies and can cause increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiate atherosclerosis in murine exposures. With the discovery of endothelial progenitor cells (EPCs), a door has been opened which may explain these observed cardiovascular effects associated with inhaled air particles and nickel exposure. In order to further quantify the effects of inhaled nickel nanoparticles and attempt to elucidate how the observed findings from other studies may occur, several whole body inhalation exposure experiments to nickel nanoparticles were performed. Methods. Following whole body exposure to approximately 500mug/m3 of nickel nanoparticles for 5 hrs, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation, and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells, circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the inhalation exposure. Plasma proteins were assessed using the 2D DIGE proteomic approach and commercially available ELISAs. Results and Conclusions. Exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation. CECs were significantly upregulated suggesting that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. This decrease in EPC function coincided with downregulation of receptors for EPC mobilization and homing. Antioxidant plasma proteins were upregulated post-exposure and transferrin was downregulated. In conclusion, these results indicate that inhalation exposure to Ni nanoparticles below the current OSHA permissible exposure limit for Ni compounds can lead to alterations in bone marrow progenitor cells that may ultimately lead to the development of various cardiovascular diseases.

Peptides and Food Intake

PubMed Central

Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

2014-01-01

The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food intake, whereas the other subpopulation coexpresses pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript that inhibits food intake. AgRP antagonizes the effects of the POMC product, α-melanocyte-stimulating hormone (α-MSH). Both populations project to areas important in the regulation of food intake, including the hypothalamic paraventricular nucleus, which also receives important inputs from other hypothalamic nuclei. PMID:24795698

The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

NASA Astrophysics Data System (ADS)

Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

2014-03-01

The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

Lactobacillus johnsonii Supplementation Attenuates Respiratory Viral Infection via Metabolic Reprogramming and Immune Cell Modulation

PubMed Central

Fonseca, Wendy; Lucey, Kaitlyn; Jang, Sihyug; Fujimura, Kei E.; Rasky, Andrew; Ting, Hung-An; Petersen, Julia; Johnson, Christine C.; Boushey, Homer A.; Zoratti, Edward; Ownby, Dennis R.; Levine, Albert M.; Bobbit, Kevin R.

2017-01-01

Summary Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii-supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii-supplementation reduced airway Th2 cytokines, dendritic cell function, increased T-regulatory cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone-marrow derived dendritic cells (BMDC) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice, or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice, or with wild-type derived BMDCs pre-treated with plasma from L. johnsonii-supplemented mice, reduced airway pathologic responses to infection in recipient animals. Thus, L. johnsonii-supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function. PMID:28295020

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

PubMed Central

Krause, Sabrina; Boeck, Christina; Gumpp, Anja M.; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

2018-01-01

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system. PMID:29535656

Dysregulation of in vitro cytokine production by monocytes during sepsis.

PubMed Central

Munoz, C; Carlet, J; Fitting, C; Misset, B; Blériot, J P; Cavaillon, J M

1991-01-01

The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections. Images PMID:1939659

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

PubMed Central

Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

2014-01-01

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

PubMed

Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

2014-01-01

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

PubMed Central

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M.; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W. Y.; Marass, Francesco; Gale, Davina; Ali, H. Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P.; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-01-01

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution. PMID:26530965

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.

PubMed

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W Y; Marass, Francesco; Gale, Davina; Ali, H Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-11-04

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Type 2 diabetes associated changes in the plasma non-esterified fatty acids, oxylipins and endocannabinoids

USDA-ARS?s Scientific Manuscript database

Type 2 diabetes (T2D) has profound effects on metabolism that can be detected in plasma. While increases in circulating non-esterified fatty acids (NEFA) are well described in T2D, effects on circulating signaling lipids have received little attention. Oxylipins and endocannabinoids are classes of ...

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy

PubMed Central

Bonechi, Martina; Galardi, Francesca; Biagioni, Chiara; De Luca, Francesca; Bergqvist, Mattias; Neumüller, Magnus; Guarducci, Cristina; Boccalini, Giulia; Gabellini, Stefano; Migliaccio, Ilenia; Di Leo, Angelo; Pestrin, Marta; Malorni, Luca

2018-01-01

The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1/PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients. PMID:29662653

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy.

PubMed

Bonechi, Martina; Galardi, Francesca; Biagioni, Chiara; De Luca, Francesca; Bergqvist, Mattias; Neumüller, Magnus; Guarducci, Cristina; Boccalini, Giulia; Gabellini, Stefano; Migliaccio, Ilenia; Di Leo, Angelo; Pestrin, Marta; Malorni, Luca

2018-03-27

The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1 / PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.

Elevated expression of CXC chemokines in pediatric osteosarcoma patients.

PubMed

Li, Yiting; Flores, Ricardo; Yu, Alexander; Okcu, M Fatih; Murray, Jeffrey; Chintagumpala, Murali; Hicks, John; Lau, Ching C; Man, Tsz-Kwong

2011-01-01

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis. The authors used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using enzyme-linked immunosorbent assays on an independent set of samples. The tumor expressions of 3 chemokines were examined in 2 sets of osteosarcoma tissue arrays. The authors also evaluated the proliferative effect of the chemokines in 4 osteosarcoma cell lines. The authors found that the plasma levels of CXCL4, CXCL6, and CXCL12 in the osteosarcoma patients were significantly higher than those in the controls, and the results were validated by an independent osteosarcoma cohort (P < .05). However, CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (P < .05). The results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma, and that the plasma levels of these 2 chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention. © 2010 American Cancer Society.

Grazing Affects Exosomal Circulating MicroRNAs in Cattle

PubMed Central

Muroya, Susumu; Ogasawara, Hideki; Hojito, Masayuki

2015-01-01

Circulating microRNAs (c-miRNAs) are associated with physiological adaptation to acute and chronic aerobic exercise in humans. To investigate the potential effect of grazing movement on miRNA circulation in cattle, here we profiled miRNA expression in centrifugally prepared exosomes from the plasma of both grazing and housed Japanese Shorthorn cattle. Microarray analysis of the c-miRNAs resulted in detection of a total of 231 bovine exosomal miRNAs in the plasma, with a constant expression level of let-7g across the duration and cattle groups. Expression of muscle-specific miRNAs such as miR-1, miR-133a, miR-206, miR-208a/b, and miR-499 were undetectable, suggesting the mildness of grazing movement as exercise. According to validation by quantitative RT-PCR, the circulating miR-150 level in the grazing cattle normalized by the endogenous let-7g level was down-regulated after 2 and 4 months of grazing (P < 0.05), and then its levels in housed and grazing cattle equalized when the grazing cattle were returned to a housed situation. Likewise, the levels of miR-19b, miR-148a, miR-221, miR-223, miR-320a, miR-361, and miR-486 were temporarily lowered in the cattle at 1 and/or 2 month of grazing compared to those of the housed cattle (P < 0.05). In contrast, the miR-451 level was up-regulated in the grazing cattle at 2 months of grazing (P = 0.044). The elevation of miR-451 level in the plasma was coincident with that in the biceps femoris muscle of the grazing cattle (P = 0.008), which suggests the secretion or intake of miR-451 between skeletal muscle cells and circulation during grazing. These results revealed that exosomal c-miRNAs in cattle were affected by grazing, suggesting their usefulness as molecular grazing markers and functions in physiological adaptation of grazing cattle associated with endocytosis, focal adhesion, axon guidance, and a variety of intracellular signaling, as predicted by bioinformatic analysis. PMID:26308447

Interventional Vitamin C-A Strategy for Attenuation of Coagulopathy and Inflammation in Hemorrhagic Trauma and Shock

DTIC Science & Technology

2017-10-01

edema, protein leak and exuberant infiltration of inflammatory cells. Significant hemorrhage and cellular damage were also evident in liver and kidney ...Treatment with VitC also reduced the expression of pro-inflammatory mediators in lungs, liver and kidneys . Preliminary lipidomic analysis showed that VitC at...Histological changes to lung, liver and kidney f. Proteomic analysis of plasma for identification of novel circulating proteins, and g. Lipidomic

A Lower Olfactory Capacity Is Related to Higher Circulating Concentrations of Endocannabinoid 2-Arachidonoylglycerol and Higher Body Mass Index in Women.

PubMed

Pastor, Antoni; Fernández-Aranda, Fernando; Fitó, Montserrat; Jiménez-Murcia, Susana; Botella, Cristina; Fernández-Real, Jose M; Frühbeck, Gema; Tinahones, Francisco J; Fagundo, Ana B; Rodriguez, Joan; Agüera, Zaida; Langohr, Klaus; Casanueva, Felipe F; de la Torre, Rafael

2016-01-01

The endocannabinoid (eCB) system can promote food intake by increasing odor detection in mice. The eCB system is over-active in human obesity. Our aim is to measure circulating eCB concentrations and olfactory capacity in a human sample that includes people with obesity and explore the possible interaction between olfaction, obesity and the eCB system. The study sample was made up of 161 females with five groups of body mass index sub-categories ranging from under-weight to morbidly obese. We assessed olfactory capacity with the "Sniffin´Sticks" test, which measures olfactory threshold-discrimination-identification (TDI) capacity. We measured plasma concentrations of the eCBs 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine or anandamide (AEA), and several eCB-related compounds, 2-acylglycerols and N-acylethanolamines. 2-AG and other 2-acylglycerols fasting plasma circulating plasma concentrations were higher in obese and morbidly obese subjects. AEA and other N-acylethanolamine circulating concentrations were lower in under-weight subjects. Olfactory TDI scores were lower in obese and morbidly obese subjects. Lower TDI scores were independently associated with higher 2-AG fasting plasma circulating concentrations, higher %body fat, and higher body mass index, after controlling for age, smoking, menstruation, and use of contraceptives. Our results show that obese subjects have a lower olfactory capacity than non-obese ones and that elevated fasting plasma circulating 2-AG concentrations in obesity are linked to a lower olfactory capacity. In agreement with previous studies we show that eCBs AEA and 2-AG, and their respective congeners have a distinct profile in relation to body mass index. The present report is the first study in humans in which olfactory capacity and circulating eCB concentrations have been measured in the same subjects.

Customized Internal Reference Controls for Improved Assessment of Circulating MicroRNAs in Disease.

PubMed

Schlosser, Kenny; McIntyre, Lauralyn A; White, R James; Stewart, Duncan J

2015-01-01

Altered levels of circulating extracellular miRNA in plasma and serum have shown promise as non-invasive biomarkers of disease. However, unlike the assessment of cellular miRNA levels for which there are accepted housekeeping genes, analogous reference controls for normalization of circulating miRNA are lacking. Here, we provide an approach to identify and validate circulating miRNA reference controls on a de novo basis, and demonstrate the advantages of these customized internal controls in different disease settings. Importantly, these internal controls overcome key limitations of external spike-in controls. Using a global RT-qPCR screen of 1066 miRNAs in plasma from pulmonary hypertension patients (PAH) and healthy subjects as a case example, we identified a large pool of initial candidate miRNAs that were systematically ranked according to their plasma level stability using a predefined algorithm. The performance of the top candidates was validated against multiple comparators, and in a second independent cohort of PAH and control subjects. The broader utility of this approach was demonstrated in a completely different disease setting with 372 miRNAs screened in plasma from septic shock patients and healthy controls. Normalization of data with specific internal reference controls significantly reduced the overall variation in circulating miRNA levels between subjects (relative to raw data), provided a more balanced distribution of up- and down-regulated miRNAs, replicated the results obtained by the benchmark geometric averaging of all detected miRNAs, and outperformed the commonly used external spike-in strategy. We demonstrate the feasibility of identifying circulating reference controls that can reduce extraneous technical variations, and improve the assessment of disease-related changes in plasma miRNA levels. This study provides a novel conceptual framework that addresses a critical and previously unmet need if circulating miRNAs are to advance as reliable diagnostic tools in medicine.

Customized Internal Reference Controls for Improved Assessment of Circulating MicroRNAs in Disease

PubMed Central

Schlosser, Kenny; McIntyre, Lauralyn A.; White, R. James; Stewart, Duncan J.

2015-01-01

Background Altered levels of circulating extracellular miRNA in plasma and serum have shown promise as non-invasive biomarkers of disease. However, unlike the assessment of cellular miRNA levels for which there are accepted housekeeping genes, analogous reference controls for normalization of circulating miRNA are lacking. Here, we provide an approach to identify and validate circulating miRNA reference controls on a de novo basis, and demonstrate the advantages of these customized internal controls in different disease settings. Importantly, these internal controls overcome key limitations of external spike-in controls. Methods Using a global RT-qPCR screen of 1066 miRNAs in plasma from pulmonary hypertension patients (PAH) and healthy subjects as a case example, we identified a large pool of initial candidate miRNAs that were systematically ranked according to their plasma level stability using a predefined algorithm. The performance of the top candidates was validated against multiple comparators, and in a second independent cohort of PAH and control subjects. The broader utility of this approach was demonstrated in a completely different disease setting with 372 miRNAs screened in plasma from septic shock patients and healthy controls. Results Normalization of data with specific internal reference controls significantly reduced the overall variation in circulating miRNA levels between subjects (relative to raw data), provided a more balanced distribution of up- and down-regulated miRNAs, replicated the results obtained by the benchmark geometric averaging of all detected miRNAs, and outperformed the commonly used external spike-in strategy. Conclusions We demonstrate the feasibility of identifying circulating reference controls that can reduce extraneous technical variations, and improve the assessment of disease-related changes in plasma miRNA levels. This study provides a novel conceptual framework that addresses a critical and previously unmet need if circulating miRNAs are to advance as reliable diagnostic tools in medicine. PMID:26010841

Plasma-derived microparticles in polycythaemia vera.

PubMed

Ahadon, M; Abdul Aziz, S; Wong, C L; Leong, C F

2018-04-01

Microparticles are membrane bound vesicles, measuring less than 1.0 um, which are released during cellular activation or during apoptosis. Studies have shown that these circulating microparticles play a role in coagulation, cell signaling and cellular interactions. Increased levels of circulating microparticles have been observed in a number of conditions where there is vascular dysfunction, thrombosis and inflammation. The objective of this study was to determine the various plasma-derived microparticles in patients with polycythaemia vera (PV) in Universiti Kebangsaan Malaysia Medical Centre and to compare them with normal control. A total of 15 patients with PV and 15 healthy volunteers were included in this cross-sectional descriptive study. Plasma samples from both patients and healthy volunteers were prepared and further processed for isolation of microparticles. Flow cytometry analyses were then carried out in all samples to determine the cellular origin of the microparticles. Full blood count parameters for both groups were also collected. Data collected were analyzed using SPSS version 12.0. Patients with PV had a significantly higher percentage of platelet derived microparticles compared to healthy controls (P <0.05). The control group had a higher level of endothelial derived microparticles but the differences were not statistically significant (P > 0.05). The median percentage of positive events for platelet derived microparticles was higher in patients with PV compared to normal healthy controls.

Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects

PubMed Central

Yeri, Ashish; Courtright, Amanda; Reiman, Rebecca; Carlson, Elizabeth; Beecroft, Taylor; Janss, Alex; Siniard, Ashley; Richholt, Ryan; Balak, Chris; Rozowsky, Joel; Kitchen, Robert; Hutchins, Elizabeth; Winarta, Joseph; McCoy, Roger; Anastasi, Matthew; Kim, Seungchan; Huentelman, Matthew; Van Keuren-Jensen, Kendall

2017-01-01

Interest in circulating RNAs for monitoring and diagnosing human health has grown significantly. There are few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. In this study, total extracellular RNA (exRNA) was isolated and sequenced from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 male college athletes ages 18–25 years. Many participants provided more than one sample, allowing us to investigate variability in an individual’s exRNA expression levels over time. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. The small RNA profile of each biofluid is distinct. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid, and profiles of miRNA are different for each biofluid. Not unexpectedly, saliva samples have high levels of exogenous sequence that can be traced to bacteria. These data significantly contribute to the current number of sequenced exRNA samples from normal healthy individuals. PMID:28303895

Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots.

PubMed

Zubairova, Laily D; Nabiullina, Roza M; Nagaswami, Chandrasekaran; Zuev, Yuriy F; Mustafin, Ilshat G; Litvinov, Rustem I; Weisel, John W

2015-12-04

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1-0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis.

Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots

PubMed Central

Zubairova, Laily D.; Nabiullina, Roza M.; Nagaswami, Chandrasekaran; Zuev, Yuriy F.; Mustafin, Ilshat G.; Litvinov, Rustem I.; Weisel, John W.

2015-01-01

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1–0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis. PMID:26635081

Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

2015-12-16

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.

Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203).

PubMed

Beninson, Lida A; Brown, Peter N; Loughridge, Alice B; Saludes, Jonel P; Maslanik, Thomas; Hills, Abigail K; Woodworth, Tyler; Craig, Wendy; Yin, Hang; Fleshner, Monika

2014-01-01

Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation.

[The physiology of glucagon-like peptide-1 and its role in the pathophysiology of type 2 diabetes mellitus].

PubMed

Escalada, Francisco Javier

2014-09-01

The hormone glucagon-like peptide-1 (GLP-1) is synthesized and secreted by L cells in the small intestine in response to food ingestion. After reaching the general circulation it has a half-life of 2-3 minutes due to degradation by the enzyme dipeptidyl peptidase-4. Its physiological role is directed to control plasma glucose concentration, though GLP-1 also plays other different metabolic functions following nutrient absorption. Biological activities of GLP-1 include stimulation of insulin biosynthesis and glucose-dependent insulin secretion by pancreatic beta cell, inhibition of glucagon secretion, delay of gastric emptying and inhibition of food intake. GLP-1 is able to reduce plasma glucose levels in patients with type 2 diabetes and also can restore beta cell sensitivity to exogenous secretagogues, suggesting that the increasing GLP-1 concentration may be an useful therapeutic strategy for the treatment of patients with type 2 diabetes. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Quantitation of methylglyoxal bis(guanylhydrazone) in blood plasma and leukemia cells of patients receiving the drug.

PubMed

Seppänen, P; Alhonen-Hongisto, L; Siimes, M; Jänne, J

1980-11-15

Methylglyoxal bis(guanylhydrazone), a cytostatic compound which apparently interferes with the metabolism and/or functions of the natural polyamines (spermidine and spermine), was effectively taken up by cultured human lymphocytic leukemia cells, rapidly resulting in the formation of a concentration gradient of up to 1,000-fold across the cell membrane in cells grown in the presence of micromolar concentrations of the drug. For an anti-proliferative effect on the leukemia cells, an intracellular concentration of more than 0.5 mM was required. The uptake of methylglyoxal bis(guanylhydrazone) was critically dependent on the growth rate of the leukemia cells. Low intracellular concentrations of the drug were present in cells growing slowly, whereas in rapidly dividing cells the intracellular concentration of the drug approached 5mM. When given as repeated intravenous infusions to two leukemic children, methylglyoxal bis(guanylhydrazone) exhibited sharp and transient peaks of plasma concentration, the drug having an apparent half-life in plasma of only 1-2 h. However, as in cultured cells, the drug was rapidly concentrated in the leukemia cells, reaching concentrations that were distinctly anti-proliferative. In contrast to the rapid disappearance of methylglyoxal bis(guanylhydrazone) from plasma, the circulation leukemia cells retained the drug for a period of several days with only minimal decrease in the initial concentrations. Methylglyoxal bis(guanylhydrazone) was given to the patients for 1 to 2 months as intravenous infusions, the timing of which was determined by regular assays of the drug concentrations in the leukemia cells. In agreement with the results obtained with the cultured cells, and intracellular concentration of about 0.5 to 1mM was apparently required for growth-inhibitory action to occur. Regular determination of the cellular drug concentrations indicated that methylglyoxal bis(quanylhydrazone) could be given as weekly infusions. This treatment schedule represents much lower dosing of the drug than the earlier daily regimens which were commonly associated with unacceptable toxicity.

Galectin-3, a biomarker linking oxidative stress and inflammation with the clinical outcomes of patients with atherothrombosis.

PubMed

Madrigal-Matute, Julio; Lindholt, Jes Sandal; Fernandez-Garcia, Carlos Ernesto; Benito-Martin, Alberto; Burillo, Elena; Zalba, Guillermo; Beloqui, Oscar; Llamas-Granda, Patricia; Ortiz, Alberto; Egido, Jesus; Blanco-Colio, Luis Miguel; Martin-Ventura, Jose Luis

2014-08-05

Galectin-3 (Gal-3) participates in different mechanisms involved in atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Thus, there have been committed intensive efforts to elucidate the function of Gal-3 in cardiovascular (CV) diseases. The role of Gal-3 as a circulating biomarker has been demonstrated in patients with heart failure, but its importance as a biomarker in atherothrombosis is still unknown. Because Gal-3 is involved in monocyte-to-macrophage transition, we used fresh isolated monocytes and the in vitro model of macrophage differentiation of THP-1 cells stimulated with phorbol myristate acetate (PMA). Gal-3 release is increased by PMA in human monocytes and macrophages, a process involving exosomes and regulated by reactive oxygen species/NADPH oxidase activity. In asymptomatic subjects (n=199), Gal-3 plasma levels are correlated with NADPH oxidase activity in peripheral blood mononuclear cells (r=0.476; P<0.001) and carotid intima-media thickness (r=0.438; P<0.001), a surrogate marker of atherosclerosis. Accordingly, Gal-3 plasma concentrations are increased in patients with carotid atherosclerosis (n=158), compared to control subjects (n=115; 14.3 [10.7 to 16.9] vs. 10.4 [8.6 to 12.5] ng/mL; P<0.001). Finally, on a 5-year follow-up study in patients with peripheral artery disease, Gal-3 concentrations are significantly and independently associated with an increased risk for CV mortality (hazard ratio=2.24, 95% confidence interval: 1.06 to 4.73, P<0.05). Gal-3 extracellular levels could reflect key underlying mechanisms involved in atherosclerosis etiology, development, and plaque rupture, such as inflammation, infiltration of circulating cells and oxidative stress. Moreover, circulating Gal-3 concentrations are associated with clinical outcomes in patients with atherothrombosis. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation.

PubMed

Perretti, M; Flower, R J

1996-06-01

1. Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N-terminus peptide, cell-associated LC1-like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane. 2. Treatment of mice with dexamethasone (Dex; 0.5-5 micrograms per mouse corresponding to approximately 0.015-1.5 mg kg-1) increased LC1 levels in neutrophils and monocytes. The 2-3 fold increase in LC1 levels was time-dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg-1 orally) decreased LC1-like immunoreactivity in all three types of circulating leukocytes by > or = 50%. 3. Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (> or = 50%) in LC1 levels compared with circulating neutrophils. A high LC1-like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane-associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane-associated LC1 to a significant extent, i.e. up to 70%. 4. In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.

[Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy].

PubMed

Ponomaryova, A A; Cherdyntseva, N V; Bondar, A A; Dobrodeev, A Y; Zavyalov, A A; Tuzikov, S A; Vlassov, V V; Choinzonov, E L; Laktionov, P P; Rykova, E Y

2017-01-01

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.

The use of decompression to simulate the effect of extravehicular activity on human lymphocyte transformation

NASA Technical Reports Server (NTRS)

Meehan, R. T.; Duncan, U.; Neale, L.; Waligora, J.; Taylor, G. R.

1986-01-01

Lymphocytes from 35 subjects participating in a chamber study simulating extravehicular activity (EVA) conditions were studied. No significant differences in H3 thymidine uptake between pre chamber and post chamber response to any mitogens autologous plasma, or among circulating mononuclear cells by flow cytometry are observed. The studies could not identify the subjects who developed venous bubbles. Data from eight subjects suggests that acute stress associated with participating in the study augments in vitro lymphocyte proliferation. Results indicate EVA exposure does not greatly influence space-flight induced alterations in immune effector cell function.

Plasma surface reflectance spectroscopy for non-invasive and continuous monitoring of extracellular component of blood

NASA Astrophysics Data System (ADS)

Sakota, Daisuke; Takatani, Setsuo

2012-04-01

To achieve the quantitative optical non-invasive diagnosis of blood during extracorporeal circulation therapies, the instrumental technique to extract extracellular spectra from whole blood was developed. In the circuit, the continuous blood flow was generated by a centrifugal blood pump. The oxygen saturation was maintained 100% by an oxygenator. The developed glass optical flow cell was attached to the outlet tubing of the oxygenator. The halogen lamp including the light from 400 to 900 nm wavelength was used for the light source. The light was guided into an optical fiber. The light emitted by the fiber was collimated and emitted to the flow cell flat surface at the incident angle of 45 degrees. The light just reflected on the boundary between inner surface of the flow cell and plasma at 45 degrees was detected by the detection fiber. The detected light was analyzed by a spectral photometer. The obtained spectrum from 400 to 600nm wavelength was not changed with respect to the hematocrit. In contrast, the signal in the spectral range was changed when the plasma free hemoglobin increased. By using two spectral range, 505+/-5 nm and 542.5+/-2.5 nm, the differential spectrum was correlated with the free hemoglobin at R2=0.99. On the other hand, as for the hematocrit, the differential spectrum was not correlated at R2=0.01. Finally, the plasma free hemoglobin was quantified with the accuracy of 22+/-19mg/dL. The result shows that the developed plasma surface reflectance spectroscopy (PSRS) can extract the plasma spectrum from flowing whole blood.

Cryoglobulinemia

MedlinePlus

... is also used. In this his procedure, blood plasma is taken out of blood circulation and abnormal cryoglobulin antibody proteins are removed. The plasma is replaced by fluid, protein, or donated plasma. ...

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

PubMed Central

Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

Upregulation of circulating components of the alternative renin-angiotensin system in inflammatory bowel disease: A pilot study.

PubMed

Garg, Mayur; Burrell, Louise M; Velkoska, Elena; Griggs, Karen; Angus, Peter W; Gibson, Peter R; Lubel, John S

2015-09-01

The relationship between intestinal inflammation and circulating components of the renin-angiotensin system (RAS) is poorly understood. Demographic and clinical data were obtained from healthy controls and patients with inflammatory bowel disease (IBD). Plasma concentrations of the classical RAS components (angiotensin-converting enzyme (ACE) and angiotensin II (Ang II)) and alternative RAS components (ACE2 and angiotensin (1-7) (Ang (1-7))) were analysed by radioimmuno- and enzymatic assays. Systemic inflammation was assessed using serum C-reactive protein (CRP), white cell count, platelet count and albumin, and intestinal inflammation by faecal calprotectin. Nineteen healthy controls (11 female; mean age 38 years, range 23-68), 19 patients with Crohn's disease (11 female; aged 45 years, range 23-76) and 15 patients with ulcerative colitis (6 female; aged 42 years, 26-64) were studied. Circulating classical RAS component levels were similar across the three groups, whereas ACE2 activity and Ang (1-7) concentrations were higher in patients with IBD compared to controls (ACE2: 21.5 vs 13.3 pmol/ml/min, p<0.05; Ang (1-7): 22.8 vs 14.1 pg/ml, p<0.001). Ang (1-7) correlated weakly with platelet and white cell counts, but not calprotectin or CRP, in patients with IBD. Circulating components of the alternative RAS are increased in patients with IBD. © The Author(s) 2014.

Survivin as a Potential Mediator to Support Autoreactive Cell Survival in Myasthenia Gravis: A Human and Animal Model Study

PubMed Central

Kusner, Linda L.; Ciesielski, Michael J.; Marx, Alexander; Kaminski, Henry J.; Fenstermaker, Robert A.

2014-01-01

The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders. PMID:25050620

Circulating and tumor-associated caspase-4: a novel diagnostic and prognostic biomarker for non-small cell lung cancer

PubMed Central

Terlizzi, Michela; Colarusso, Chiara; De Rosa, Ilaria; De Rosa, Nicolina; Somma, Pasquale; Curcio, Carlo; Sanduzzi, Alessandro Zamparelli; Micheli, Pietro; Molino, Antonio; Saccomanno, Antonello; Salvi, Rosario; Aquino, Rita P.; Pinto, Aldo; Sorrentino, Rosalinda

2018-01-01

Late diagnosis limits therapeutic options and survival rate of non-small cell lung cancer (NSCLC) patients. Therefore the identification of biomarkers represents an emerging medical need. A highly sensitive and specific test was developed to identify/quantify a novel/selective diagnostic biomarker for NSCLC patients, caspase-4. This test was validated by using i) plasma from 125 NSCLC patients and 79 healthy (non-pathological) subjects, ii) plasma from 139 smokers and iii) from 70 chronic-obstructive pulmonary disease (COPD) patients. Caspase-4 quantification was also assessed in the lung tumor mass of 98 paired NSCLC patients compared to 10 non-tumor lung tissues (i.e. tuberculosis). Circulating caspase-4 was detected in both healthy and NSCLC patients; however at different range values: 2.603–3.372 ng/ml for NSCLC patients (95% CI) compared to 0.3994-0.6219 ng/ml for healthy subjects (95% CI). The sensitivity of the test ranged from 97.07% to 100%; the specificity was 88.1% with a positive predictive value of 92.54%, accuracy of 95.19% and AUC of 0.971. Smokers (95% CI, 0.3947–0.6197 ng/ml) and COPD patients (95% CI, 1.703–2.995 ng/ml) showed intermediate values of circulating caspase-4. Tissue levels of caspase-4 in the tumor mass showed that 72 (72.7%) out of 99 patients were positive. More importantly, higher levels (cut-off value = 0.307 ng/ml) of caspase-4 in the tumor mass were associated to reduced overall survival (median 0.92 years) compared to NSCLC patients with lower levels (median 3.02 years). We report for the first time caspase-4 as a novel diagnostic and prognostic biomarker, opening new therapeutic perspectives for NSCLC patients. PMID:29721208

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

PubMed Central

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

2016-01-01

Human milk (HM) contains regulatory biomolecules including miRNAs, the origin and functional significance of which are still undetermined. We used TaqMan OpenArrays to profile 681 mature miRNAs in HM cells and fat, and compared them with maternal peripheral blood mononuclear cells (PBMCs) and plasma, and bovine and soy infant formulae. HM cells and PBMCs (292 and 345 miRNAs, respectively) had higher miRNA content than HM fat and plasma (242 and 219 miRNAs, respectively) (p < 0.05). A strong association in miRNA profiles was found between HM cells and fat, whilst PBMCs and plasma were distinctly different to HM, displaying marked inter-individual variation. Considering the dominance of epithelial cells in mature milk of healthy women, these results suggest that HM miRNAs primarily originate from the mammary epithelium, whilst the maternal circulation may have a smaller contribution. Our findings demonstrate that unlike infant formulae, which contained very few human miRNA, HM is a rich source of lactation-specific miRNA, which could be used as biomarkers of the performance and health status of the lactating mammary gland. Given the recently identified stability, uptake and functionality of food- and milk-derived miRNA in vivo, HM miRNA are likely to contribute to infant protection and development. PMID:26854194

A novel plasma circular RNA circFARSA is a potential biomarker for non-small cell lung cancer.

PubMed

Hang, Dong; Zhou, Jing; Qin, Na; Zhou, Wen; Ma, Hongxia; Jin, Guangfu; Hu, Zhibin; Dai, Juncheng; Shen, Hongbing

2018-06-01

Emerging evidence indicates that circular RNAs (circRNAs) are implicated in cancer development. This study aimed to evaluate whether circulating circRNAs may serve as novel biomarkers for non-small cell lung cancer (NSCLC). We used RNA sequencing (RNA-seq) and quantitative real-time PCR to explore cancer-related circRNAs. Bioinformatics and functional analyses were performed to reveal biological effects of circRNAs on lung cancer cells. A total of 5471 distinct circRNAs were identified by total RNA-seq, in which 185 were differentially expressed between cancerous and adjacent normal tissues. A circRNA derived from exon 5-7 of the FARSA gene, termed circFARSA, was observed to increase in cancerous tissues (P = 0.016), and was more abundant in patients' plasma than controls (P < 0.001). Overexpression of circFARSA in A549 cell line significantly promoted cell migration and invasion. In silico analysis suggested that circFARSA might sponge miR-330-5p and miR-326, thereby relieving their inhibitory effects on oncogene fatty acid synthase. Summarily, this study revealed circRNA profile of NSCLC for the first time and provided the evidence of plasma circFARSA as a potential noninvasive biomarker for this malignancy. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk.

PubMed

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E; Geddes, Donna T; Kakulas, Foteini

2016-02-08

Human milk (HM) contains regulatory biomolecules including miRNAs, the origin and functional significance of which are still undetermined. We used TaqMan OpenArrays to profile 681 mature miRNAs in HM cells and fat, and compared them with maternal peripheral blood mononuclear cells (PBMCs) and plasma, and bovine and soy infant formulae. HM cells and PBMCs (292 and 345 miRNAs, respectively) had higher miRNA content than HM fat and plasma (242 and 219 miRNAs, respectively) (p < 0.05). A strong association in miRNA profiles was found between HM cells and fat, whilst PBMCs and plasma were distinctly different to HM, displaying marked inter-individual variation. Considering the dominance of epithelial cells in mature milk of healthy women, these results suggest that HM miRNAs primarily originate from the mammary epithelium, whilst the maternal circulation may have a smaller contribution. Our findings demonstrate that unlike infant formulae, which contained very few human miRNA, HM is a rich source of lactation-specific miRNA, which could be used as biomarkers of the performance and health status of the lactating mammary gland. Given the recently identified stability, uptake and functionality of food- and milk-derived miRNA in vivo, HM miRNA are likely to contribute to infant protection and development.

Sustained Zero-Order Release of Intact Ultra-Stable Drug-Loaded Liposomes from an Implantable Nanochannel Delivery System

PubMed Central

Celia, Christian; Ferrati, Silvia; Bansal, Shyam; van de Ven, Anne L.; Ruozi, Barbara; Zabre, Erika; Hosali, Sharath; Paolino, Donatella; Sarpietro, Maria Grazia; Fine, Daniel; Fresta, Massimo; Ferrari, Mauro

2014-01-01

Metronomic chemotherapy supports the idea that long-term, sustained, constant administration of chemotherapeutics, currently not achievable, could be effective against numerous cancers. Particularly appealing are liposomal formulations, used to solubilize hydrophobic therapeutics and minimize side effects, while extending drug circulation time and enabling passive targeting. As liposome alone cannot survive in circulation beyond 48 hrs, sustaining their constant plasma level for many days is a challenge. To address this, we developed, as a proof of concept, an implantable nanochannel delivery system and ultra-stable PEGylated lapatinib loaded-liposomes, and we demonstrate the release of intact vesicles for over 18 days. Further, we investigate intravasation kinetics of subcutaneously delivered liposomes and verify their biological activity post nanochannel release on BT474 breast cancer cells. The key innovation of this work is the combination of two nanotechnologies to exploit the synergistic effect of liposomes, demonstrated as passive-targeting vectors and nanofluidics to maintain therapeutic constant plasma levels. In principle, this approach could maximize efficacy of metronomic treatments. PMID:23881575

Brain regions involved in the development of acute phase responses accompanying fever in rabbits.

PubMed Central

Morimoto, A; Murakami, N; Nakamori, T; Sakata, Y; Watanabe, T

1989-01-01

1. The effects of microinjection of rabbit endogenous pyrogen and human recombinant interleukin-1 alpha on rectal temperature and acute phase responses were extensively examined in forty different brain regions of rabbits. The acute phase responses that were investigated were the changes in plasma levels of iron, zinc and copper concentration and the changes in circulating leucocyte count. 2. The rostral hypothalamic regions, such as nucleus broca ventralis, preoptic area and anterior hypothalamic region, responded to the microinjection of endogenous pyrogen or interleukin-1 by producing both fever and acute phase responses. 3. The microinjection of endogenous pyrogen or interleukin-1 into the rostral hypothalamic regions significantly decreased the plasma levels of iron and zinc concentration 8 and 24 h after injection. The circulating leucocyte count increased 8 h after injection. However, neither the injections of endogenous pyrogen nor interleukin-1 affected the number of red blood cells. 4. The present results show that the rostral hypothalamic regions respond directly to endogenous pyrogen or interleukin-1 with the consequent development of fever and acute phase responses. PMID:2514261

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

PubMed

Lee-Sundlov, Melissa M; Ashline, David J; Hanneman, Andrew J; Grozovsky, Renata; Reinhold, Vernon N; Hoffmeister, Karin M; Lau, Joseph Ty

2017-01-01

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

PubMed Central

Stamler, J S; Jaraki, O; Osborne, J; Simon, D I; Keaney, J; Vita, J; Singel, D; Valeri, C R; Loscalzo, J

1992-01-01

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone. PMID:1502182

Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

PubMed

Stamler, J S; Jaraki, O; Osborne, J; Simon, D I; Keaney, J; Vita, J; Singel, D; Valeri, C R; Loscalzo, J

1992-08-15

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.

Quantitative analyses and transcriptomic profiling of circulating messenger RNAs as biomarkers of rat liver injury.

PubMed

Wetmore, Barbara A; Brees, Dominique J; Singh, Reetu; Watkins, Paul B; Andersen, Melvin E; Loy, James; Thomas, Russell S

2010-06-01

Serum aminotransferases have been the clinical standard for evaluating liver injury for the past 50-60 years. These tissue enzymes lack specificity, also tracking injury to other tissues. New technologies assessing tissue-specific messenger RNA (mRNA) release into blood should provide greater specificity and permit indirect assessment of gene expression status of injured tissue. To evaluate the potential of circulating mRNAs as biomarkers of liver injury, rats were treated either with hepatotoxic doses of D-(+)-galactosamine (DGAL) or acetaminophen (APAP) or a myotoxic dose of bupivacaine HCl (BPVC). Plasma, serum, and liver samples were obtained from each rat. Serum alanine aminotransferase and aspartate aminotransferase were increased by all three compounds, whereas circulating liver-specific mRNAs were only increased by the hepatotoxicants. With APAP, liver-specific mRNAs were significantly increased in plasma at doses that had no effect on serum aminotransferases or liver histopathology. Characterization of the circulating mRNAs by sucrose density gradient centrifugation revealed that the liver-specific mRNAs were associated with both necrotic debris and microvesicles. DGAL treatment also induced a shift in the size of plasma microvesicles, consistent with active release of microvesicles following liver injury. Finally, gene expression microarray analysis of the plasma following DGAL and APAP treatment revealed chemical-specific profiles. The comparative analysis of circulating liver mRNAs with traditional serum transaminases and histopathology indicated that the circulating liver mRNAs were more specific and more sensitive biomarkers of liver injury. Further, the possibility of identifying chemical-specific transcriptional profiles from circulating mRNAs could open a range of possibilities for identifying the etiology of drug/chemical-induced liver injury.

Idiotype vaccination in patients with myeloma reduced circulating myeloma cells (CMC).

PubMed

Abdalla, A O; Kokhaei, P; Hansson, L; Mellstedt, H; Osterborg, A

2008-06-01

Circulating myeloma cells (CMC), exhibiting the same immunoglobulin heavy-chain gene rearrangements as the plasma cells, are part of the myeloma clone. In this study, we evaluated the effect of idiotype (Id) vaccination on CMC. Eleven patients were immunized with the autologous Id in combinations with granulocyte-macrophage colony-stimulating factor and interleukin 12, and followed for CMC by quantitative real-time allele-specific PCR. Id-specific T cells were monitored by proliferation assay, enzyme-linked immunospot (interferon-gamma) assay, and quantitative real-time PCR for cytokines. Regulatory T (T(reg)) cells were analyzed by flow cytometry. CMC were detected in 9 of 11 patients at start of vaccination. In four patients, CMC declined and two had a complete molecular remission. Further two patients had stable levels of CMC during follow-up, while in three patients CMC progressively increased. Six patients had a vaccine-induced Id-specific T-cell response. A significant correlation was observed between reduced/stable levels of CMC and the Id-specific T cells (P < 0.02). The frequency of T(reg) cells was decreased in immune responders, but increased in immune nonresponders (P < 0.05). No significant change in the serum M-protein concentration was, however, observed in any patient. Id vaccination reduced CMC, which correlated with vaccine-induced Id-specific T cells. Further studies are warranted to analyze the clinical significance of CMC and clinical effects of Id vaccination.

In vivo flow cytometry and time-resolved near-IR angiography and lymphography

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

2007-05-01

Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.

Bacteroidaceae in Thromboembolic Disease: Effects of Cell Wall Components on Blood Coagulation In Vivo and In Vitro

PubMed Central

Bjornson, H. S.; Hill, E. O.

1973-01-01

The effects of Bacteroides sp., Fusobacterium mortiferum, Bacteroides fragilis, and Sphaerophorus necrophorus on various parameters of blood coagulation in vivo and in vitro were determined and compared to the coagulation effects of Escherichia coli and Salmonella minnesota, wild type and R595. Intravenous injection of washed cells, culture filtrate, lipopolysaccharide, or lipid A of the anaerobic gram-negative microorganisms into mice resulted in acceleration of coagulation. Lipopolysaccharide and lipid A of the anaerobic microorganisms had no apparent effect on circulating platelets in mice or rabbits and did not cause aggregation of human platelets in vitro. Washed cells, lipopolysaccharide, and lipid A of Bacteroides sp. and F. mortiferum also significantly accelerated the clotting time of recalcified platelet poor normal human plasma and C6-deficient rabbit plasma. Lipid A, but not lipopolysaccharide, of E. coli and washed cells of S. minnesota R595 accelerated coagulation by a similar mechanism. These results indicated that Bacteroides sp. and F. mortiferum can accelerate blood coagulation in vivo and in vitro by a mechanism which does not involve platelets or terminal components of complement. PMID:4594118

Lactobacillus johnsonii supplementation attenuates respiratory viral infection via metabolic reprogramming and immune cell modulation.

PubMed

Fonseca, W; Lucey, K; Jang, S; Fujimura, K E; Rasky, A; Ting, H-A; Petersen, J; Johnson, C C; Boushey, H A; Zoratti, E; Ownby, D R; Levine, A M; Bobbit, K R; Lynch, S V; Lukacs, N W

2017-11-01

Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii supplementation reduced airway T helper type 2 cytokines and dendritic cell (DC) function, increased regulatory T cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone marrow-derived DCs (BMDCs) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T-cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice or with wild-type derived BMDCs pretreated with plasma from L. johnsonii-supplemented mice reduced airway pathological responses to infection in recipient animals. Thus L. johnsonii supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.

PubMed

Warton, Kristina; Yuwono, Nicole L; Cowley, Mark J; McCabe, Mark J; So, Alwin; Ford, Caroline E

2017-10-01

Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Detection of FAM172A expressed in circulating tumor cells is a feasible method to predict high-risk subgroups of colorectal cancer.

PubMed

Cui, Chun-Hui; Chen, Ri-Hong; Zhai, Duan-Yang; Xie, Lang; Qi, Jia; Yu, Jin-Long

2017-06-01

Previous studies used to enumerate circulating tumor cells to predict prognosis and therapeutic effect of colorectal cancer. However, increasing studies have shown that only circulating tumor cells enumeration was not enough to reflect the heterogeneous condition of tumor. In this study, we classified different metastatic-potential circulating tumor cells from colorectal cancer patients and measured FAM172A expression in circulating tumor cells to improve accuracy of clinical diagnosis and treatment of colorectal cancer. Blood samples were collected from 45 primary colorectal cancer patients. Circulating tumor cells were enriched by blood filtration using isolation by size of epithelial tumor cells, and in situ hybridization with RNA method was used to identify and discriminate subgroups of circulating tumor cells. Afterwards, FAM172A expression in individual circulating tumor cells was measured. Three circulating tumor cell subgroups (epithelial/biophenotypic/mesenchymal circulating tumor cells) were identified using epithelial-mesenchymal transition markers. In our research, mesenchymal circulating tumor cells significantly increased along with tumor progression, development of distant metastasis, and vascular invasion. Furthermore, FAM172A expression rate in mesenchymal circulating tumor cells was significantly higher than that in epithelial circulating tumor cells, which suggested that FAM172A may correlate with malignant degree of tumor. This hypothesis was further verified by FAM172A expression in mesenchymal circulating tumor cells, which was strictly related to tumor aggressiveness factors. Mesenchymal circulating tumor cells and FAM172A detection may predict highrisk stage II colorectal cancer. Our research proved that circulating tumor cells were feasible surrogate samples to detect gene expression and could serve as a predictive biomarker for tumor evaluation.

Selective simulation of allelic expression: effectf antibodies to allotypic markers on lymphoid cells.

PubMed

Frensdorff, A; Jones, P P; Berwald-Netter, Y; Cebra, J J; Mage, R

1971-01-29

Peripheral blood leukocytes from rabbits which were heterozygous (b(5)/b(9)) for markers on their immunoglobulin light chains were maintained in vitro for up to 24 hours in the presence or absence of antibody to b9. After culture they were transferred into lethally irradiated b(4)/b(4)hosts. Recipients of cells exposed to antibodies to allotype markers showed a striking increase in concentration of circulating b9 molecules and number of b9 plasma cells in their spleens compared pared to control animals receiving untreated cells from the same donor. There was no appreciable difyerence between the two groups of recipients with respect to their content of b5 molecules and immunocytes.

Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Egloff, Caroline; Crump, Doug, E-mail: doug.crump@ec.gc.ca; Porter, Emily

The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highestmore » doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy. • TEP elicited more adverse molecular and phenotypic effects than TBOEP.« less

Long Noncoding RNA H19 Promotes Neuroinflammation in Ischemic Stroke by Driving Histone Deacetylase 1-Dependent M1 Microglial Polarization.

PubMed

Wang, Jue; Zhao, Haiping; Fan, Zhibin; Li, Guangwen; Ma, Qingfeng; Tao, Zhen; Wang, Rongliang; Feng, Juan; Luo, Yumin

2017-08-01

Long noncoding RNA H19 is repressed after birth, but can be induced by hypoxia. We aim to investigate the impact on and underlying mechanism of H19 induction after ischemic stroke. Circulating H19 levels in stroke patients and mice subjected to middle cerebral artery occlusion were assessed using real-time polymerase chain reaction. H19 siRNA and histone deacetylase 1 (HDAC1) plasmid were used to knock down H19 and overexpress HDAC1, respectively. Microglial polarization and ischemic outcomes were assessed in middle cerebral artery occlusion mice and BV2 microglial cells subjected to oxygen-glucose deprivation. Circulating H19 levels were significantly higher in stroke patients compared with healthy controls, indicating high diagnostic sensitivity and specificity. Moreover, plasma H19 levels showed a positive correlation with National Institute of Health Stroke Scale score and tumor necrosis factor-α levels. After middle cerebral artery occlusion in mice, H19 levels increased in plasma, white blood cells, and brain. Intracerebroventricular injection of H19 siRNA reduced infarct volume and brain edema, decreased tumor necrosis factor-α and interleukin-1β levels in brain tissue and plasma, and increased plasma interleukin-10 concentrations 24 hours poststroke. Additionally, H19 knockdown attenuated brain tissue loss and neurological deficits 14 days poststroke. BV2 cell-based experiments showed that H19 knockdown blocked oxygen-glucose deprivation-driven M1 microglial polarization, decreased production of tumor necrosis factor-α and CD11b, and increased the expression of Arg-1 and CD206. Furthermore, H19 knockdown reversed oxygen-glucose deprivation-induced upregulation of HDAC1 and downregulation of acetyl-histone H3 and acetyl-histone H4. In contrast, HDAC1 overexpression negated the effects of H19 knockdown. Our findings indicate that H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke. © 2017 American Heart Association, Inc.

Expression profiles and clinical value of plasma exosomal Tim-3 and Galectin-9 in non-small cell lung cancer.

PubMed

Gao, Jianwei; Qiu, Xiangyu; Li, Xinying; Fan, Hang; Zhang, Fang; Lv, Tangfeng; Song, Yong

2018-04-06

Exosomes are membrane-bound, virus-sized vesicles present in circulating blood. Tumor cells are avid producers of exosomes, which are thought to mimic molecular features of parent tumor cells. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) is a the next-generation immune checkpoint that can be activated by its ligand Galectin-9 to negatively regulate the anti-tumor immune response. However, the characteristics of plasma exosomal Tim-3 and Galectin-9 (Exo-T/G) in cancer remained unknown. This study was conducted to investigate the expression patterns and clinical value of plasma exosomal total protein (Exo-pro) and Exo-T/G in non-small cell lung cancer (NSCLC). Plasma was collected from 103 NSCLC patients including 60 early stages and 43 advanced stages disease samples as well as 56 healthy subjects. Exosomes were isolated from plasma by commercial exosome precipitation solution and identified by western blotting of CD63 and transmission electron microscopy. Exo-pro concentration was measured by the BCA assay. Enzyme-linked immunosorbent assay was used to quantify Exo-T/G. Additionally, 34 NSCLC samples were applied to directly detect plasma TIM-3 (Plas-T) and Galectin-9 (Plas-G). Our results showed that Exo-pro, Exo-T, and Exo-G were significantly increased in NSCLC plasma compared to that in the healthy samples. High levels of Exo-T and Exo-G were all positively correlated with several malignant parameters, including larger tumor size, advanced stages, and more distant metastasis. High levels of Exo-pro and Exo-T were also correlated with more lymph node metastasis. Additionally, plasma from lung squamous cell carcinoma showed higher Exo-T and Exo-G compared with that from lung adenocarcinoma. ALK-positive patients showed to have decreased Exo-T and Exo-G levels. Pearson's correlation analysis revealed a significant correlation between Exo-pro and Exo-T/G, Exo-T and Exo-G, Exo-T and Plas-T, Exo-G and Plas-G, and Plas-T and Plas-G. Together, our data revealed that Exo-pro, especially Exo-T and Exo-G could be potential biomarkers for NSCLC. Further studies focusing on pure tumor-derived exosomes isolated from plasma were needed. Copyright © 2018 Elsevier Inc. All rights reserved.

Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides

PubMed Central

Calenda, Giulia; Tong, Yuehong; Kanika, Nirmala D.; Tar, Moses T.; Suadicani, Sylvia O.; Zhang, Xinhua; Melman, Arnold; Rougeot, Catherine

2011-01-01

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man. PMID:21784987

Reversal of diabetic vasculopathy in a rat model of type 1 diabetes by opiorphin-related peptides.

PubMed

Calenda, Giulia; Tong, Yuehong; Kanika, Nirmala D; Tar, Moses T; Suadicani, Sylvia O; Zhang, Xinhua; Melman, Arnold; Rougeot, Catherine; Davies, Kelvin P

2011-10-01

Diabetes results in a myriad of vascular complications, often referred to as diabetic vasculopathy, which encompasses both microvascular [erectile dysfunction (ED), retinopathy, neuropathy, and nephropathy] and macrovascular complications (hypertension, coronary heart disease, and myocardial infarction). In diabetic animals and patients with ED, there is decreased opiorphin or opiorphin-related gene expression in corporal tissue. Both opiorphin and the rat homologous peptide sialorphin are found circulating in the plasma. In the present study, we investigated if diabetes induced changes in plasma sialorphin levels and if changes in these levels could modulate the biochemistry and physiology of vascular smooth muscle. We show that circulating sialorphin levels are reduced in a rat model of type I diabetes. Intracorporal injection of plasmids expressing sialorphin into diabetic rats restores sialorphin levels to those seen in the blood of nondiabetic animals and results in both improved erectile function and blood pressure. Sialorphin modulated the ability of C-type natriuretic peptide to relax both corporal and aortic smooth muscle strips and of bradykinin to regulate intracellular calcium levels in both corporal and aortic smooth muscle cells. We have previously shown that expression of genes encoding opiorphins is increased when erectile function is improved. Our findings thus suggest that by affecting circulating levels of opiorphin-related peptides, proper erectile function is not only an indicator but also a modulator of overall vascular health of a man.

Optimization study of normal conductor tokamak for commercial neutron source

NASA Astrophysics Data System (ADS)

Fujita, T.; Sakai, R.; Okamoto, A.

2017-05-01

The optimum conceptual design of tokamak with normal conductor coils was studied for minimizing the cost for producing a given neutron flux by using a system code, PEC. It is assumed that the fusion neutrons are used for burning transuranics from the fission reactor spent fuel in the blanket and a fraction of the generated electric power is circulated to opearate the tokamak with moderate plasma fusion gain. The plasma performance was assumed to be moderate ones; {β\\text{N}}~∼ ~3{--}4 in the aspect ratio A~=~2{--}3 and {{H}98y2}~=~1 . The circulating power is an important factor affecting the cost. Though decreasing the aspect ratio is useful to raise the plasma beta and decrease the toroidal field, the maximum field in the coil starts to rise in the very low aspect ratio range and then the circulating power increases with decrease in the plasma aspect ratio A below A~∼ ~2 , while the construction cost increases with A . As a result, the cost per neutron has its minimum around A~∼ ~2.2 , namely, between ST and the conventional tokamak. The average circulating power fraction is expected to be ~51%.

Effects of endurance exercise training on inflammatory circulating progenitor cell content in lean and obese adults.

PubMed

Niemiro, Grace M; Allen, Jacob M; Mailing, Lucy J; Khan, Naiman A; Holscher, Hannah D; Woods, Jeffrey A; De Lisio, Michael

2018-06-19

Chronic inflammation underlies many of the health decrements associated with obesity. Circulating progenitor cells can sense and respond to inflammatory stimuli, increasing the local inflammatory response within tissues. Here we show that 6 weeks of endurance exercise training significantly decreases inflammatory circulating progenitor cells in obese adults. These findings provide novel cellular mechanisms for the beneficial effects of exercise in obese adults. Circulating progenitor cells (CPCs) and subpopulations are normally found in the bone marrow, but can migrate to peripheral tissues to participate in local inflammation and/or remodelling. The purpose of this study was to compare the CPC response, particularly the inflammatory-primed haematopoietic stem and progenitor (HSPC) subpopulation, to a 6 week endurance exercise training (EET) intervention between lean and obese adults. Seventeen healthy weight (age: 23.9 ± 5.4 years, body mass index (BMI): 22.0 ± 2.6 kg m -2 ) and 10 obese (age: 29.0 ± 8.0 years, BMI: 33.1 ± 6.0 kg m -2 ) previously sedentary adults participated in an EET. Blood was collected before and after EET for quantification of CPCs and subpopulations via flow cytometry, colony forming unit assays and plasma concentrations of C-X-C motif chemokine 12 (CXCL12), granulocyte-colony stimulating factor (G-CSF), and chemokine (C-C motif) ligand 2 (CCL2). Exercise training reduced the number of circulating HSPCs and adipose tissue-derived mesenchymal stem cells (AT-MSCs). EET increased the colony forming potential of granulocytes and macrophages irrespective of BMI. EET reduced the number of HSPCs expressing the chemokine receptor CCR2 and the pro-inflammatory marker TLR4. EET-induced changes in adipose tissue-derived MSCs and bone marrow-derived MSCs were negatively related to changes in absolute fitness. Our results indicate that EET, regardless of BMI status, decreases CPCs and subpopulations, particularly those primed for contribution to tissue inflammation. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Persistently Low Natural Killer Cell Activity and Circulating Levels of Plasma Beta Endorphin: Risk Factors for Infectious Disease

DTIC Science & Technology

1990-01-01

influenza, pneumonia, cold sores, gum infections, mononucleosis, strep throat , and gastro-intcstinal illnesses, plus the direct reporting of symptoms such...other upper respiratory symptoms ) over the follow-up period. Specifically, older age predicted lower URI morbidity; younger age was associated with the...work. They were instructed to record daily symptoms of infectious illness experienced, as well as the amount of sleep per night, daily exercise, and

A Memory B Cell Crossmatch Assay for Quantification of Donor-Specific Memory B Cells in the Peripheral Blood of HLA-Immunized Individuals.

PubMed

Karahan, G E; de Vaal, Y J H; Krop, J; Wehmeier, C; Roelen, D L; Claas, F H J; Heidt, S

2017-10-01

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow-residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA-specific memory B cells are scarce and insufficient in quantifying the complete donor-specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor-specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme-linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II-specific memory B cells per million IgG-producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor-specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross-match assay to quantify donor-specific memory B cells in patients with a history of sensitizing events. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Increased plasma levels of big-endothelin-2 and big-endothelin-3 in patients with end-stage renal disease.

PubMed

Miyauchi, Yumi; Sakai, Satoshi; Maeda, Seiji; Shimojo, Nobutake; Watanabe, Shigeyuki; Honma, Satoshi; Kuga, Keisuke; Aonuma, Kazutaka; Miyauchi, Takashi

2012-10-15

Big endothelins (pro-endothelin; inactive-precursor) are converted to biologically active endothelins (ETs). Mammals and humans produce three ET family members: ET-1, ET-2 and ET-3, from three different genes. Although ET-1 is produced by vascular endothelial cells, these cells do not produce ET-3, which is produced by neuronal cells and organs such as the thyroid, salivary gland and the kidney. In patients with end-stage renal disease, abnormal vascular endothelial cell function and elevated plasma ET-1 and big ET-1 levels have been reported. It is unknown whether big ET-2 and big ET-3 plasma levels are altered in these patients. The purpose of the present study was to determine whether endogenous ET-1, ET-2, and ET-3 systems including big ETs are altered in patients with end-stage renal disease. We measured plasma levels of ET-1, ET-3 and big ET-1, big ET-2, and big ET-3 in patients on chronic hemodialysis (n=23) and age-matched healthy subjects (n=17). In patients on hemodialysis, plasma levels (measured just before hemodialysis) of both ET-1 and ET-3 and big ET-1, big ET-2, and big ET-3 were markedly elevated, and the increase was higher for big ETs (Big ET-1, 4-fold; big ET-2, 6-fold; big ET-3: 5-fold) than for ETs (ET-1, 1.7-fold; ET-3, 2-fold). In hemodialysis patients, plasma levels of the inactive precursors big ET-1, big ET-2, and big ET-3 levels are markedly increased, yet there is only a moderate increase in plasma levels of the active products, ET-1 and ET-3. This suggests that the activity of endothelin converting enzyme contributing to circulating levels of ET-1 and ET-3 may be decreased in patients on chronic hemodialysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Plasma Cytokines Correlated With Disease Characteristics, Progression-Free Survival, and Overall Survival in Testicular Germ-Cell Tumor Patients.

PubMed

Svetlovska, Daniela; Miskovska, Viera; Cholujova, Dana; Gronesova, Paulina; Cingelova, Silvia; Chovanec, Michal; Sycova-Mila, Zuzana; Obertova, Jana; Palacka, Patrik; Rajec, Jan; Kalavska, Katarina; Usakova, Vanda; Luha, Jan; Ondrus, Dalibor; Spanik, Stanislav; Mardiak, Jozef; Mego, Michal

2017-06-01

Cytokines are the communicators of immune system and are involved in all immune responses. The aim of this study was to assess the correlation among plasma cytokines, patient and tumor characteristics, and clinical outcome in chemonaive testicular germ-cell tumor (TGCT) patients. This study included 92 metastatic chemotherapy-naive TGCT patients treated with platinum-based chemotherapy from July 2010 to March 2014. Plasma was isolated before first administration of chemotherapy, and the concentration of 51 plasma cytokines were analyzed using multiplex bead arrays. At a median follow-up of 33.2 months (range, 0.1-54.8 months), 10.9% of patients experienced disease progression, and 7.6% died. Several cytokines were associated with different baseline clinicopathologic features. Elevated plasma levels of interferon (IFN)-α2, interleukin (IL)-2Rα, IL-16, hepatocyte growth factor (HGF), and monocyte chemotactic protein (MCP)-3 were significantly associated with worse progression-free survival and overall survival (OS). Moreover, elevated levels of stem-cell growth factor (SCGF)-β were also associated with worse OS. Patients with elevated levels of all 6 cytokines experienced significantly worse outcomes compared to patients who had fewer than 6 cytokines elevated (hazard ratio = 12.06; 95% confidence interval, 7.39-19.49; P = .002 for progression-free survival, and hazard ratio = 39.65; 95% confidence interval, 25.03-62.18; P < .00001 for OS, respectively). Results were independent of International Germ Cell Cancer Collaborative Group criteria. We found a correlation among progression free-survival, OS, and circulating cytokines in TGCT. This suggests the existence an association between plasma cytokines and baseline clinicopathologic features in TGCT. Plasma cytokines could be used for identification of high-risk patients who are candidates for new therapeutic approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

Prediction of the efficacy of immunotherapy by measuring the integrity of cell-free DNA in plasma in colorectal cancer.

PubMed

Kitahara, Masahiro; Hazama, Shoichi; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Kanekiyo, Shinsuke; Inoue, Yuka; Nakajima, Masao; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Iida, Michihisa; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Ueno, Tomio; Yamamoto, Shigeru; Yoshino, Shigefumi; Nagano, Hiroaki

2016-12-01

We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor DNA of heterogeneous mutant EGFR genes in lung cancer.

PubMed

Imamura, Fumio; Uchida, Junji; Kukita, Yoji; Kumagai, Toru; Nishino, Kazumi; Inoue, Takako; Kimura, Madoka; Oba, Shigeyuki; Kato, Kikuya

2016-04-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatic effects on EGFR-mutant non-small-cell lung cancer (NSCLC). However, most patients experience disease recurrences, approximately half of which are T790M-mediated. Monitoring EGFR status with re-biopsy has spatiotemporal limitations. EGFR circulating tumor DNA (ctDNA) in serial plasma samples was amplified and 10(5) of them were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 10(5)EGFR cell free DNA (cfDNA). PM scores of various EGFR mutations showed dynamic, case-specific changes during EGFR-TKI treatments in 52 patients. The effects of the treatment on EGFR ctDNA were evaluated in 38 patients with elevated pre-treatment PM scores. The ctDNA responses correlated well with radiologic responses in radiologic good responders, whereas correlation was poor in non-responders. In addition to the peaks for the most prevalent ctDNA, small peaks of ctDNA with different types of activating EGFR mutations or the T790M mutation (early T790M ctDNA) appeared transiently in 10.5% and 26.3%, respectively. Early T790M ctDNA disappeared in all patients, including 7 who eventually developed acquired resistance accompanied by elevated levels of T790M ctDNA. Monitoring ctDNA is useful in evaluating treatment responses and monitoring driver oncogene status in NSCLC. ctDNA revealed clonal heterogeneity and genetic processes of cancer evolution in individual patients. The simple presence of the T790M mutation may be insufficient to confer EGFR-TKI resistance to tumor cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule.

PubMed

Witek, Małgorzata A; Aufforth, Rachel D; Wang, Hong; Kamande, Joyce W; Jackson, Joshua M; Pullagurla, Swathi R; Hupert, Mateusz L; Usary, Jerry; Wysham, Weiya Z; Hilliard, Dawud; Montgomery, Stephanie; Bae-Jump, Victoria; Carey, Lisa A; Gehrig, Paola A; Milowsky, Matthew I; Perou, Charles M; Soper, John T; Whang, Young E; Yeh, Jen Jen; Martin, George; Soper, Steven A

2017-01-01

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.

Capturing tumor heterogeneity and clonal evolution in solid cancers using circulating tumor DNA analysis.

PubMed

Perdigones, Nieves; Murtaza, Muhammed

2017-06-01

Circulating tumor DNA analysis has emerged as a potential noninvasive alternative to tissue biopsies for tumor genotyping in patients with metastatic cancer. This is particularly attractive in cases where tissue biopsies are contraindicated or repeat genotyping after progression on treatment is required. However, tissue and plasma analysis results are not always concordant and clinical interpretation of discordant results is not completely understood. Discordant results could arise due to analytical limits of assays used for tumor and plasma DNA analysis or due to low overall contribution of tumor-specific DNA in plasma. Once these factors are ruled out, tissue-plasma concordance and quantitative levels of somatic mutations in plasma can capture tumor heterogeneity. During longitudinal follow-up of patients, this feature can be leveraged to track subclonal evolution and to guide combination or sequential adaptive treatment. Here, we summarize recent results evaluating the opportunities and limitations of circulating tumor DNA analysis in the context of tumor heterogeneity and subclonal evolution in patients with advanced cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

PubMed

Paris, Daniel H; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N; Day, Nicholas P J; Zeerleder, Sacha

2015-01-01

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos

PubMed Central

Paris, Daniel H.; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N.; Day, Nicholas P. J.; Zeerleder, Sacha

2015-01-01

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus. PMID:26317419

Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients.

PubMed

Danese, S; Katz, J A; Saibeni, S; Papa, A; Gasbarrini, A; Vecchi, M; Fiocchi, C

2003-10-01

The CD40/CD40L system, a key regulator and amplifier of immune reactivity, is activated in inflammatory bowel disease (IBD) mucosa. To determine whether plasma levels of sCD40L are elevated in Crohn's disease (CD) and ulcerative colitis (UC) patients compared with normal controls, to investigate the cellular source of sCD40L, and to explore CD40L induction mechanisms. CD, UC, and normal control subjects were studied. The concentration of sCD40L in plasma and supernatants of freshly isolated platelets and autologous peripheral blood T cells (PBT) was measured by ELISA. Surface CD40L expression level was measured by flow cytometry in resting and thrombin activated platelets, and unstimulated and CD3/CD28 stimulated PBT before and after coculture with human intestinal microvascular endothelial cells (HIMEC). Compared with normal controls, plasma sCD40L levels were significantly higher in both CD and UC patients and proportional to the extent of mucosal inflammation. Platelets from IBD patients displayed a significantly higher surface CD40L expression than those from control subjects, and released greater amounts of sCD40L than autologous PBT. Contact with IL-1beta activated HIMEC induced significant upregulation of CD40L surface expression and release by platelets. Elevated levels of sCD40L in the circulation of IBD patients reflect enhanced surface expression and release of CD40L by platelets. This phenomenon translates to an increased platelet activation state apparently induced by passage through an inflamed mucosal microvascular bed, a conclusion supported by the positive correlation of plasma sCD40L levels with the extent of anatomical involvement by IBD. These results suggest that platelet-endothelial interactions critically contribute to activation of the CD40 pathway in IBD.

Effects of the somatostatin analog lanreotide on the circulating levels of chromogranin-A, prostate-specific antigen, and insulin-like growth factor-1 in advanced prostate cancer patients.

PubMed

Berruti, A; Dogliotti, L; Mosca, A; Tarabuzzi, R; Torta, M; Mari, M; Gorzegno, G; Fontana, D; Angeli, A

2001-05-15

The concept that neuroendocrine cells detected within prostate adenocarcinoma produce paracrine factors, that may exert a proliferative effect on exocrine prostate tumor cells, provides a rationale for the use of somatostatin analogs with the aim to counteract or delay the tumor progression. This study was designed to provide preliminary information on the effect of the administration of a long-acting somatostatin analog, lanreotide, on plasma levels of chromogranin A (CgA). Secondary aims were the evaluation of changes in circulating prostate-specific antigen (PSA) and insulin-like growth factor-1 (IGF-1). Lanreotide (Ipstyl 30 mg; Ipsen, Milan, Italy) was administered intramuscularly every 14 days for 2 months to nine heavily pretreated prostate cancer patients with hormone refractory disease. All patients had, at baseline conditions, CgA values above the normal range. Androgen deprivation was maintained during the study period, while other concomitant antineoplastic treatments were not allowed. Serum PSA levels and plasma CgA and IGF-1 values were measured every week. Lanreotide treatment was very well tolerated and no patient experienced major toxicity. Plasma CgA values at baseline: mean 109 U/liter, standard deviation +/- 85 decreased significantly after treatment as follows: 42 U/liter, +/- 17.8; 27.2 U/liter +/- 13.6; 31.4 U/liter, +/- 17.8 and 27.6 U/liter, +/- 17.0; after 7, 14, 21, and 28 days, respectively (P < 0.01, Friedman ANOVA). Serum PSA did not change. Baseline IGF-1 was found to be above the detection limit in four cases, all of them showing a decrease after lanreotide. Lanreotide administration to prostate cancer patients induces a decrease in plasma CgA and IGF-1 levels, without any influence on serum PSA values. Prostate 47:205-211, 2001. Copyright 2001 Wiley-Liss, Inc.

Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane

PubMed Central

Qin, Lizheng; Liu, Xibao; Sun, Qifei; Fan, Zhipeng; Xia, Dengsheng; Ding, Gang; Ong, Hwei Ling; Adams, David; Gahl, William A.; Zheng, Changyu; Qi, Senrong; Jin, Luyuan; Zhang, Chunmei; Gu, Liankun; He, Junqi; Deng, Dajun; Ambudkar, Indu S.; Wang, Songlin

2012-01-01

In vivo recycling of nitrate (NO3−) and nitrite (NO2−) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO3− is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO3− to NO2−, which enters circulation and leads to NO generation. The transporters for NO3− in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO3−/H+ cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO3− or sialic acid (SA), but not by Br−, and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO3−-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H+-dependent NO3− conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO3− secretion in saliva after intake of a NO3−-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO3−/H+ transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis. PMID:22778404

Release of Cell-free MicroRNA Tumor Biomarkers into the Blood Circulation with Pulsed Focused Ultrasound: A Noninvasive, Anatomically Localized, Molecular Liquid Biopsy

PubMed Central

Chevillet, John R.; Khokhlova, Tatiana D.; Giraldez, Maria D.; Schade, George R.; Starr, Frank; Wang, Yak-Nam; Gallichotte, Emily N.; Wang, Kai; Hwang, Joo Ha

2017-01-01

Purpose To compare the abilities of three pulsed focused ultrasound regimes (that cause tissue liquefaction, permeabilization, or mild heating) to release tumor-derived microRNA into the circulation in vivo and to evaluate release dynamics. Materials and Methods All rat experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Reverse-transcription quantitative polymerase chain reaction array profiling was used to identify candidate microRNA biomarkers in a rat solid tumor cell line. Rats subcutaneously grafted with these cells were randomly assigned among three pulsed focused ultrasound treatment groups: (a) local tissue liquefaction via boiling histotripsy, (b) tissue permeabilization via inertial cavitation, and (c) mild (<10°C) heating of tissue, as well as a sham-treated control group. Blood specimens were drawn immediately prior to treatment and serially over 24 hours afterward. Plasma microRNA was quantified with reverse-transcription quantitative polymerase chain reaction, and statistical significance was determined with one-way analysis of variance (Kruskal-Wallis and Friedman tests), followed by the Dunn multiple-comparisons test. Results After tissue liquefaction and cavitation treatments (but not mild heating), plasma quantities of candidate biomarkers increased significantly (P value range, <.0001 to .04) relative to sham-treated controls. A threefold to 32-fold increase occurred within 15 minutes after initiation of pulsed focused ultrasound tumor treatment, and these increases persisted for 3 hours. Histologic examination confirmed complete liquefaction of the targeted tumor area with boiling histotripsy, in addition to areas of petechial hemorrhage and tissue disruption by means of cavitation-based treatment. Conclusion Mechanical tumor tissue disruption with pulsed focused ultrasound–induced bubble activity significantly increases the plasma abundance of tumor-derived microRNA rapidly after treatment. © RSNA, 2016 Online supplemental material is available for this article. PMID:27802108

Toward Standardization of Epstein-Barr Virus DNA Load Monitoring: Unfractionated Whole Blood as Preferred Clinical Specimen

PubMed Central

Stevens, Servi J. C.; Pronk, Inge; Middeldorp, Jaap M.

2001-01-01

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 × 106 copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for β-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation. PMID:11283029

Taoren-Honghua herb pair and its main components promoting blood circulation through influencing on hemorheology, plasma coagulation and platelet aggregation.

PubMed

Liu, Li; Duan, Jin-ao; Tang, Yuping; Guo, Jianming; Yang, Nianyun; Ma, Hongyue; Shi, Xuqin

2012-01-31

Persicae Semen (Taoren) and Carthami Flos (Honghua) used in pair which is named as Taoren-Honghua (TH) herb pair has been used in traditional Chinese medicine (TCM) for promoting blood circulation to dissipate blood stasis for many years in China. This paper investigated the effects of TH and its main components amygdalin and hydroxysafflor yellow A (HSYA) on hemorheological disorders of blood stasis in rats. Rats were randomly divided into seven groups (control group, model group, TH group, amygdalin group, HSYA group, amygdalin+HSYA group, and aspirin group) with eight animals in each, whose gender was equally distributed throughout groups. All treatments were performed by gavage and administered seven times with an interval of 12h. After the fifth administration, the model rats except those in control group with blood stasis were established by being placed in ice-cold water during the interval between two injections of adrenaline hydrochloride (Adr); and blood samples were collected 30min after the last administration on the following day. TH could significantly decrease whole blood viscosity (WBV), plasma viscosity (PV) and packed cell volume (PCV). It also significantly prolonged thrombin time (TT) and thromboplastin time (APTT), increased prothrombin time (PT) and lowered fibrinogen content (FIB). HSYA which significantly decreased WBV and PV had no effect on plasma coagulation parameters. Amygdalin could significantly decrease PV, prolong APTT and decrease FIB, showing few effects on WBV. TH and its main components amygdalin and HSYA could significantly reduce platelet aggregation and protect vascular endothelial cells. Based on the above results, amygdalin and HSYA were responsible for the main curative effects of TH and usually had synergetic effects, such as decreasing PV and platelet aggregation percentage. The study may provide scientific information to further understanding of the mechanism(s) of TH and its main components in activating blood circulation to dissipate blood. It may also create valuable insight into the possible effects and utilization of TH and its components as a feasible alternative therapeutic agent for patients with hemorheological disorders. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Evaluation of neopterin as a biomarker for the monitoring of Gaucher disease patients.

PubMed

Drugan, Cristina; Drugan, Tudor C; Miron, Nicolae; Grigorescu-Sido, Paula; Naşcu, Ioana; Cătană, Cristina

2016-07-01

Biomarker research is an important area of investigation in Gaucher disease, caused by an inherited deficiency of a lysosomal enzyme, glucocerebrosidase. We evaluated the usefulness of neopterin, as a novel biomarker reflecting chronic inflammation and immune system activation in Gaucher disease and analysed its evolution in response to enzyme replacement therapy (ERT). Circulating plasma neopterin levels in 31 patients with non-neuronopathic Gaucher disease were measured before and after the onset of ERT and were compared with those of 18 healthy controls. Plasma chitotriosidase activity was also monitored, as a reference biomarker, against which we evaluated the evolution of neopterin. Neopterin levels were significantly increased in treatment-naïve patients (mean 11.90 ± 5.82 nM) compared with controls (6.63 ± 5.59 nM, Mann-Whitney U test P = 0.001), but returned to normal levels (6.92 ± 4.66 nM) following ERT. Investigating the diagnostic value of neopterin by receiver operating characteristic analysis, we found a cut-off value of 7.613 nM that corresponds to an area under the curve of 0.780 and indicates a good discrimination capacity, with a sensitivity of 0.774 and a specificity of 0.778. Our results suggest that measurement of circulating neopterin may be considered as a novel test for the confirmation of diagnosis and monitoring of the efficacy of therapeutic intervention in Gaucher disease. Plasma neopterin levels reflect the global accumulation and activation of Gaucher cells and the extent of chronic immune activation in this disorder. Neopterin may be an alternative storage cell biomarker in Gaucher disease, especially in chitotriosidase-deficient patients.

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

PubMed Central

Krug, A K; Enderle, D; Karlovich, C; Priewasser, T; Bentink, S; Spiel, A; Brinkmann, K; Emenegger, J; Grimm, D G; Castellanos-Rizaldos, E; Goldman, J W; Sequist, L V; Soria, J -C; Camidge, D R; Gadgeel, S M; Wakelee, H A; Raponi, M; Noerholm, M; Skog, J

2018-01-01

Abstract Background A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials NCT01526928 PMID:29216356

CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain

PubMed Central

Courret, Nathalie; Darche, Sylvie; Sonigo, Pierre; Milon, Geneviève; Buzoni-Gâtel, Dominique; Tardieux, Isabelle

2006-01-01

The protozoan parasite Toxoplasma gondii enters hosts through the intestinal mucosa and colonizes distant tissues such as the brain, where its progeny persists for a lifetime. We investigated the role of CD11c- and CD11b-expressing leukocytes in T gondii transport during the early step of parasitism from the mouse small intestine and during subsequent parasite localization in the brain. Following intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer patches, and the mesenteric lymph nodes were parasitized while in the blood, parasites were associated with the CD11c- CD11b+ monocytes. Using adoptive transfer experiments, we demonstrated that these parasitized cells triggered a parasitic process in the brain of naive recipient mice. Ex vivo analysis of parasitized leukocytes showed that single tachyzoites remained at the cell periphery, often surrounded by the host cell plasma membrane, but did not divide. Using either a dye that labels circulating leukocytes or an antibody known to prevent CD11b+ circulating leukocytes from leaving the microvascular bed lumen, and chimeric mice in which the hematopoietic cells expressed the green fluorescent protein, we established that T gondii zoites hijacked CD11b+ leukocytes to reach the brain extravascular space. PMID:16051744

Hemopoiesis in the pig-tailed monkey Macaca nemestrina during chronic altitude exposure.

NASA Technical Reports Server (NTRS)

Buderer, M. C.; Pace, N.

1972-01-01

Study of monkeys for 180 days at 3800 m altitude to examine their hemopoietic response. Plasma volume was found to be reduced while red cell volume increased steadily for four to five months. Reduction in mean corpuscular hemoglobin content was observed from day 30 to day 120 at altitude. Total plasma protein concentration was unchanged at altitude, but marked reduction in the albumin/globulin ratio occurred. Total circulating plasma protein and albumin were reduced in amount, whereas nonalbumin protein was unchanged. These results imply loss of albumin coupled with a corresponding loss of water from the blood and maintenance of normal plasma osmotic pressure. The body/venous hematocrit ratio was found to be reduced at altitude, possibly as a consequence of the expanded capillary volume of the body. The hemopoietic responses of the pig-tailed monkey at altitude require at least several months for completion, and closely resemble those seen in man; thus, the monkey can serve well for long-term studies of high-altitude acclimatization.

Evaluating the effect of stressors on thiaminase activity in alewife

USGS Publications Warehouse

Lepak, J.M.; Kraft, C.E.; Honeyfield, D.C.; Brown, S.B.

2008-01-01

No consistent explanation has been found for the variability in the thiaminase activity of alewives Alosa pseudoharengus despite the role of alewife thiaminase in large-scale salmonine mortality in the Laurentian Great Lakes. We conducted experiments to evaluate the effect of two stressors, reduced salt content in the water and food limitation, on alewife thiaminase activity. Alewives were subjected to treatments in replicated tanks in which conductivity was lowered (<100 ??S/cm) for 8 d and feeding was limited for 39 d. Circulating white blood cells, plasma cortisol, plasma glucose, and whole-body thiaminase were measured in individual alewives to assess their response to these experimental treatments. Alewives from the controls had significantly larger numbers of circulating white blood cells than those in the salt-reduced and food-limited treatments (24,000 and 19,000 cells/??L and 11,000 and 9,000 cells/??L for alewives from the two control and salt-reduced treatment tanks, respectively, and 34,000 and 30,000 cells/??L and 21,000 and 16,000 cells/??L for alewives from the two control and food-limited treatment tanks). No significant differences in alewife thiaminase activity were found between treatment fish and their controls. The mean thiaminase activity in the alewives studied increased from 6,900 to 16,000 pmol??g -1??min-1 from the time of their collection in Cayuga Lake to the start of laboratory experiments 1.5-2.5 years later; the latter value was more than twice that of previously reported levels of thiaminase activity from alewives collected in the wild. These data suggest that the variability in alewife thiaminase is not related to stress from salt reduction or food limitation, but laboratory holding conditions significantly increased thiaminase through a mechanism not evaluated by our experimental treatments. ?? Copyright by the American Fisheries Society 2008.

Circulating Plasma Levels of MicroRNA-21 and MicroRNA-221 Are Potential Diagnostic Markers for Primary Intrahepatic Cholangiocarcinoma

PubMed Central

Kemeny, Nancy; Kingham, T. Peter; Allen, Peter J.; D’Angelica, Michael I.; DeMatteo, Ronald P.; Betel, Doron; Klimstra, David; Jarnagin, William R.; Ventura, Andrea

2016-01-01

Background MicroRNAs (miRNAs) are potential biomarkers in various malignancies. We aim to characterize miRNA expression in intrahepatic cholangiocarcinoma (ICC) and identify circulating plasma miRNAs with potential diagnostic and prognostic utility. Methods Using deep-sequencing techniques, miRNA expression between tumor samples and non-neoplastic liver parenchyma were compared. Overexpressed miRNAs were measured in plasma from an independent cohort of patients with cholangiocarcinoma using RT-qPCR and compared with that healthy volunteers. The discriminatory ability of the evaluated plasma miRNAs between patients and controls was evaluated with receiving operating characteristic (ROC) curves. Results Small RNAs from 12 ICC and 11 tumor-free liver samples were evaluated. Unsupervised hierarchical clustering using the miRNA expression data showed clear grouping of ICC vs. non-neoplastic liver parenchyma. We identified 134 down-regulated and 128 upregulated miRNAs. Based on overexpression and high fold-change, miR21, miR200b, miR221, and miR34c were measured in plasma from an independent cohort of patients with ICC (n = 25) and healthy controls (n = 7). Significant overexpression of miR-21 and miR-221 was found in plasma from ICC patients. Furthermore, circulating miR-21 demonstrated a high discriminatory ability between patients with ICC and healthy controls (AUC: 0.94). Conclusion Among the differentially expressed miRNAs in ICC, miR-21 and miR-221 are overexpressed and detectable in the circulation. Plasma expression levels of these miRNAs, particularly miR-21, accurately differentiates patients with ICC from healthy controls and could potentially serve as adjuncts in diagnosis. Prospective validation and comparison with other hepatobiliary malignancies is required to establish their potential role as diagnostic and prognostic biomarkers. PMID:27685844

PGE2 contributes to TGF-β induced T regulatory cell function in human non-small cell lung cancer

PubMed Central

Baratelli, Felicita; Lee, Jay M; Hazra, Saswati; Lin, Ying; Walser, Tonya C; Schaue, Dorthe; Pak, Peter S; Elashoff, David; Reckamp, Karen; Zhang, Ling; Fishbein, Michael C; Sharma, Sherven; Dubinett, Steven M

2010-01-01

CD4+CD25bright regulatory T cells (Treg) play an important role in cancer-mediated immunosuppression. We and others have previously shown that prostaglandin E2 (PGE2) and transforming growth factor beta (TGF-β) induce CD4+CD25brightFOXP3+Treg. Based on these studies, we investigated the requirement for PGE2 in Treg induction by TGF-β. TGF-β stimulation of human CD4+ T cells induced COX-2-dependent production of PGE2. PGE2-neutralizing antibody treatment significantly reduced the suppressive function of TGF-β-induced Treg (TGF-β-Treg) in vitro. TGF-β concentration measured in the plasma of non-small cell lung cancer (NSCLC) patients directly correlated with the frequency of circulating CD4+CD25brightFOXP3+T cells. Flow cytometry analysis showed increased FOXP3 expression in circulating CD4+CD25+HLA-DR- cells of lung cancer patients compared to control subjects. Immunohistochemical analysis revealed co-expression of TGF-β, COX-2, and FOXP3 in serial sections from resected lung tumor tissues. All together these observations suggest interplay between TGF-β and COX-2 in the induction of Treg activities. Interrupting TGF-β and PGE2 signaling may be important in therapeutic interventions that aim to limit Tregfunction in lung cancer. PMID:20733946

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

PubMed

Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

2017-04-28

Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Characterization and Plasma Measurement of the WE-14 Peptide in Patients with Pheochromocytoma

PubMed Central

Guillemot, Johann; Guérin, Marlène; Thouënnon, Erwan; Montéro-Hadjadje, Maité; Leprince, Jérôme; Lefebvre, Hervé; Klein, Marc; Muresan, Mihaela; Anouar, Youssef; Yon, Laurent

2014-01-01

Granins and their derived peptides are valuable circulating biological markers of neuroendocrine tumors. The aim of the present study was to investigate the tumoral chromogranin A (CgA)-derived peptide WE-14 and the potential advantage to combine plasma WE-14 detection with the EM66 assay and the existing current CgA assay for the diagnosis of pheochromocytoma. Compared to healthy volunteers, plasma WE-14 levels were 5.4-fold higher in patients with pheochromocytoma, but returned to normal values after surgical resection of the tumor. Determination of plasma CgA and EM66 concentrations in the same group of patients revealed that the test assays for these markers had an overall 84% diagnostic sensitivity, which is identical to that determined for WE-14. However, we found that WE-14 measurement improved the diagnostic sensitivity when combined with the results of CgA or EM66 assays. By combining the results of the three assays, the sensitivity for the diagnosis of pheochromocytoma was increased to 95%. In fact, the combination of WE-14 with either CgA or EM66 test assays achieved 100% sensitivity for the diagnosis of paragangliomas and sporadic or malignant pheochromocytomas if taken separately to account for the heterogeneity of the tumor. These data indicate that WE-14 is produced in pheochromocytoma and secreted into the general circulation, and that elevated plasma WE-14 levels are correlated with the occurrence of this chromaffin cell tumor. In addition, in association with other biological markers, such as CgA and/or EM66, WE-14 measurement systematically improves the diagnostic sensitivity for pheochromocytoma. These findings support the notion that granin-processing products may represent complementary tools for the diagnosis of neuroendocrine tumors. PMID:24523932

Accuracy of circulating histones in predicting persistent organ failure and mortality in patients with acute pancreatitis.

PubMed

Liu, T; Huang, W; Szatmary, P; Abrams, S T; Alhamdi, Y; Lin, Z; Greenhalf, W; Wang, G; Sutton, R; Toh, C H

2017-08-01

Early prediction of acute pancreatitis severity remains a challenge. Circulating levels of histones are raised early in mouse models and correlate with disease severity. It was hypothesized that circulating histones predict persistent organ failure in patients with acute pancreatitis. Consecutive patients with acute pancreatitis fulfilling inclusion criteria admitted to Royal Liverpool University Hospital were enrolled prospectively between June 2010 and March 2014. Blood samples were obtained within 48 h of abdominal pain onset and relevant clinical data during the hospital stay were collected. Healthy volunteers were enrolled as controls. The primary endpoint was occurrence of persistent organ failure. The predictive values of circulating histones, clinical scores and other biomarkers were determined. Among 236 patients with acute pancreatitis, there were 156 (66·1 per cent), 57 (24·2 per cent) and 23 (9·7 per cent) with mild, moderate and severe disease respectively, according to the revised Atlanta classification. Forty-seven healthy volunteers were included. The area under the receiver operating characteristic (ROC) curve (AUC) for circulating histones in predicting persistent organ failure and mortality was 0·92 (95 per cent c.i. 0·85 to 0·99) and 0·96 (0·92 to 1·00) respectively; histones were at least as accurate as clinical scores or biochemical markers. For infected pancreatic necrosis and/or sepsis, the AUC was 0·78 (0·62 to 0·94). Histones did not predict or correlate with local pancreatic complications, but correlated negatively with leucocyte cell viability (r = -0·511, P = 0·001). Quantitative assessment of circulating histones in plasma within 48 h of abdominal pain onset can predict persistent organ failure and mortality in patients with acute pancreatitis. Early death of immune cells may contribute to raised circulating histone levels in acute pancreatitis. © 2017 The Authors. BJS published by John Wiley & Sons Ltd on behalf of BJS Society Ltd.

Circulating betatrophin is elevated in patients with type 1 and type 2 diabetes.

PubMed

Yamada, Hodaka; Saito, Tomoyuki; Aoki, Atsushi; Asano, Tomoko; Yoshida, Masashi; Ikoma, Aki; Kusaka, Ikuyo; Toyoshima, Hideo; Kakei, Masafumi; Ishikawa, San-E

2015-01-01

There is evidence that betatrophin, a hormone derived from adipose tissue and liver, affects the proliferation of pancreatic beta cells in mice. The aim of this study was to examine circulating betatrophin concentrations in Japanese healthy controls and patients with type 1 and type 2 diabetes. A total of 76 subjects (12 healthy controls, 34 type 1 diabetes, 30 type 2 diabetes) were enrolled in the study. Circulating betatrophin was measured with an ELISA kit and clinical parameters related to betatrophin were analyzed statistically. Circulating betatrophin (Log transformed) was significantly increased in patients with diabetes compared with healthy subjects (healthy controls, 2.29 ± 0.51; type 1 diabetes, 2.94 ± 0.44; type 2 diabetes, 3.17 ± 0.18; p<0.001, 4.1 to 5.4 times in pg/mL order). Age, HbA1c, fasting plasma glucose and Log triglyceride were strongly associated with Log betatrophin in all subjects (n=76) in correlation analysis. In type 1 diabetes, there was a correlation between Log betatrophin and Log CPR. These results provide the first evidence that circulating betatrophin is significantly elevated in Japanese patients with diabetes. The findings of this pilot study also suggest a possibility of association between the level of betatrophin and the levels of glucose and triglycerides.

Effects of Aged Stored Autologous Red Blood Cells on Human Endothelial Function

PubMed Central

Kanias, Tamir; Triulzi, Darrel; Donadee, Chenell; Barge, Suchitra; Badlam, Jessica; Jain, Shilpa; Belanger, Andrea M.; Kim-Shapiro, Daniel B.

2015-01-01

Rationale: A major abnormality that characterizes the red cell “storage lesion” is increased hemolysis and reduced red cell lifespan after infusion. Low levels of intravascular hemolysis after transfusion of aged stored red cells disrupt nitric oxide (NO) bioavailabity, via accelerated NO scavenging reaction with cell-free plasma hemoglobin. The degree of intravascular hemolysis post-transfusion and effects on endothelial-dependent vasodilation responses to acetylcholine have not been fully characterized in humans. Objectives: To evaluate the effects of blood aged to the limits of Food and Drug Administration–approved storage time on the human microcirculation and endothelial function. Methods: Eighteen healthy individuals donated 1 U of leukopheresed red cells, divided and autologously transfused into the forearm brachial artery 5 and 42 days after blood donation. Blood samples were obtained from stored blood bag supernatants and the antecubital vein of the infusion arm. Forearm blood flow measurements were performed using strain-gauge plethysmography during transfusion, followed by testing of endothelium-dependent blood flow with increasing doses of intraarterial acetylcholine. Measurements and Main Results: We demonstrate that aged stored blood has higher levels of arginase-1 and cell-free plasma hemoglobin. Compared with 5-day blood, the transfusion of 42-day packed red cells decreases acetylcholine-dependent forearm blood flows. Intravascular venous levels of arginase-1 and cell-free plasma hemoglobin increase immediately after red cell transfusion, with more significant increases observed after infusion of 42-day-old blood. Conclusions: We demonstrate that the transfusion of blood at the limits of Food and Drug Administration–approved storage has a significant effect on the forearm circulation and impairs endothelial function. Clinical trial registered with www.clinicaltrials.gov (NCT 01137656) PMID:26222884

Vinpocetine and pyritinol: a new model for blood rheological modulation in cerebrovascular disorders—a randomized controlled clinical study.

PubMed

Alkuraishy, Hayder M; Al-Gareeb, Ali I; Albuhadilly, Ali K

2014-01-01

Blood and plasma viscosity are the major factors affecting blood flow and normal circulation. Whole blood viscosity is mainly affected by plasma viscosity, red blood cell deformability/aggregation and hematocrit, and other physiological factors. Thirty patients (twenty males + ten females) with age range 50-65 years, normotensive with history of cerebrovascular disorders, were selected according to the American Heart Stroke Association. Blood viscosity and other rheological parameters were measured after two-day abstinence from any medications. Dual effects of vinpocetine and pyritinol exhibit significant effects on all hemorheological parameters (P < 0.05), especially on low shear whole blood viscosity (P < 0.01), but they produced insignificant effects on total serum protein and high shear whole blood viscosity (P > 0.05). Therefore, joint effects of vinpocetine and pyritinol improve blood and plasma viscosity in patients with cerebrovascular disorders.

Vinpocetine and Pyritinol: A New Model for Blood Rheological Modulation in Cerebrovascular Disorders—A Randomized Controlled Clinical Study

PubMed Central

Alkuraishy, Hayder M.; Al-Gareeb, Ali I.; Albuhadilly, Ali K.

2014-01-01

Blood and plasma viscosity are the major factors affecting blood flow and normal circulation. Whole blood viscosity is mainly affected by plasma viscosity, red blood cell deformability/aggregation and hematocrit, and other physiological factors. Thirty patients (twenty males + ten females) with age range 50–65 years, normotensive with history of cerebrovascular disorders, were selected according to the American Heart Stroke Association. Blood viscosity and other rheological parameters were measured after two-day abstinence from any medications. Dual effects of vinpocetine and pyritinol exhibit significant effects on all hemorheological parameters (P < 0.05), especially on low shear whole blood viscosity (P < 0.01), but they produced insignificant effects on total serum protein and high shear whole blood viscosity (P > 0.05). Therefore, joint effects of vinpocetine and pyritinol improve blood and plasma viscosity in patients with cerebrovascular disorders. PMID:25548768

Erythrocyte deformation in ischemic acute tubular necrosis and amelioration by splenectomy in the dog.

PubMed

Mandal, A K; Taylor, C A; Bell, R D; Hillman, N M; Jarnot, M D; Cunningham, J D; Phillips, L G

1991-11-01

Bilateral renal artery occlusion (RAO) for 120 minutes in dogs results in acute tubular necrosis (ATN) and peritubular capillary (PTC) congestion with rapidly deteriorating renal function. We have shown that prior splenectomy minimizes RAO-induced renal functional and histopathologic changes. The purpose of this study was to examine whether this renal protection is due to prevention of red blood cell echinocyte formation and resultant renal PTC congestion. Echinocytes (burr cells) are poorly deformable, impart high viscosity to the blood, and may hinder reperfusion by increasing resistance to renal capillary blood flow. Splenectomized (SPLX) or sham-SPLX dogs were treated with bilateral RAO for 120 minutes. After RAO, renal function and renal blood flow were monitored, and peripheral blood red blood cells were examined at 1 hour and at 24-hour intervals for 96 hours. Renal biopsies were taken 1 hour after RAO and the kidneys removed 96 hours after RAO. The RBCs and renal tissues were studied using scanning electron microscopy. Renal function was assessed by endogenous creatinine clearance. Sham-SPLX animals showed a marked and sustained decrease in creatinine clearance, consistently elevated serum creatinine levels and fractional excretion of sodium, and diffuse ATN and PTC congestion with echinocytes. These animals had a peak in circulating echinocytes 1 hour after RAO (p less than 0.05), which showed an excellent negative correlation with creatinine clearance (r = -0.999; p less than 0.001). On the contrary, SPLX animals had essentially no change in serum creatinine or fractional excretion of sodium, minimal tubular changes, no PTC congestion, and no rise in circulating echinocytes during the 96-hour observation. In vitro treatment of the postischemic red blood cells from sham animals with adenosine-inosine or fresh postischemic plasma from the SPLX animals showed almost complete reversal to discocytes (normal red blood cells), whereas in vitro treatment of postischemic red blood cells from the SPLX animals with fresh postischemic plasma from the sham animals resulted in a marked echinocytic response. We conclude that 1) a marked echinocyte response in the immediate postischemic period is an important mechanism in initiating ischemic ATN, 2) an echinocyte inducing factor may reside in the plasma of spleen-intact animals, and 3) mitigation of ATN and PTC congestion by splenectomy is, at least in part, consequential to attenuated echinocytic response in the immediate postischemic period.

A longitudinal analysis of circulating stress-related proteins and chronic ethanol self-administration in cynomolgus macaques

PubMed Central

Helms, Christa M.; Messaoudi, Ilhem; Jeng, Sophia; Freeman, Willard M.; Vrana, Kent E.; Grant, Kathleen A.

2011-01-01

Background Alcoholics have alterations in endocrine and immune function and increased susceptibility to stress-related disorders. A longitudinal analysis of chronic ethanol intake on homeostatic mechanisms is, however, incompletely characterized in primates. Methods Plasma proteins (n = 60; Luminex) and hormones (adrenocorticotropic hormone, ACTH; cortisol) were repeatedly measured in adult male cynomolgus monkeys (Macaca fascicularis, n = 10) during a 32-month experimental protocol at baseline, during induction of water and ethanol (4% w/v in water) self-administration, after 4 months and after 12 months of 22-h daily concurrent access to ethanol and water. Results Significant changes were observed in ACTH, cortisol and 45/60 plasma proteins: a majority (28/45) were suppressed as a function of ethanol self-administration, eight proteins were elevated and nine showed biphasic changes. Cortisol and ACTH were greatest during induction, and correlations between these hormones and plasma proteins varied across the experiment. Pathway analyses implicated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) as possible mediators of ethanol-induced effects on immune-related proteins in primates. Conclusion Chronic ethanol consumption in primates leads to an allostatic state of physiological compromise with respect to circulating immune- and stress-related proteins in NF-κB- and STAT/JAK-related pathways in correlation with altered endocrine activity. PMID:22141444

Studies of Tissue Perfusion Failure at LAC+USCMC and the Incorporation of the Results into a National Trauma Database

DTIC Science & Technology

2005-04-01

Context Link] 29. Gibson JG, Seligman AM, Peacock WC, et al: The circulating red cell and plasma volume and the distribution of blood in large and...BS; Gandhi, Ashutosh BS; Chien, Li-Chien MD; Lu, Kevin MD; Martin, Matthew J. MD; Chan, Linda S. PhD; Demetriades, Demetrios MD, PhD; Ahmadpour...Physicians Outcome Prediction of Emergency Patients by Noninvasive Hemodynamic Monitoring* William C. Shoemaker, MD; Charles C. J. Wo, BS; Linda

Circulating miR-132-3p as a Candidate Diagnostic Biomarker for Malignant Mesothelioma

PubMed Central

Gawrych, Katarzyna; Casjens, Swaantje; Brik, Alexander; Lehnert, Martin; Taeger, Dirk; Pesch, Beate; Kollmeier, Jens; Bauer, Torsten T.; Johnen, Georg; Brüning, Thomas

2017-01-01

The use of circulating microRNAs as biomarkers has opened new opportunities for diagnosis of cancer because microRNAs exhibit tumor-specific expression profiles. The aim of this study was the identification of circulating microRNAs in human plasma as potential biomarkers for the diagnosis of malignant mesothelioma. For discovery, TaqMan Low Density Array Human MicroRNA Cards were used to analyze 377 microRNAs in plasma samples from 21 mesothelioma patients and 21 asbestos-exposed controls. For verification, individual TaqMan microRNA assays were used for quantitative real-time PCR in plasma samples from 22 mesothelioma patients and 44 asbestos-exposed controls. The circulating miR-132-3p showed different expression levels between mesothelioma patients and asbestos-exposed controls. For discrimination, sensitivity of 86% and specificity of 61% were calculated. Circulating miR-132-3p in plasma was not affected by hemolysis and no impact of age or smoking status on miR-132-3p levels could be observed. For the combination of miR-132-3p with the previously described miR-126, sensitivity of 77% and specificity of 86% were calculated. The results of this study indicate that miR-132-3p might be a new promising diagnostic biomarker for malignant mesothelioma. It is indicated that the combination of miR-132-3p with other individual biomarkers improves the biomarker performance. PMID:28321148

Sex-hormone-binding globulin.

PubMed

Anderson, D C

1974-01-01

A review was made to understand how plasma binding protein might influence sex-hormone action in target tissues. Steroids are predominately bound to plasma proteins and only unbound steroids enter the cells. Sex-hormone-binding globulin (SHBG) binds to both the main circulating steroid T and E2 but changes in SHBG concentrations exert significant results. Increased SHBG levels increase estrogen production and decreases T activity; whereas, increased androgens increase T action and inhibit SHBG production. These disturbances in hormone maintenance may lead to abnormal adult sex differentiation such as hirsutism and forms of hynaecomastia. By developing SHBG concentration measurement methods-responses of hirsutism to glucocorticoid or estrogem may be assessed. In addition, the effect of thyroid hormones on SHBG may also have therapeutic implications in endocrine disease.

Evaluation of Different Blood Collection Tubes and Blood Storage Conditions for the Preservation and Stability of Cell-Free Circulating DNA for the Analysis of the Methylated mSEPT9 Colorectal Cancer Screening Marker.

PubMed

Distler, Jurgen; Tetzner, Reimo; Weiss, Gunter; König, Thomas; Schlegel, Anne; Bagrowski, Michal

2016-01-01

For the subsequent analysis of the methylated mSEPT9 colorectal cancer screening marker in plasma, different blood collection tubes and blood storage conditions were investigated. The study demonstrated that methylated Septin 9 ( m SEPT9) can be consistently detected in plasma samples derived from whole blood samples collected with S-Monovette® K3E and BD Vacutainer ® K2EDTA tubes stored at 2-8 °C for a maximum of 24 h and for samples collected in S-Monovette CPDA tubes stored at 18-25 °C for up to 48 h.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia.

PubMed

Cortés-Puch, Irene; Wang, Dong; Sun, Junfeng; Solomon, Steven B; Remy, Kenneth E; Fernandez, Melinda; Feng, Jing; Kanias, Tamir; Bellavia, Landon; Sinchar, Derek; Perlegas, Andreas; Solomon, Michael A; Kelley, Walter E; Popovsky, Mark A; Gladwin, Mark T; Kim-Shapiro, Daniel B; Klein, Harvey G; Natanson, Charles

2014-02-27

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia

PubMed Central

Wang, Dong; Sun, Junfeng; Solomon, Steven B.; Remy, Kenneth E.; Fernandez, Melinda; Feng, Jing; Kanias, Tamir; Bellavia, Landon; Sinchar, Derek; Perlegas, Andreas; Solomon, Michael A.; Kelley, Walter E.; Popovsky, Mark A.; Gladwin, Mark T.; Kim-Shapiro, Daniel B.; Klein, Harvey G.; Natanson, Charles

2014-01-01

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes. PMID:24366359

Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain.

PubMed

Rummel, Christoph; Sachot, Christelle; Poole, Stephen; Luheshi, Giamal N

2006-11-01

Interleukin (IL)-6 is an important humoral mediator of fever following infection and inflammation and satisfies a number of criteria for a circulating pyrogen. However, evidence supporting such a role is diminished by the moderate or even absent ability of the recombinant protein to induce fever and activate the cyclooxygenase-2 (COX-2) pathway in the brain, a prerequisite step in the initiation and maintenance of fever. In the present study, we investigated the role of endogenous circulating IL-6 in a rodent model of localized inflammation, by neutralizing its action using a specific antiserum (IL-6AS). Rats were injected with LPS (100 microg/kg) or saline into a preformed air pouch in combination with an intraperitoneal injection of either normal sheep serum or IL-6AS (1.8 ml/rat). LPS induced a febrile response, which was accompanied by a significant rise in plasma IL-6 and nuclear STAT3 translocation in endothelial cells throughout the brain 2 h after treatment, including areas surrounding the sensory circumventricular organs and the median preoptic area (MnPO), important regions in mediating fever. These responses were abolished in the presence of the IL-6AS, which also significantly inhibited the LPS-induced upregulation of mRNA expression or immunoreactivity (IR) of the inducible form of COX, the rate-limiting enzyme for PGE2-synthesis. Interestingly, nuclear signal transducer and activator of transcription (STAT)3-positive cells colocalized with COX-2-IR, signifying that IL-6-activated cells are directly involved in PGE2 production. These observations suggest that IL-6 is an important circulating pyrogen that activates the COX-2-pathway in cerebral microvasculature, most likely through a STAT3-dependent pathway.

Rapid isolation of blood plasma using a cascaded inertial microfluidic device

PubMed Central

Robinson, M.; Hinsdale, T.; Coté, G.

2017-01-01

Blood, saliva, mucus, sweat, sputum, and other biological fluids are often hindered in their ability to be used in point-of-care (POC) diagnostics because their assays require some form of off-site sample pre-preparation to effectively separate biomarkers from larger components such as cells. The rapid isolation, identification, and quantification of proteins and other small molecules circulating in the blood plasma from larger interfering molecules are therefore particularly important factors for optical blood diagnostic tests, in particular, when using optical approaches that incur spectroscopic interference from hemoglobin-rich red blood cells (RBCs). In this work, a sequential spiral polydimethylsiloxane (PDMS) microfluidic device for rapid (∼1 min) on-chip blood cell separation is presented. The chip utilizes Dean-force induced migration via two 5-loop Archimedean spirals in series. The chip was characterized in its ability to filter solutions containing fluorescent beads and silver nanoparticles and further using blood solutions doped with a fluorescent protein. Through these experiments, both cellular and small molecule behaviors in the chip were assessed. The results exhibit an average RBC separation efficiency of ∼99% at a rate of 5.2 × 106 cells per second while retaining 95% of plasma components. This chip is uniquely suited for integration within a larger point-of-care diagnostic system for the testing of blood plasma, and the use of multiple filtering spirals allows for the tuning of filtering steps, making this device and the underlying technique applicable for a wide range of separation applications. PMID:28405258

Renal lesions associated with plasma cell dyscrasias: practical approach to diagnosis, new concepts, and challenges.

PubMed

Herrera, Guillermo A

2009-02-01

Patients with plasma cell dyscrasias (myeloma) may exhibit a variety of renal manifestations as a result of damage from circulating light- and heavy-chain immunoglobulin components produced by the neoplastic plasma cells. The renal alterations can occur in any of the renal compartments, and in a significant number of the cases more than one compartment is affected. Research in the laboratory has helped considerably in providing a solid conceptual understanding of how renal damage occurs. To detail advances that have been made in the diagnosis of these conditions and to provide an account of research accomplishments that have solidified diagnostic criteria. The new knowledge that has been acquired serves to provide a solid platform for the future design of new therapeutic interventions aimed at ameliorating or abolishing the progressive renal damage that typically takes place. Translational efforts have substantially contributed to elucidate mechanistically the molecular events responsible for the renal damage. The spectrum of renal manifestations associated with plasma cell dyscrasias has expanded significantly in the last 10 years. Diagnostic criteria have also been refined. This information has been summarized from work done at several institutions. A number of significant challenges remain in the diagnosis of these conditions, some of which will be discussed in this article. Dealing with these challenges will require additional translational efforts and close cooperation between basic researchers, clinicians, and pathologists in order to improve the diagnostic tools available to renal pathologists and to acquire a more complete understanding of clinical and pathologic manifestations associated with these conditions.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

PubMed

Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P

2017-09-01

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched

PubMed Central

García, José Miguel; García, Vanesa; Peña, Cristina; Domínguez, Gemma; Silva, Javier; Diaz, Raquel; Espinosa, Pablo; Citores, Maria Jesús; Collado, Manuel; Bonilla, Félix

2008-01-01

Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin. PMID:18456845

Plasma Level of Interleukin-35 as an Independent Prognostic Indicator in Hepatocellular Carcinoma.

PubMed

Qiu, Xiangting; Wang, Xinhua; Song, Yucui; Chen, Lingling

2016-12-01

Hepatocellular carcinoma is a major type of liver cancer with poor prognosis. The aim of the study was to determine the prognostic significance of plasma interleukin-35 level in hepatocellular carcinoma. A total of 153 hepatocellular carcinoma patients and 153 healthy controls were enrolled. Blood samples were obtained at admission. Plasma interleukin-35 level was analyzed by enzyme-linked immunosorbent assay. Distribution of T cell subset and expression of Fas/FasL protein were detected by flow cytometry. The patients were followed up for 2 years. Poor prognosis was defined as death of hepatocellular carcinoma. The plasma levels of interleukin-35 were significantly higher in the patients than the controls (25.1 ± 13.1, 9.3 ± 6.3 pg/mL, P < 0.001). After adjusted for multiple confounding factors, the multivariate logistic regression analyses reported that high level of interleukin-35 (≥25.0 pg/mL) was associated with the poor prognosis in the patients (OR 6.63, 95 % CI 3.27-13.47). Compared with the patients with low level of interleukin-35 (<25.0 pg/mL), the patients with high level of interleukin-35 showed higher frequencies of CD4+CD25+FoxP3+ and CD3+Foxp3+ regulatory T cells (P < 0.001 and P < 0.001) and also showed higher apoptosis levels of CD8+ T cells (P < 0.001). Circulating interleukin-35 concentration might be an independent prognostic indicator in hepatocellular carcinoma. Such prognostic significance could be partly involved in the activation of regulatory T cell and the apoptosis of CD8+ T cell.

Peptide YY kinetics and effects on blood pressure and circulating pancreatic and gastrointestinal hormones and metabolites in man.

PubMed

Adrian, T E; Sagor, G R; Savage, A P; Bacarese-Hamilton, A J; Hall, G M; Bloom, S R

1986-10-01

Peptide YY (PYY) is a 36 amino acid peptide produced by mucosal endocrine cells of the ileum and colon which inhibits acid secretion and intestinal transit in man. To assess its effects on metabolites and digestive hormones PYY was infused into 18 fasting normal subjects at three dose levels (0.06, 0.19, and 0.57 pmol kg-1 min-1), each for a period of 1 h. During the infusions mean plasma PYY levels increased by 8, 25, and 73 pmol/liter, respectively. The mean disappearance half-time on stopping the infusions was 9.2 +/- 0.4 (SEM) min. The mean MCR was 7.3 +/- 0.7 ml kg-1 min-1 and the apparent volume of distribution was calculated to be 94 +/- 9 ml kg-1. During the highest dose infusion there was a significant increase in both systolic and diastolic blood pressure, of 8.6 +/- 3.7 mmHg (P less than 0.05) and 10.9 +/- 3.0 mmHg (P less than 0.01), respectively. PYY caused a significant 50% reduction in plasma pancreatic polypeptide concentrations (P less than 0.05) and a 55% reduction in circulating motilin levels (P less than 0.05). PYY had no significant effect on circulating concentrations of insulin, glucagon, gastrin, gastric inhibitory peptide, neurotensin, enteroglucagon, or vasoactive intestinal peptide. PYY also had no significant effect on circulating concentrations of glucose, lactate, glycerol, or nonesterified fatty acids. This recently discovered human intestinal hormonal peptide thus has significant effects both on gastrointestinal hormones (motilin and pancreatic polypeptide) and blood pressure in man, but appears not to influence glucose or lipid metabolism.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Red blood cells for transfusion in patients with sepsis: respective roles of unit age and exposure to recipient plasma.

PubMed

Chadebech, Philippe; Bodivit, Gwellaouen; Razazi, Keyvan; de Vassoigne, Christophe; Pellé, Laurence; Burin-des-Roziers, Nicolas; Bocquet, Thibault; Bierling, Philippe; Djoudi, Rachid; Mekontso-Dessap, Armand; Pirenne, France

2017-08-01

Red blood cell (RBC) storage in blood banks is not exempt from cellular injury. Alterations not observed on RBCs freshly isolated from units can rapidly appear in circulation. The transfusion of old blood units, even if this is a controversial issue, could therefore have adverse effects on the recipient. We wanted to determine the respective effects of storage duration and recipient plasma on RBCs for transfusion into patients with severe sepsis. Eleven stored RBC units were sampled at various time points, approximately Days 3 to 8 (referred to as fresh RBCs) and Days 38 to 42 (old RBCs) and tested in coincubation experiments with plasma obtained from 13 patients with severe sepsis and 17 healthy donors as controls. RBCs were tested after 24 or 48 hours at 37°C for the detection of senescence markers (phosphatidylserine exposure, calcium influx, and reactive oxygen species detection and decrease in size) with or without exposure to plasma. We confirmed that a 42-day refrigerated storage of RBCs alone (without any incubation in plasma) had no significant effect on RBCs and no senescence marker detected. By contrast, ex vivo exposure to plasma samples altered both fresh and old RBCs, with a much larger effect for old RBCs, regardless of the plasma used (sepsis vs. control). We show that the main factor affecting the senescence of RBCs for transfusion into patients with severe sepsis is the age of the stored units rather than the clinical status of the recipient. © 2017 AABB.

Circulation of Plasma in the Jovian Magnetosphere as Inferred from the Galileo Magnetometer Observations

NASA Astrophysics Data System (ADS)

Yu, Z. J.; Russell, C. T.; Kivelson, M. G.; Khurana, K. K.

2000-10-01

Massloading of the jovian magnetosphere by the addition of ions at the moon Io is the ultimate engine of the circulation of the magnetospheric plasma. In steady state the radial density profile enables the radial outflow velocity to be calculated from the mass addition rate. Some of these ions are lost from the field lines through pitch angle diffusion. Expected loss rates can be calculated from the fluctuation level in the magnetic field. Radial velocities can be calculated from observations of the Europa wake and force balance in the magnetodisk. The resulting transport times are shorter than the pitch angle scattering loss times so that most of the plasma is transported to the tail and lost by magnetic island formation. In turn the island formation process (reconnection) depletes magnetic field lines making them buoyant and allowing them to "float" back to the inner magnetosphere. In the torus these depleted flux tubes can be seen as thin tubes with stronger than the ambient field strength, implying plasma pressures about 2% of the magnetic field and ion temperatures principally in the range 30-150 eV. When the depleted flux tubes reach the orbit of Io where the energy density of the plasma drops these depleted flux tubes become indistinguishable from the ambient plasma, completing the circulation loop.

Uroguanylin levels in intestine and plasma are regulated by nutritional status in a leptin-dependent manner.

PubMed

Folgueira, C; Sanchez-Rebordelo, E; Barja-Fernandez, S; Leis, R; Tovar, S; Casanueva, F F; Dieguez, C; Nogueiras, R; Seoane, L M

2016-03-01

Uroguanylin (UGN) is a 16 amino acid peptide produced mainly by intestinal epithelial cells. Nutrients intake increases circulating levels of prouroguanylin that is processed and converted to UGN to activate the guanylyl cyclase 2C receptor (GUCY2C). Given that the UGN-GUCY2C system has been proposed as a novel gut-brain endocrine axis regulating energy balance, the aim of the present study was to investigate the regulation of UGN protein levels in duodenum and circulating levels in lean and obese mice under different nutritional conditions and its potential interaction with leptin. Swiss, C57BL/6 wild-type and ob/ob male adult mice under different nutritional conditions were used: fed ad libitum standard diet (control); 48 h fasting (fasted); 48 h fasting followed by 24 h of feeding (refed); and fed high-fat diet (45 %) during 10 weeks. In addition, peripheral leptin administration was performed. Intestinal uroguanylin expression was studied by Western blot analysis; plasma levels were measured by ELISA. Food deprivation significantly reduced plasma UGN levels, which were correlated with the lower protein levels of UGN in duodenum. These effects were reverted after refeeding and leptin challenge. Consistently, in ob/ob mice UGN expression was decreased, whereas leptin treatment up-regulated UGN levels in duodenum in these genetically modified mice compared to WT. Diet-induced obese mice displayed increased UGN levels in intestine and plasma in comparison with lean mice. Our findings suggest that UGN levels are correlated with energy balance status and that the regulation of UGN by nutritional status is leptin-dependent.

The role of phosphatidylserine in recognition and removal of erythrocytes.

PubMed

Kuypers, F A; de Jong, K

2004-03-01

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

PubMed Central

Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

2017-01-01

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

Plasma Sheet Circulation Pathways

NASA Technical Reports Server (NTRS)

Moore, Thomas E.; Delcourt, D. C.; Slinker, S. P.; Fedder, J. A.; Damiano, P.; Lotko, W.

2008-01-01

Global simulations of Earth's magnetosphere in the solar wind compute the pathways of plasma circulation through the plasma sheet. We address the pathways that supply and drain the plasma sheet, by coupling single fluid simulations with Global Ion Kinetic simulations of the outer magnetosphere and the Comprehensive Ring Current Model of the inner magnetosphere, including plasmaspheric plasmas. We find that the plasma sheet is supplied with solar wind plasmas via the magnetospheric flanks, and that this supply is most effective for northward IMF. For southward IMF, the innermost plasma sheet and ring current region are directly supplied from the flanks, with an asymmetry of single particle entry favoring the dawn flank. The central plasma sheet (near midnight) is supplied, as expected, from the lobes and polar cusps, but the near-Earth supply consists mainly of slowly moving ionospheric outflows for typical conditions. Work with the recently developed multi-fluid LFM simulation shows transport via plasma "fingers" extending Earthward from the flanks, suggestive of an interchange instability. We investigate this with solar wind ion trajectories, seeking to understand the fingering mechanisms and effects on transport rates.

miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

PubMed Central

Goedeke, Leigh; Rotllan, Noemi; Ramírez, Cristina M.; Aranda, Juan F.; Canfrán-Duque, Alberto; Araldi, Elisa; Fernández-Hernando, Ana; Langhi, Cedric; de Cabo, Rafael; Baldán, Ángel; Suárez, Yajaira; Fernández-Hernando, Carlos

2015-01-01

Rationale Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and Results Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels. PMID:26520906

Circulating RNAs as new biomarkers for detecting pancreatic cancer

PubMed Central

Kishikawa, Takahiro; Otsuka, Motoyuki; Ohno, Motoko; Yoshikawa, Takeshi; Takata, Akemi; Koike, Kazuhiko

2015-01-01

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers. PMID:26229396

Circulating levels of miR-7, miR-152 and miR-192 respond to vitamin D supplementation in adults with prediabetes and correlate with improvements in glycemic control.

PubMed

Nunez Lopez, Yury O; Pittas, Anastassios G; Pratley, Richard E; Seyhan, Attila A

2017-11-01

Vitamin D may play an important role in modifying the risk of type 2 diabetes. Supplementation with cholecalciferol has been shown to improve β cell function and to attenuate the rise in glycated hemoglobin in people at high risk of diabetes. We examined whether circulating microRNAs (miRNAs) reflect disease progression and/or respond to vitamin D supplementation. We measured plasma levels of select miRNAs implicated in diabetes in people with prediabetes treated either with placebo (n=21) or 2000 U of cholecalciferol daily (n=21) for 4 months in the Calcium and Vitamin D for Diabetes Mellitus trial and compared the baseline-adjusted changes after correcting for age, body mass index, race, time of study entry (season) and baseline disposition index. Circulating levels of miR-7 (sixfold reduction, P=.01), miR-152 (1.5-fold increase, P=.03), and miR-192 (1.7-fold reduction, P=.026) displayed significant treatment-by-time interactions between the placebo- and the vitamin-D-treated groups. Plasma levels of miR-7 were reduced in the vitamin D and increased in the placebo group. The change in miR-152 positively correlated with the change in levels of the circulating metabolite 25-hydroxyvitamin D (r=0.33, P=.046) and negatively correlated with the change in glycated hemoglobin (r=-0.37, P=.024). The change in miR-192 positively correlated with the change in fasting glucose (r=0.41, P<.011). In conclusion, reduction of circulating miR-7 and miR-192, accompanied by elevation of miR-152, reflects a beneficial metabolic response to vitamin D treatment in people with prediabetes. These miRNAs may be useful biomarkers in diabetes prevention trials and other studies of vitamin D. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients

PubMed Central

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-01-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients. PMID:29067441

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

PubMed

Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

2015-11-27

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices.

PubMed

Josephs, S F; Loudovaris, T; Dixit, A; Young, S K; Johnson, R C

1999-01-01

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.

Targeting B cells in immune-mediated inflammatory disease: a comprehensive review of mechanisms of action and identification of biomarkers.

PubMed

Dörner, Thomas; Kinnman, Nils; Tak, Paul P

2010-03-01

B cell-depletion therapy, particularly using anti-CD20 treatment, has provided proof of concept that targeting B cells and the humoral response may result in clinical improvements in immune-mediated inflammatory disease. In this review, the mechanisms of action of B cell-targeting drugs are investigated, and potential biomarkers associated with response to treatment in patients with autoimmune diseases are identified. Most available data relate to B cell depletion using anti-CD20 therapy (rituximab) in patients with rheumatoid arthritis (RA). Treatment leads to significant clinical benefit, but apparently fails to deplete long-lived plasma cells, and discontinuation is associated with relapse. Biomarkers commonly used in studies of B cell-targeted therapies include rheumatoid factor, anti-citrullinated peptide antibodies, and immunoglobulin (Ig) levels. More recently, there has been interest in markers such as B cell phenotype analysis, and B lymphocyte stimulator (BLyS)/a proliferation-inducing ligand (APRIL), the latter particularly in studies of the IgG Fc-transmembrane activator and CAML interactor (TACI) fusion protein (atacicept) and anti-BLyS therapy (belimumab). Data from clinical trials of B cell-depleting agents in RA suggest that specific autoantibodies, BLyS, APRIL, and circulating and synovial B lineage cell levels may have potential as biomarkers predictive of response to treatment. Further trials validating these markers against clinical outcomes in RA are required. In patients with systemic lupus erythematosus, Fc receptors and levels of circulating immune cells (including B cells and natural killer cells) may be relevant markers. 2010 Elsevier Inc. All rights reserved.

Recombinant granulocyte colony-stimulating factor administered enterally to neonates is not absorbed.

PubMed

Calhoun, Darlene A; Maheshwari, Akhil; Christensen, Robert D

2003-08-01

Granulocyte colony-stimulating factor (G-CSF) is present in liquids swallowed by the fetus and neonate; specifically, amniotic fluid, colostrum, and human milk. The swallowed G-CSF has local effects on enteric cells, which express the G-CSF receptor. However, some portion of the G-CSF ingested by the fetus and neonate might be absorbed into the circulation and have systemic actions, such as stimulating neutrophil production. To assess this possibility we sought to determine if circulating G-CSF concentrations of neonates increase after enteral administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). This was a single-center, prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhG-CSF (100 microg/kg) and 1 dose of placebo. Plasma G-CSF concentrations were measured at 2 and 4 hours after administration of the test solution. No significant change in plasma G-CSF concentration was observed after the enteral administration of rhG-CSF. On this basis, we conclude that orally administered rhG-CSF is not absorbed in significant quantities, and we speculate that the G-CSF swallowed by the fetus and neonate has local but not systemic effects.

A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening.

PubMed

Jing, Rong-Rong; Wang, Hui-Min; Cui, Ming; Fang, Meng-Kang; Qiu, Xiao-Jun; Wu, Xin-Hua; Qi, Jin; Wang, Yue-Guo; Zhang, Lu-Rong; Zhu, Jian-Hua; Ju, Shao-Qing

2011-09-01

Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format. Copyright © 2011 The Canadian Society of Clinical Chemists. All rights reserved.

Fibrin monomer increases platelet adherence to tumor cells in a flowing system: a possible role in metastasis?

PubMed

Biggerstaff, J P; Seth, N B; Meyer, T V; Amirkhosravi, A; Francis, J L

1998-12-15

Considerable evidence exists linking hemostasis and malignancy. Platelet adhesion to tumor cells has been implicated in the metastatic process. Plasma fibrinogen (Fg) and fibrin (Fn) monomer, increased in cancer, may play a role in tumor biology. Binding of Fn monomer to tumor cells and its effect on platelet-tumor cell adhesion in a flowing system were studied. Fn monomer was produced by adding thrombin (1 micro/mL) to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro (GPRP) amide. Fn monomer binding to live A375 cells was visualized by confocal laser scanning microscopy (CLSM). Adherent cells were perfused for 1h with Fn monomer, washed and stained in situ with anti-human Fn (American Biogenetic Sciences, Inc.) followed by goat anti-mouse IgG(FITC). Platelet adherence to Fn monomer treated A375 cells was performed under flow conditions by passing platelets (5x10(4)/microl 0.25 mL/min; labeled with the carbocyanine dye DiI) over the tumor cells for 30 min. CLSM images were obtained after washing. There was considerable binding of Fn monomer, but not Fg alone. Platelets adhered relatively weakly to untreated A375 cells and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or thrombin. However, pre-treatment with Fn monomer resulted in extensive platelet binding to tumor cells, suggesting that coagulation activation and the subsequent increase in circulating Fn monomer may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Circulating exosomes potentiate tumor malignant properties in a mouse model of chronic sleep fragmentation

PubMed Central

Khalyfa, Abdelnaby; Almendros, Isaac; Gileles-Hillel, Alex; Akbarpour, Mahzad; Trzepizur, Wojciech; Mokhlesi, Babak; Huang, Lei; Andrade, Jorge; Farré, Ramon; Gozal, David

2016-01-01

Background Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. We hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties. Results SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post. SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways. Methods Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before (Pre) and after adherent treatment for 6 wks (Post) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets. Conclusions Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA. PMID:27419627

Hamiltonian structure of the guiding center plasma model

NASA Astrophysics Data System (ADS)

Burby, J. W.; Sengupta, W.

2018-02-01

The guiding center plasma model (also known as kinetic MHD) is a rigorous sub-cyclotron-frequency closure of the Vlasov-Maxwell system. While the model has been known for decades and it plays a fundamental role in describing the physics of strongly magnetized collisionless plasmas, its Hamiltonian structure has never been found. We provide explicit expressions for the model's Poisson bracket and Hamiltonian and thereby prove that the model is an infinite-dimensional Hamiltonian system. The bracket is derived in a manner which ensures that it satisfies the Jacobi identity. We also report on several previously unknown circulation theorems satisfied by the guiding center plasma model. Without knowledge of the Hamiltonian structure, these circulation theorems would be difficult to guess.

Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA.

PubMed

Lehmann-Werman, Roni; Magenheim, Judith; Moss, Joshua; Neiman, Daniel; Abraham, Ofri; Piyanzin, Sheina; Zemmour, Hai; Fox, Ilana; Dor, Talya; Grompe, Markus; Landesberg, Giora; Loza, Bao-Li; Shaked, Abraham; Olthoff, Kim; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2018-06-21

Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.

Cell-Free circulating DNA: a new biomarker for the acute coronary syndrome.

PubMed

Cui, Ming; Fan, Mengkang; Jing, Rongrong; Wang, Huimin; Qin, Jingfeng; Sheng, Hongzhuan; Wang, Yueguo; Wu, Xinhua; Zhang, Lurong; Zhu, Jianhua; Ju, Shaoqing

2013-01-01

In recent studies, concentrations of cell-free circulating DNA (cf-DNA) have been correlated with clinical characteristics and prognosis in several diseases. The relationship between cf-DNA concentrations and the acute coronary syndrome (ACS) remains unknown. Moreover, no data are available for the detection cf-DNA in ACS by a branched DNA (bDNA)-based Alu assay. The aim of the present study was to investigate cf-DNA concentrations in ACS and their relationship with clinical features. Plasma cf-DNA concentrations of 137 ACS patients at diagnosis, of 60 healthy individuals and of 13 patients with stable angina (SA) were determined using a bDNA-based Alu assay. ACS patients (median 2,285.0, interquartile range 916.4-4,857.3 ng/ml), especially in ST-segment elevation myocardial infarction patients (median 5,745.4, interquartile range 4,013.5-8,643.9 ng/ml), showed a significant increase in plasma cf-DNA concentrations compared with controls (healthy controls: median 118.3, interquartile range 81.1-221.1 ng/ml; SA patients: median 202.3, interquartile range 112.7-256.1 ng/ml) using a bDNA-based Alu assay. Moreover, we found positive correlations between cf-DNA and Gensini scoring and GRACE (Global Registry of Acute Coronary Events) scoring in ACS. cf-DNA may be a valuable marker for diagnosing and predicting the severity of coronary artery lesions and risk stratification in ACS. Copyright © 2013 S. Karger AG, Basel.

Diet-resistant obesity is characterized by a distinct plasma proteomic signature and impaired muscle fiber metabolism

PubMed Central

Thrush, A B; Antoun, G; Nikpay, M; Patten, D A; DeVlugt, C; Mauger, J-F; Beauchamp, B L; Lau, P; Reshke, R; Doucet, É; Imbeault, P; Boushel, R; Gibbings, D; Hager, J; Valsesia, A; Slack, R S; Al-Dirbashi, O Y; Dent, R; McPherson, R; Harper, M-E

2018-01-01

Background/Objectives: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. Subjects/Methods: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. Results: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. Conclusions: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women. PMID:29151592

Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection.

PubMed

Titanji, Kehmia; De Milito, Angelo; Cagigi, Alberto; Thorstensson, Rigmor; Grützmeier, Sven; Atlas, Ann; Hejdeman, Bo; Kroon, Frank P; Lopalco, Lucia; Nilsson, Anna; Chiodi, Francesca

2006-09-01

Circulating memory B cells are severely reduced in the peripheral blood of HIV-1-infected patients. We investigated whether dysfunctional serologic memory to non-HIV antigens is related to disease progression by evaluating the frequency of memory B cells, plasma IgG, plasma levels of antibodies to measles, and Streptococcus pneumoniae, and enumerating measles-specific antibody-secreting cells in patients with primary, chronic, and long-term nonprogressive HIV-1 infection. We also evaluated the in vitro production of IgM and IgG antibodies against measles and S pneumoniae antigens following polyclonal activation of peripheral blood mononuclear cells (PBMCs) from patients. The percentage of memory B cells correlated with CD4+ T-cell counts in patients, thus representing a marker of disease progression. While patients with primary and chronic infection had severe defects in serologic memory, long-term nonprogressors had memory B-cell frequency and levels of antigen-specific antibodies comparable with controls. We also evaluated the effect of antiretroviral therapy on these serologic memory defects and found that antiretroviral therapy did not restore serologic memory in primary or in chronic infection. We suggest that HIV infection impairs maintenance of long-term serologic immunity to HIV-1-unrelated antigens and this defect is initiated early in infection. This may have important consequences for the response of HIV-infected patients to immunizations.

Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

PubMed

Biggerstaff, J P; Seth, N; Amirkhosravi, A; Amaya, M; Fogarty, S; Meyer, T V; Siddiqui, F; Francis, J L

1999-01-01

There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins alphaIIb beta3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28 +/- 8 platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Ontogenic and nutritional changes in circulating insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins in growing ewe and ram lambs.

PubMed

Gatford, K L; Quinn, K J; Walton, P E; Grant, P A; Hosking, B J; Egan, A R; Owens, P C

1997-10-01

The ontogeny of the IGF endocrine system was investigated in 15 young lambs before and after weaning at 62 days of age. Before weaning, plasma IGF-I concentrations were higher in rams than ewes, and plasma concentrations of IGF-II and IGF-binding protein-3 (IGFBP-3) also tended to be higher in rams than in ewes. Feed intake of ewes and rams was restricted after weaning to remove sex differences in feed intake. Plasma concentrations of IGF-I and IGFBP-3 did not differ between rams and ewes at 100 days of age, but plasma IGF-II was higher in rams than in ewes at this time. Since circulating concentrations of GH were higher in rams than in ewes at 100 days of age, this implies that the restricted feed intake blocked the IGF-I and IGFBP-3 responses to GH. We conclude that sex differences in circulating IGF-I and IGFBP-3 concentrations in the growing lamb alter with age, and are not present when nutrition is restricted.

High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

PubMed Central

Brenu, Ekua W.; Ashton, Kevin J.; Batovska, Jana; Staines, Donald R.; Marshall-Gradisnik, Sonya M.

2014-01-01

Background MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. Results Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients. Conclusion Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers. PMID:25238588

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

PubMed Central

Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck

2010-01-01

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973

Tumoricidal responses in spontaneous canine neoplasms after extracorporeal perfusion over immobilized protein A.

PubMed

Terman, D S

1981-01-01

I describe morphologic, histologic, immunohistochemical, and serologic changes in dogs with spontaneous breast adenocarcinoma, squamous cell carcinoma, hemangiopericytoma, and fibrosarcoma after extracorporeal perfusion of plasma over heat-killed and formalin-stabilized Staphylococcus aureus Cowans I (SAC), which was embedded in a membrane filtration system. In 12 dogs with breast adenocarcinoma, tumor necrosis was observed within 12 hours after perfusion; 24 hours after perfusion, multiple visible lesions in 6 of 6 dogs exhibited necrosis, but there was no reaction in uninvolved normal mammary tissue. In 8 dogs, healing of large ulcerated areas of cutaneous tumor was observed within 8 to 18 days after perfusion. Similar tumoricidal responses were observed in dogs with other neoplasms after SAC perfusion. Tumor cell necrosis oserved within 4 hours after extracorporeal perfusion was associated with immunohistochemical deposits of IgG and C'3 and ultrastructural evidence of lytic lesions on tumor cell membranes. No tumoricidal effects were observed after perfusion over Staphylococcus aureus Woods (SAW) (non-protein A bearing) in 3 dogs that previously or subsequently responded to SAC perfusion. No tumoricidal reactions were noted after phlebotomy of up to 50% of plasma volume in 6 tumor-bearing dogs that subsequently responded to SAC perfusion. SAC but not SAW perfusion was followed by increases in circulating tumor associated antibodies (TAA) for up to 48 hours after perfusion. Immune complexes increased after perfusion and remained elevated fo 72 hours. Findings suggest that the acute tumoricial responses are not due to mere removal of circulating immune reactants and may be initiated by TAA that are rendered operational after extracorporeal perfusion over SAC. The rapidity, specificity, and magnitude of the observed tumoricidal effects in various canine neoplastic diseases suggests that this may have potentially broad-based therapeutic and biologic implications for canine neoplasia.

Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease.

PubMed

Ramachandran, Sweta; Lowenthal, Alexander; Ritner, Carissa; Lowenthal, Shiri; Bernstein, Harold S

2017-01-01

Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.

Changes in the pattern of plasma extracellular vesicles after severe trauma

PubMed Central

Kuravi, Sahithi J.; Yates, Clara M.; Foster, Mark; Hampson, Peter; Watson, Chris; Midwinter, Mark

2017-01-01

Background Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. Methods and findings 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1–2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. Conclusions EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes. PMID:28837705

Label-free counting of circulating cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Quanyu; Yang, Ping; Wang, Qiyan; Pang, Kai; Zhou, Hui; He, Hao; Wei, Xunbin

2018-02-01

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.

Diurnal rhythms in peripheral blood immune cell numbers of domestic pigs.

PubMed

Engert, Larissa C; Weiler, Ulrike; Pfaffinger, Birgit; Stefanski, Volker; Schmucker, Sonja S

2018-02-01

Diurnal rhythms within the immune system are considered important for immune competence. Until now, they were mostly studied in humans and rodents. However, as the domestic pig is regarded as suitable animal model and due to its importance in agriculture, this study aimed to characterize diurnal rhythmicity in porcine circulating leukocyte numbers. Eighteen pigs were studied over periods of up to 50 h. Cosinor analyses revealed diurnal rhythms in cell numbers of most investigated immune cell populations in blood. Whereas T cell, dendritic cell, and eosinophil counts peaked during nighttime, NK cell and neutrophil counts peaked during daytime. Relative amplitudes of cell numbers in blood differed in T helper cell subtypes with distinctive differentiation states. Mixed model analyses revealed that plasma cortisol concentration was negatively associated with cell numbers of most leukocyte types, except for NK cells and neutrophils. The observed rhythms mainly resemble those found in humans and rodents. Copyright © 2017 Elsevier Ltd. All rights reserved.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

PubMed

Jordan, Nicole Vincent; Bardia, Aditya; Wittner, Ben S; Benes, Cyril; Ligorio, Matteo; Zheng, Yu; Yu, Min; Sundaresan, Tilak K; Licausi, Joseph A; Desai, Rushil; O'Keefe, Ryan M; Ebright, Richard Y; Boukhali, Myriam; Sil, Srinjoy; Onozato, Maristela L; Iafrate, Anthony J; Kapur, Ravi; Sgroi, Dennis; Ting, David T; Toner, Mehmet; Ramaswamy, Sridhar; Haas, Wilhelm; Maheswaran, Shyamala; Haber, Daniel A

2016-09-01

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER + /HER2 - primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2 + and HER2 - subpopulations: HER2 + circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2 - circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2 + and HER2 - circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2 + and HER2 - circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2 + state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2 - phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

Serum Progranulin Concentrations May Be Associated With Macrophage Infiltration Into Omental Adipose Tissue

PubMed Central

Youn, Byung-Soo; Bang, Sa-Ik; Klöting, Nora; Park, Ji Woo; Lee, Namseok; Oh, Ji-Eun; Pi, Kyung-Bae; Lee, Tae Hee; Ruschke, Karen; Fasshauer, Mathias; Stumvoll, Michael; Blüher, Matthias

2009-01-01

OBJECTIVE—Progranulin is an important molecule in inflammatory response. Chronic inflammation is frequently associated with central obesity and associated disturbances; however, the role of circulating progranulin in human obesity, type 2 diabetes, and dyslipidemia is unknown. RESEARCH DESIGN AND METHODS—For the measurement of progranulin serum concentrations, we developed an enzyme-linked immunosorbent assay (ELISA). Using this ELISA, we assessed circulating progranulin in a cross-sectional study of 209 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance and in 60 individuals with normal (NGT) or impaired (IGT) glucose tolerance or type 2 diabetes before and after a 4-week physical training program. Progranulin mRNA and protein expression was measured in paired samples of omental and subcutaneous adipose tissue (adipocytes and cells of the stromal vascular fraction) from 55 lean or obese individuals. Measurement of Erk activation and chemotactic activity induced by progranulin in vitro was performed using THP-1–based cell migration assays. RESULTS—Progranulin serum concentrations were significantly higher in individuals with type 2 diabetes compared with NGT and in obese subjects with predominant visceral fat accumulation. Circulating progranulin significantly correlates with BMI, macrophage infiltration in omental adipose tissue, C-reactive protein (CRP) serum concentrations, A1C values, and total cholesterol. Multivariable linear regression analyses revealed CRP levels as the strongest independent predictor of circulating progranulin. The extent of in vitro progranulin-mediated chemotaxis is similar to that of monocyte chemoattractant protein-1 but independent of Gα. Moreover, in type 2 diabetes, but not in IGT and NGT individuals, physical training for 4 weeks resulted in significantly decreased circulating progranulin levels. CONCLUSIONS—Elevated progranulin serum concentrations are associated with visceral obesity, elevated plasma glucose, and dyslipidemia. We identified progranulin as a novel marker of chronic inflammation in obesity and type 2 diabetes that closely reflects omental adipose tissue macrophage infiltration. Physical training significantly reduces elevated circulating progranulin in patients with type 2 diabetes. PMID:19056610

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

PubMed Central

Muir, Roshell; Metcalf, Talibah; Tardif, Virginie; Takata, Hiroshi; Phanuphak, Nittaya; Kroon, Eugene; Colby, Donn J.; Trichavaroj, Rapee; Valcour, Victor; Robb, Merlin L.; Michael, Nelson L.; Ananworanich, Jintanat; Trautmann, Lydie; Haddad, Elias K.

2016-01-01

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. PMID:27463374

Enhanced Chemokine Receptor Recycling and Impaired S1P1 Expression Promote Leukemic Cell Infiltration of Lymph Nodes in Chronic Lymphocytic Leukemia.

PubMed

Patrussi, Laura; Capitani, Nagaja; Martini, Veronica; Pizzi, Marco; Trimarco, Valentina; Frezzato, Federica; Marino, Filippo; Semenzato, Gianpietro; Trentin, Livio; Baldari, Cosima T

2015-10-01

Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome. ©2015 American Association for Cancer Research.

Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

PubMed

Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

2014-01-01

Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patients from healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients' plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for various maladies.

Comparison of Circulating, Hepatocyte Specific Messenger RNA and microRNA as Biomarkers for Chronic Hepatitis B and C

PubMed Central

Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

2014-01-01

Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patientsfrom healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients’ plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for various maladies. PMID:24643113

Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.

PubMed

Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni

2018-06-01

Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.

Angiogenin: a novel inhibitor of neutrophil lactoferrin release during extracorporeal circulation.

PubMed

Schmaldienst, Sabine; Oberpichler, André; Tschesche, Harald; Hörl, Walter H

2003-01-01

Degranulation of polymorphonuclear leukocytes (PMNL) occurs during extracorporeal circulation. A degranulation-inhibiting protein identical to angiogenin was recently isolated from high-flux dialyzer ultrafiltrate. This protein inhibits the release of lactoferrin and metalloproteinases from PMNL in vitro. In the present study, we investigated end-stage renal disease patients undergoing regular hemodialysis treatment with either high-flux dialyzers (n = 51) or low-flux dialyzers (n = 44), and chronically uremic patients undergoing hemodiafiltration (n = 30). Hemodialysis therapy with low-flux polysulfone or cellulose triacetate membranes caused no or only minimal reduction (

The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

PubMed

Marwarha, Gurdeep; Berry, Daniel C; Croniger, Colleen M; Noy, Noa

2014-01-01

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

PubMed Central

Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

2015-01-01

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

Plasma Autoantibodies against Heat Shock Protein 70, Enolase 1 and Ribonuclease/Angiogenin Inhibitor 1 as Potential Biomarkers for Cholangiocarcinoma

PubMed Central

Rucksaken, Rucksak; Pairojkul, Chawalit; Pinlaor, Porntip; Khuntikeo, Narong; Roytrakul, Sittiruk; Selmi, Carlo; Pinlaor, Somchai

2014-01-01

The diagnosis of cholangiocarcinoma (CCA) is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1) and CCA cell lines (M055, M214 and M139) were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70), enolase 1 (ENO1) and ribonuclease/angiogenin inhibitor 1 (RNH1) obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity). Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001). In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05). Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA. PMID:25058392

Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 51Cr-erythrocyte tracers

USGS Publications Warehouse

Gingerich, W.H.; Pityer, R.A.

1989-01-01

Total, packed cell and, plasma volume estimates were made for the whole body and selected tissues of rainbow trout by the simultaneous injection of radiolabelled trout erythrocyte (51Cr-RBC) and radioiodinated bovine serum albumin (125I-BSA) tracers. Blood volumes were estimated with both markers separately by the tracer-hematocrit method and as the combination of the 51Cr-RBC packed cell and 125I-BSA plasma volumes. Mean whole body blood volume was significantly less when calculated from the 51Cr-RBC tracer data (3.52±0.78 ml/100 g; ±SD) than when calculated with the 125I-BSA tracer (5.06±0.86 ml/100 g) or as the sum of the two volumes combined (4.49±0.60 ml/100 g). The whole body hematocrit (28±5%), estimated as the quotient of the 51Cr-RBC volume divided by the sum of the 125I-BSA and the 51Cr-RBC volumes, also was significantly less than the dorsal aortic microhematocrit (36±4%). Estimates of total blood volumes in most tissues were significantly smaller when calculated from the51Cr-RBC data than when calculated by the other two methods. Tissue blood volumes were greatest in highly vascularized and well perfused tissues and least in poorly vascularized tissues. The relative degree of vascularization among tissues generally remained the same regardless of whether the red cell or the plasma tracer was used to calculated blood volume. It is not clear whether the expanded plasma volume is the result of the distribution of erythrocyte-poor blood into the secondary circulation or the result of extravascular exchange of plasma proteins.

Extracellular microvesicle microRNAs in children with sickle cell anaemia with divergent clinical phenotypes.

PubMed

Khalyfa, Abdelnaby; Khalyfa, Ahamed A; Akbarpour, Mahzad; Connes, Phillippe; Romana, Marc; Lapping-Carr, Gabrielle; Zhang, Chunling; Andrade, Jorge; Gozal, David

2016-09-01

Sickle cell anaemia (SCA) is the most frequent genetic haemoglobinopathy, which exhibits a highly variable clinical course characterized by hyper-coagulable and pro-inflammatory states, as well as endothelial dysfunction. Extracellular microvesicles are released into biological fluids and play a role in modifying the functional phenotype of target cells. We hypothesized that potential differences in plasma-derived extracellular microvesicles (EV) function and cargo from SCA patients may underlie divergent clinical trajectories. Plasma EV from SCA patients with mild, intermediate and severe clinical disease course were isolated, and primary endothelial cell cultures were exposed. Endothelial cell activation, monocyte adhesion, barrier disruption and exosome cargo (microRNA microarrays) were assessed. EV disrupted the endothelial barrier and induced expression of adhesion molecules and monocyte adhesion in a SCA severity-dependent manner compared to healthy children. Microarray approaches identified a restricted signature of exosomal microRNAs that readily distinguished severe from mild SCA, as well as from healthy children. The microRNA candidates were further validated using quantitative real time polymerase chain reaction assays, and revealed putative gene targets. Circulating exosomal microRNAs may play important roles in predicting the clinical course of SCA, and in delineation of individually tailored, mechanistically-based clinical treatment approaches of SCA patients in the near future. © 2016 John Wiley & Sons Ltd.

Reducing the Viscosity of Blood by Pulsed Magnetic Field

NASA Astrophysics Data System (ADS)

Tao, R.; Huang, K.

2010-03-01

Blood viscosity is a major player in heart disease. When blood is viscous, in addition to a high blood pressure required for the blood circulation, blood vessel walls are also easy to be damaged. While this issue is very important, currently the only method to reduce the blood viscosity is to take medicine, such as aspirin. Here we report our new finding that the blood viscosity can be reduced by pulsed magnetic field. Blood is a suspension of red blood cells (erythrocytes), white blood cells (leukocytes) and platelets in plasma, a complex solution of gases, salts, proteins, carbohydrates, and lipids. The base liquid, plasma, has low viscosity. The effective viscosity of whole blood increases mainly due to the red blood cells, which have a volume fraction about 40% or above. Red blood cells contain iron and are sensitive to magnetic field. Therefore, when we apply a strong magnetic field, the red cells make their diameters align in the field direction to form short chains. This change in rheology reduces the effective viscosity as high as 20-30%. While this reduction is not permanent, it lasts for several hours and repeatable. The reduction rate can be controlled by selecting suitable magnetic field and duration of field application to make blood viscosity within the normal range.

Pivotal role of oxidative stress in tumor metastasis under diabetic conditions in mice.

PubMed

Ikemura, Mai; Nishikawa, Makiya; Kusamori, Kosuke; Fukuoka, Miho; Yamashita, Fumiyoshi; Hashida, Mitsuru

2013-09-10

Diabetic patients are reported to have a high incidence and mortality of cancer, but little is known about the linkage. In this study, we investigated whether high oxidative stress is involved in the acceleration of tumor metastasis in diabetic mice. Murine melanoma B16-BL6 cells stably labeled with firefly luciferase (B16-BL6/Luc) were inoculated into the tail vein of streptozotocin (STZ)-treated or untreated mice. A luciferase assay demonstrated that tumor cells were present largely in the lung of untreated mice, whereas large numbers of tumor cells were detected in both the lung and liver of STZ-treated mice. Repeated injections of polyethylene glycol-conjugated catalase (PEG-catalase), a long-circulating derivative, reduced the elevated fasting blood glucose levels and plasma lipoperoxide levels of STZ-treated mice, but had no significant effects on these parameters in untreated mice. In addition, the injections significantly reduced the number of tumor cells in the lung and liver in both untreated and STZ-treated mice. Culture of B16-BL6/Luc cells in medium containing over 45 mg/dl glucose hardly affected the proliferation of the cells, whereas the addition of plasma of STZ-treated mice to the medium significantly increased the number of cells. Plasma samples of STZ-treated mice receiving PEG-catalase exhibited no such effect on proliferation. These findings indicate that a hyperglycemia-induced increase in oxidative stress is involved in the acceleration of tumor metastasis, and the removal of systemic hydrogen peroxide by PEG-catalase can inhibit the progression of diabetic conditions and tumor metastasis in diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

The technique of therapeutic apheresis. Removal of abnormal blood elements may succeed when all else fails.

PubMed

McLeod, B C

1991-05-01

Therapeutic apheresis is a generic term that refers to removal of abnormal blood cells and plasma constituents. The terms "plasmapheresis," "leukapheresis," and "erythrocytapheresis" describe the specific blood element that is removed. Apheresis therapies can be performed in the ICU to manage a number of neurologic, hematologic, and autoimmune disorders, including myasthenia gravis, Guillain-Barré syndrome, sickle-cell disease, and Goodpasture's syndrome. Apheresis procedures generally require two points of contact with the circulation--one for blood withdrawal and one for return; the withdrawal site should sustain a flow rate of at least 50 mL/min. Although apheresis is generally quite safe, hemodynamic instability, hypocalcemia, and dilutional coagulopathy can occur.

Morphology of ovaries in laron dwarf mice, with low circulating plasma levels of insulin-like growth factor-1 (IGF-1), and in bovine GH-transgenic mice, with high circulating plasma levels of IGF-1

PubMed Central

2012-01-01

Background It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals, and one of the signs of aging is progressive gonadal dysfunction. Methods To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on ovaries, we analyzed ovaries isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice, with low circulating plasma levels of IGF-1, and 6-month-old bovine growth hormone transgenic (bGHTg) mice, with high circulating plasma levels of IGF-1. The ages of the Laron dwarf mutants employed in our studies were selected based on their overall survival (up to ~ 4 years for Laron dwarf mice and ~ 1 year for bGHTg mice). Results Morphological analysis of the ovaries of mice that reached ~50% of their maximal life span revealed a lower biological age for the ovaries isolated from 2-year-old Laron dwarf mice than their normal-lifespan wild type littermates. By contrast, the ovarian morphology of increased in size 6 month old bGHTg mice was generally normal. Conclusion Ovaries isolated from 2-year-old Laron dwarf mice exhibit a lower biological age compared with ovaries from normal WT littermates at the same age. At the same time, no morphological features of accelerated aging were found in 0.5-year-old bGHTg mice compared with ovaries from normal the same age-matched WT littermates. PMID:22747742

Functional Consequences of Mannose and Asialoglycoprotein Receptor Ablation*

PubMed Central

Mi, Yiling; Coonce, Marcy; Fiete, Dorothy; Steirer, Lindsay; Dveksler, Gabriela; Townsend, R. Reid; Baenziger, Jacques U.

2016-01-01

The mannose receptor (ManR, Mrc1) and asialoglycoprotein receptor (ASGR, Asgr1 and Asgr2) are highly abundant endocytic receptors expressed by sinusoidal endothelial cells and parenchymal cells in the liver, respectively. We genetically manipulated either receptor individually or in combination, revealing phenotypic changes in female and male mice associated with changes in circulating levels of many glycoproteins. Both receptors rise and fall in response to progesterone during pregnancy. Thirty percent of Asgr2−/− and 65% of Mrc1−/−Asgr2−/− mice are unable to initiate parturition at the end of pregnancy, whereas Mrc1−/− mice initiate normally. Twenty five percent of Mrc1−/−Asgr2−/− male mice develop priapism when mating due to thrombosis of the penile vein, but neither Mrc1−/− nor Asgr2−/− mice do so. The half-life for luteinizing hormone (LH) clearance increases in Mrc1−/− and Mrc1−/−Asgr2−/− mice but not in Asgr2−/− mice; however, LH and testosterone are elevated in all three knockouts. The ManR clears LH thus regulating testosterone production, whereas the ASGR appears to mediate clearance of an unidentified glycoprotein that increases LH levels. More than 40 circulating glycoproteins are elevated >3.0-fold in pregnant Mrc1−/−Asgr2−/− mice. Pregnancy-specific glycoprotein 23, undetectable in WT mice (<50 ng/ml plasma), reaches levels of 1–10 mg/ml in the plasma of Mrc1−/−Asgr2−/− and Asgr2−/− mice, indicating it is cleared by the ASGR. Elevation of multiple coagulation factors in Mrc1−/−Asgr2−/− mice may account for priapism seen in males. These male and female phenotypic changes underscore the key roles of the ManR and ASGR in controlling circulating levels of numerous glycoproteins critical for regulating reproductive hormones and blood coagulation. PMID:27405760

Endothelial and circulating C19MC microRNAs are biomarkers of infantile hemangioma

PubMed Central

Strub, Graham M.; Kirsh, Andrew L.; Whipple, Mark E.; Kuo, Winston P.; Keller, Rachel B.; Kapur, Raj P.; Majesky, Mark W.; Perkins, Jonathan A.

2016-01-01

Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1–expressing (GLUT-1–expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response. PMID:27660822

Transfer of vaginal chloramphenicol to circulating blood in pregnant women and its relationship with their maternal background and neonatal health.

PubMed

Harauchi, Satoe; Osawa, Takashi; Kubono, Naoko; Itoh, Hiroaki; Naito, Takafumi; Kawakami, Junichi

2017-07-01

Few clinical studies have determined the quantitative transfer of vaginal chloramphenicol to circulating blood in pregnant women. This study aimed to evaluate the plasma concentration of chloramphenicol in pregnant women treated with trans-vaginal tablets and its relationship with maternal background and neonatal health. Thirty-seven pregnant women treated with 100 mg of trans-vaginal chloramphenicol once daily for bacterial vaginosis and its suspected case were enrolled. The plasma concentration of chloramphenicol was determined using liquid chromatography coupled to tandem mass spectrometry at day 2 or later after starting the medication. The correlations between the maternal plasma concentration of chloramphenicol and the background and neonatal health at birth were investigated. Chloramphenicol was detected from all maternal plasma specimens and its concentration ranged from 0.043 to 73.1 ng/mL. The plasma concentration of chloramphenicol declined significantly with the administration period. The plasma concentration of chloramphenicol was lower at the second than the first blood sampling. No correlations were observed between the maternal plasma concentration of chloramphenicol and background such as number of previous births, gestational age at dosing, and clinical laboratory data. Neonatal infant health parameters such as birth-weight, Apgar score at birth, and gestational age at the time of childbearing were not related to the maternal plasma concentration of chloramphenicol. Vaginal chloramphenicol transfers to circulating blood in pregnant women. The maternal plasma concentration of chloramphenicol varied markedly and was associated with the administration day, but not with maternal background or her neonatal health. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Plasma circulating fibrinogen stability and moderate beer consumption.

PubMed

Gorinstein, Shela; Caspi, Abraham; Zemser, Marina; Libman, Imanuel; Goshev, Ivan; Trakhtenberg, Simon

2003-12-01

MODERATE BEER CONSUMPTION (MBC) IS CARDIOPROTECTIVE: it positively influences plasma lipid levels and plasma antioxidant activity in beer-consuming individuals. The connection between MBC and blood coagulation is not clearly defined. Forty-two volunteers were equally divided into experimental (EG) and control (CG) groups following coronary bypass surgery. For 30 consecutive days, only patients of the EG consumed 330 mL of beer per day (about 20 g of alcohol). A comprehensive clinical investigation of 42 patients was done. Blood samples were collected before and after the investigation for a wide range of laboratory tests. The plasma fibrinogen was denatured with 8 M urea and intrinsic fluorescence (IF), hydrophobicity and differential scanning calorimetry (DSC) were used to reveal possible qualitative changes. After 30 days of moderate beer consumption, positive changes in the plasma lipid levels, plasma anticoagulant and plasma antioxidant activities were registered in patients of the EG group. In 17 out of 21 patients of the same group, differences in plasma circulating fibrinogen's (PCF), secondary and tertiary structures were found. The stability of fibrinogen, expressed in thermodynamic parameters, has shown that the loosening of the structure takes place under ethanol and urea denaturation. Also fluorescence stability of PCF was decreased. No changes in the lipid levels, anticoagulant and antioxidant activity or changes in PCF were detected in patients of CG. In conclusion, for the first time after a short term of moderate beer consumption some qualitative changes in the plasma circulating fibrinogen were detected: differences in the emission peak response, fluorescence intensity and all thermodynamic data. Together, with the decrease in the PCF concentration it may lead to an elevation of the blood anticoagulant activity.

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

NASA Astrophysics Data System (ADS)

Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

PubMed

Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

2015-07-01

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p <0.001). A colony of circulating endothelial progenitor cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p <0.001). The culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p <0.001). The circulating endothelial progenitor cell level correlated positively with the number of patient colonies (r = 0.762, p <0.001). Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p <0.001). Earlier emergence of circulating endothelial progenitor cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Altered Sex Hormone Concentrations and Gonadal mRNA Expression Levels of Activin Signaling Factors in Hatchling Alligators From a Contaminated Florida Lake

PubMed Central

MOORE, BRANDON C.; KOHNO, SATOMI; COOK, ROBERT W.; ALVERS, ASHLEY L.; HAMLIN, HEATHER J.; WOODRUFF, TERESA K.; GUILLETTE, LOUIS J.

2014-01-01

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin βA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly. PMID:20166196

21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and enumeration...

21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and enumeration...

21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and enumeration...

21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and enumeration...

21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and enumeration...

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction.

PubMed

Javeed, Naureen; Sagar, Gunisha; Dutta, Shamit K; Smyrk, Thomas C; Lau, Julie S; Bhattacharya, Santanu; Truty, Mark; Petersen, Gloria M; Kaufman, Randal J; Chari, Suresh T; Mukhopadhyay, Debabrata

2015-04-01

Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. ©2014 American Association for Cancer Research.

The insulin receptor.

PubMed

Kaplan, S A

1984-03-01

Cells are endowed with specific cognitive molecules that function as receptors for hormones, neurotransmitters, and other intercellular messengers. The receptor molecules may be present in the plasma membrane, cytoplasm, or nucleus. When occupied by the messenger, the receptor is coupled to the cellular machinery that responds to the message-bearing molecules. For some hormones the events following attachment of the messenger to the receptor are well known. An example is the generation of cAMP after combination of glucagon with its receptor and the series of steps culminating in activation of phosphorylase. In the case of many other messengers, including insulin, the nature of these coupling steps is not known. Receptors are subject to the regulatory processes of synthesis, degradation, and conformational change; alterations in receptor properties may have significant effects on the qualitative and quantitative responses of the cell to the extracellular messenger. The insulin receptor is located in the plasma membrane, is composed of two pairs of subunits, and has a molecular weight of about 350,000. It is located in cells such as adipocytes, hepatocytes, and skeletal muscle cells as well as in cells not considered to be typical target organ cells. Insulin receptors in nonfetal cells are downregulated by exposure of the cells to high concentrations of insulin. Other factors that regulate insulin binding include muscular exercise, diet, thyroid hormones, glucocorticoids, androgens, estrogens, and cyclic nucleotides. The fetus has high concentrations of insulin receptors in several tissues. These begin to appear early in fetal life and may outnumber those found in adult tissues. Fetal insulin receptors are unusual in that they may not undergo downregulation but may experience the opposite when exposed to insulin in high concentrations. Thus the offspring of a mother with poorly controlled diabetes may be placed in double jeopardy by fetal hyperinsulinemia and augmented insulin binding by the receptors. Many disorders in children and adults are associated with changes in the properties of the insulin receptor. In general, the alterations have been measured in receptor-bearing cells that are readily accessible, such as circulating monocytes and erythrocytes. The receptors on these cells generally reflect the status of receptors on the major target organs of insulin, although exceptions are known, and conclusions drawn from studies of receptors on circulating cells must be made with caution.(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired pancreatic polypeptide release in chronic pancreatitis with steatorrhoea.

PubMed

Adrian, T E; Besterman, H S; Mallinson, C N; Garalotis, C; Bloom, S R

1979-02-01

Pancreatic polypeptide (PP) is a newly discovered hormonal peptide localised in a distinct endocrine cell type in the pancreas. PP circulates in plasma and in normal subjects levels rise substantially on the ingestion of food (mean rise 138 pmol/l). In 10 patients with chronic pancreatitis with exocrine deficiency the PP response to a test breakfast was greatly reduced (mean rise 20 pmol/l, P less than 0.001). PP response to the meal was normal in 10 patients with active coeliac disease and 12 patients with acute tropical sprue with steatorrhoea.

Impaired pancreatic polypeptide release in chronic pancreatitis with steatorrhoea.

PubMed Central

Adrian, T E; Besterman, H S; Mallinson, C N; Garalotis, C; Bloom, S R

1979-01-01

Pancreatic polypeptide (PP) is a newly discovered hormonal peptide localised in a distinct endocrine cell type in the pancreas. PP circulates in plasma and in normal subjects levels rise substantially on the ingestion of food (mean rise 138 pmol/l). In 10 patients with chronic pancreatitis with exocrine deficiency the PP response to a test breakfast was greatly reduced (mean rise 20 pmol/l, P less than 0.001). PP response to the meal was normal in 10 patients with active coeliac disease and 12 patients with acute tropical sprue with steatorrhoea. PMID:428832

Role of the protective antigen octamer in the molecular mechanism of anthrax lethal toxin stabilization in plasma

PubMed Central

Kintzer, Alexander F.; Sterling, Harry J.; Tang, Iok I.; Abdul-Gader, Ali; Miles, Andrew J.; Wallace, B. A.; Williams, Evan R.; Krantz, Bryan A.

2010-01-01

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three-proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of either LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT may assemble on host cell surfaces or extracellularly in plasma. We show that under physiological conditions in bovine plasma that LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration allowing them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that may circulate freely in the blood. PMID:20433851

Lymphoid organs function as major reservoirs for human immunodeficiency virus.

PubMed Central

Pantaleo, G; Graziosi, C; Butini, L; Pizzo, P A; Schnittman, S M; Kotler, D P; Fauci, A S

1991-01-01

The total number of human immunodeficiency virus type 1 (HIV-1)-infected circulating CD4+ T lymphocytes is considered to be a reflection of the HIV burden at any given time during the course of HIV infection. However, the low frequency of HIV-infected circulating CD4+ T lymphocytes and the low level or absence of plasma viremia in the early stages of infection do not correlate with the progressive immune dysfunction characteristic of HIV infection. In this study, we have determined whether HIV-infected circulating CD4+ T lymphocytes are a correct reflection of the total pool of HIV-infected CD4+ T cells (i.e., HIV burden). To this end, HIV burden has been comparatively analyzed in peripheral blood and lymphoid tissues (lymph nodes, adenoids, and tonsils) from the same patients. The presence of HIV-1 DNA in mononuclear cells isolated simultaneously from peripheral blood and lymphoid tissues of the same patients was determined by polymerase chain reaction amplification. We found that the frequency of HIV-1-infected cells in unfractionated or sorted CD4+ cell populations isolated from lymphoid tissues was significantly higher (0.5-1 log10 unit) than the frequency in peripheral blood. Comparable results were obtained in five HIV seropositive patients in the early stages of disease and in one patient with AIDS. These results demonstrate that a heavy viral load does reside in the lymphoid organs, indicating that they may function as major reservoirs for HIV. In addition, the finding of a heavy viral load in the lymphoid organs of patients in the early stages of disease may explain the progressive depletion of CD4+ T lymphocytes and the immune dysfunction associated with the early stages of HIV infection. Images PMID:1682922

High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation.

PubMed

Zhenyukh, Olha; Civantos, Esther; Ruiz-Ortega, Marta; Sánchez, Maria Soledad; Vázquez, Clotilde; Peiró, Concepción; Egido, Jesús; Mas, Sebastián

2017-03-01

Leucine, isoleucine and valine are essential aminoacids termed branched-chain amino acids (BCAA) due to its aliphatic side-chain. In several pathological and physiological conditions increased BCAA plasma concentrations have been described. Elevated BCAA levels predict insulin resistance development. Moreover, BCAA levels higher than 2mmol/L are neurotoxic by inducing microglial activation in maple syrup urine disease. However, there are no studies about the direct effects of BCAA in circulating cells. We have explored whether BCAA could promote oxidative stress and pro-inflammatory status in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. In cultured PBMCs, 10mmol/L BCAA increased the production of reactive oxygen species (ROS) via both NADPH oxidase and the mitochondria, and activated Akt-mTOR signalling. By using several inhibitors and activators of these molecular pathways we have described that mTOR activation by BCAA is linked to ROS production and mitochondrial dysfunction. BCAA stimulated the activation of the redox-sensitive transcription factor NF-κB, which resulted in the release of pro-inflammatory molecules, such as interleukin-6, tumor necrosis factor-α, intracellular adhesion molecule-1 or CD40L, and the migration of PBMCs. In conclusion, elevated BCAA blood levels can promote the activation of circulating PBMCs, by a mechanism that involving ROS production and NF-κB pathway activation. These data suggest that high concentrations of BCAA could exert deleterious effects on circulating blood cells and therefore contribute to the pro-inflammatory and oxidative status observed in several pathophysiological conditions. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Reduced levels of S-nitrosothiols in plasma of patients with systemic sclerosis and Raynaud's phenomenon.

PubMed

Kundu, Devi; Abraham, David; Black, Carol M; Denton, Christopher P; Bruckdorfer, K Richard

2014-12-01

S-Nitrosothiols (RSNOs) are bioactive forms of nitric oxide which are involved in cell signalling and redox regulation of vascular function. Circulating S-nitrosothiols are predominantly in the form of S-nitrosoalbumin. In this study plasma concentrations of S-nitrosothiols were measured in patients with systemic sclerosis (SSc) where NO metabolism is known to be abnormal. Venous blood was collected from 16 patients with Raynaud's phenomenon (RP), 45 with systemic sclerosis (SSc) (34 patients had limited SSc (IcSSc) and 11 diffuse cutaneous disease (dcSSc)). Twenty six healthy subjects were used as controls. Plasma S-nitrosothiol concentrations were measured by chemiluminescence. The measurements were related to the extent of biological age, capillary/skin scores and disease duration. Plasma RSNO levels in patients with Raynaud's phenomenon (RP) and in those with SSc was significantly lower compared to the concentrations in control subjects. In SSc, plasma S-nitrosothiols were often below the level of detection (1nM). Low S-nitrosothiol concentrations were observed in the blood of patients with SSc and patients with RP indicating a profound disturbance of nitric oxide metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation.

PubMed

Konduru, Nagarjun V; Murdaugh, Kimberly M; Swami, Archana; Jimenez, Renato J; Donaghey, Thomas C; Demokritou, Philip; Brain, Joseph D; Molina, Ramon M

2016-08-01

Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated (65)ZnO or silica-coated (65)ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for (65)Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated (65)ZnO were shorter (initial - <1 min; terminal - 2.5 min) than uncoated (65)ZnO (initial - 1.9 min; terminal - 38 min). Interestingly, the silica-coated (65)ZnO group had higher (65)Zn associated with red blood cells and higher initial uptake in the liver. The (65)Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.

Plasma insulin-like growth factors, insulin-like binding protein-3, and outcome in metastatic colorectal cancer: results from intergroup trial N9741.

PubMed

Fuchs, Charles S; Goldberg, Richard M; Sargent, Daniel J; Meyerhardt, Jeffrey A; Wolpin, Brian M; Green, Erin M; Pitot, Henry C; Pollak, Michael

2008-12-15

Insulin-like growth factor (IGF)-I and IGF-II stimulate neoplastic cell growth and inhibit apoptosis, whereas IGF-binding protein-3 (IGFBP-3) inhibits the bioavailability of IGF-I and has independent proapoptotic activity. We examined the influence of baseline plasma levels of IGF-I, IGF-II, IGFBP-3, and C-peptide on outcome among patients receiving first-line chemotherapy for metastatic colorectal cancer. The plasma levels of IGF-I, IGF-II, IGFBP-3, and C-peptide as well as data on prognostic factors and body size were measured at baseline among 527 patients participating in a randomized trial of first-line chemotherapy for metastatic colorectal cancer. Higher baseline plasma IGFBP-3 levels were associated with a significantly greater chemotherapy response rate (P = 0.03) after adjusting for other prognostic factors, whereas neither IGF-I nor IGF-II levels significantly predicted tumor response. Higher levels of IGF-I, IGF-II, and IGFBP-3 were all univariately associated with improved overall survival (P = 0.0001 for all). In a model that mutually adjusted for IGF-I and IGFBP-3, as well as other prognostic factors, increasing baseline-circulating IGFBP-3 was associated with a significantly longer time to tumor progression (P = 0.03), whereas circulating IGF-I was not associated with disease progression (P = 0.95). Levels of C-peptide were not associated with any measure of patient outcome. Among colorectal cancer patients receiving first-line chemotherapy, increasing levels of IGFBP-3, an endogenous antagonist to IGF-I, are associated with an improved objective treatment response and a prolonged time to cancer progression. The IGF pathway may represent an important target for future treatment strategies.

Metformin Suppresses Systemic Autoimmunity in Roquinsan/san Mice through Inhibiting B Cell Differentiation into Plasma Cells via Regulation of AMPK/mTOR/STAT3.

PubMed

Lee, Seon-Yeong; Moon, Su-Jin; Kim, Eun-Kyung; Seo, Hyeon-Beom; Yang, Eun-Ji; Son, Hye-Jin; Kim, Jae-Kyung; Min, Jun-Ki; Park, Sung-Hwan; Cho, Mi-La

2017-04-01

Circulating autoantibodies and immune complex deposition are pathological hallmarks of systemic lupus erythematosus (SLE). B cell differentiation into plasma cells (PCs) and some T cell subsets that function as B cell helpers can be therapeutic targets of SLE. Mechanistic target of rapamycin (mTOR) signaling is implicated in the formation of B cells and germinal centers (GCs). We assessed the effect of metformin, which inhibits mTOR, on the development of autoimmunity using Roquin san/san mice. Oral administration of metformin inhibited the formation of splenic follicles and inflammation in kidney and liver tissues. It also decreased serum levels of anti-dsDNA Abs without affecting serum glucose levels. Moreover, metformin inhibited CD21 high CD23 low marginal zone B cells, B220 + GL7 + GC B cells, B220 - CD138 + PCs, and GC formation. A significant reduction in ICOS + follicular helper T cells was found in the spleens of the metformin-treated group compared with the vehicle-treated group. In addition, metformin inhibited Th17 cells and induced regulatory T cells. These alterations in B and T cell subsets by metformin were associated with enhanced AMPK expression and inhibition of mTOR-STAT3 signaling. Furthermore, metformin induced p53 and NF erythroid-2-related factor-2 activity in splenic CD4 + T cells. Taken together, metformin-induced alterations in AMPK-mTOR-STAT3 signaling may have therapeutic value in SLE by inhibiting B cell differentiation into PCs and GCs. Copyright © 2017 by The American Association of Immunologists, Inc.

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

PubMed

Boutry, Claire; Matsumoto, Hideki; Bos, Cécile; Moinard, Christophe; Cynober, Luc; Yin, Yulong; Tomé, Daniel; Blachier, François

2012-10-01

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3 mg kg(-1) lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.

Mobilization of Peripheral Blood Stem Cells and Changes in the Concentration of Plasma Factors Influencing their Movement in Patients with Panic Disorder.

PubMed

Jabłoński, Marcin; Mazur, Jolanta Kucharska; Tarnowski, Maciej; Dołęgowska, Barbara; Pędziwiatr, Daniel; Kubiś, Ewa; Budkowska, Marta; Sałata, Daria; Wysiecka, Justyna Pełka; Kazimierczak, Arkadiusz; Reginia, Artur; Ratajczak, Mariusz Z; Samochowiec, Jerzy

2017-04-01

In this paper we examined whether stem cells and factors responsible for their movement may serve as new biological markers of anxiety disorders. The study was carried out on a group of 30 patients diagnosed with panic disorder (examined before and after treatment), compared to 30 healthy individuals forming the control group. We examined the number of circulating HSCs (hematopoetic stem cells) (Lin-/CD45 +/CD34 +) and HSCs (Lin-/CD45 +/AC133 +), the number of circulating VSELs (very small embryonic-like stem cells) (Lin-/CD45-/CD34 +) and VSELs (Lin-/CD45-/AC133 +), as well as the concentration of complement components: C3a, C5a and C5b-9, SDF-1 (stromal derived factor) and S1P (sphingosine-1-phosphate). Significantly lower levels of HSCs (Lin-/CD45 +/AC133 +) have been demonstrated in the patient group compared to the control group both before and after treatment. The level of VSELs (Lin-/CD45-/CD133 +) was significantly lower in the patient group before treatment as compared to the patient group after treatment.The levels of factors responsible for stem cell movement were significantly lower in the patient group compared to the control group before and after treatment. It was concluded that the study of stem cells and factors associated with their movement can be useful in the diagnostics of panic disorder, as well as differentiating between psychotic and anxiety disorders.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis1

PubMed Central

Whitaker, Paul; Meng, Xiaoli; Lavergne, Sidonie N.; El-Ghaiesh, Sabah; Monshi, Manal; Earnshaw, Caroline; Peckham, Daniel; Gooi, Jimmy; Conway, Steve; Pirmohamed, Munir; Jenkins, Rosalind E.; Naisbitt, Dean J.; Park, B. Kevin

2011-01-01

A mechanistic understanding of the relationship between the chemistry of drug antigen formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating antigens derived from piperacillin in patients undergoing therapy and the nature of the drug derived-epitopes on protein which can function as an antigen to stimulate T-cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time- and concentration-dependent, with selective modification of Lys541 observed at low concentrations, whereas at higher concentrations up to 13/59 lysine residues were modified, four of which (Lys190, 195, 432 and 541) were detected in patients’ plasma. Piperacillin-specific T-lymphocyte responses (proliferation, cytokines and granzyme-B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T-cells with characterized synthetic conjugates. Analysis of minimally-modified T-cell stimulatory albumin conjugates revealed peptide sequences incorporating Lys190, 432 and 541 as principal functional epitopes for T-cells. This study has characterized the multiple haptenic structures on albumin in patients, and showed that they constitute functional antigenic determinants for T-cells. PMID:21606251

THE USE OF RADIOACTIVE LYSINE IN STUDIES OF PROTEIN METABOLISM

PubMed Central

Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple, G. H.

1949-01-01

Racemic lysine labeled with C14 in the epsilon carbon position was fed to dogs. The distribution of C14 in blood and tissue fractions is recorded. In normal dogs sacrificed at 24 hours, approximately one-third of the C14 was found in the urine, one-third in expired air, and one-third in the body, mostly in protein, predomantly as lysine residues. The rate of C14 excretion as CO2, hour by hour, paralleled closely the amount of non-protein C14 in the blood plasma. The liver, kidney, pancreas, and spleen all have high values for C14 in 24 hour and 17 day experiments. The gastrointestinal tract is significantly high in the 24 hour experiments. Plasma protein from animals previously fed C14 containing lysine and thus in turn labeled, was transfused into other dogs and the rate of disappearance of albumin and globulin fractions from the circulation of the recipient dog followed. The results lead to the conclusion that as a whole, plasma proteins are utilized and replaced at a rate of at least 10 per cent per 24 hours. This minimum rate is substantially faster than turnover rates commonly accepted and emphasizes the rôle played by the plasma proteins in the protein economy of the body. The exact rate determination is made uncertain by the lack of knowledge of the magnitude of the amount of protein in solution in extracellular and lymph spaces and its rate of equilibrium with circulating plasma proteins. Evidence from these transfusion studies indicates that plasma globulin is metabolized at a significantly faster rate than plasma albumin. This is confirmed by the observation that following the feeding of labeled lysine to dogs, C14 is first incorporated in globulin in high concentration but that later it also disappears more rapidly from the globulin fraction. These data suggest that the period of bone marrow maturation of the red cell during which time its related hemoglobin is synthesized does not exceed 3 to 5 days. PMID:18140663

Diagnostic and prognostic value of preoperative combined GFAP, IGFBP-2, and YKL-40 plasma levels in patients with glioblastoma.

PubMed

Gállego Pérez-Larraya, Jaime; Paris, Sophie; Idbaih, Ahmed; Dehais, Caroline; Laigle-Donadey, Florence; Navarro, Soledad; Capelle, Laurent; Mokhtari, Karima; Marie, Yannick; Sanson, Marc; Hoang-Xuan, Khê; Delattre, Jean-Yves; Mallet, Alain

2014-12-15

Circulating proteins released by tumor cells have recently been investigated as potential single surrogate biomarkers for glioblastoma multiforme (GBM). The aim of the current hypothesis-generating study was to evaluate the diagnostic and prognostic role of preoperative insulin-like growth factor-binding protein 2 (IGFBP-2), chitinase-3-like protein 1 (YKL-40), and glial fibrillary acidic protein (GFAP) plasma levels in patients with GBM, both as single markers and as a combined profile. Plasma samples from 111 patients with GBM and a subset of 40 patients with nonglial brain tumors were obtained preoperatively. Plasma from 99 healthy controls was also analyzed. IGFBP-2, YKL-40, and GFAP levels were determined using enzyme-linked immunoadsorbent assay tests. Their association with histological and radiological variables was assessed. Circulating levels of all 3 proteins were found to be significantly higher in patients with GBM compared with healthy controls (P < .01). Only YKL-40 and GFAP were found to demonstrate significant differences between patients with GBM and nonglial brain tumors (P = .04). GFAP was undetectable (<0.02 ng/mL) in all patients without GBM. A receiver operating characteristic analysis accounting for a 2-step diagnostic procedure including the 3 biomarkers afforded an area under the curve of 0.77 for differentiating patients with GBM from those with nonglial brain tumors. There was a significant correlation between tumor volume and plasma IGFBP-2 level (Spearman Rho correlation coefficient, 0.22; P = .025) and GFAP (Spearman Rho correlation coefficient, 0.36; P < .001) among patients with GBM. Preoperative plasma IGFBP-2 levels were found to be independently associated with worse overall survival among patients with GBM (hazard ratio, 1.3; P = .05). A combined profile of preoperative IGFBP-2, GFAP, and YKL-40 plasma levels could serve as an additional diagnostic tool for patients with inoperable brain lesions suggestive of GBM. In addition, IGFBP-2 levels appear to constitute an independent prognostic factor in patients with GBM. © 2014 American Cancer Society.

Circulating microRNAs as novel biomarkers of ALK-positive nonsmall cell lung cancer and predictors of response to crizotinib therapy.

PubMed

Li, Liang-Liang; Qu, Li-Li; Fu, Han-Jiang; Zheng, Xiao-Fei; Tang, Chuan-Hao; Li, Xiao-Yan; Chen, Jian; Wang, Wei-Xia; Yang, Shao-Xing; Wang, Lin; Zhao, Guan-Hua; Lv, Pan-Pan; Zhang, Min; Lei, Yang-Yang; Qin, Hai-Feng; Wang, Hong; Gao, Hong-Jun; Liu, Xiao-Qing

2017-07-11

Circulating microRNAs are potential diagnostic and predictive biomarkers, but have not been investigated for patients with anaplastic lymphoma kinase (ALK)-positive lung cancer. In this exploratory study, we sought to identify potential plasma biomarkers for ALK-positive non-small cell lung cancer (NSCLC). A microRNA microarray was used to select ALK-related microRNAs in ALK-positive NSCLC (n = 3), ALK-negative NSCLC (n = 3), and healthy subjects (n = 3). Plasma levels of 21 microRNAs were differentially expressed for ALK-positive and ALK-negative NSCLC, including 14 down-regulated and 7 up-regulated microRNAs. We also identified 5s rRNA as the most stable endogenous control gene using geNorm and NormFinder algorithms. Candidate microRNAs in plasma from ALK-positive (n = 41) and ALK-negative NSCLC patients (n = 32) were quantified using real-time reverse transcriptase quantitative polymerase chain reaction. The expression levels of miR-28-5p, miR-362-5p, and miR-660-5p were all down-regulated in ALK-positive NSCLC, compared with ALK-negative NSCLC. The areas under the receiver operating characteristic curves of miR-28-5p, miR-362-5p, miR-660-5p, and 3-microRNAs panel were 0.873, 0.673, 0.760, and 0.876, respectively. The positive predictive values of miR-28-5p, miR-362-5p, and miR-660-5p were 96.43%, 80.77%, and 83.87%, respectively. Increased plasma levels of miR-660-5p after crizotinib treatment predicted good tumor response (p = 0.012). The pre-crizotinib levels of miR-362-5p were significantly associated with progression-free survival (p = 0.015). Thus, in this preliminary investigation, we identified a potential panel of 3 microRNAs for distinguishing between patients with ALK-positive and ALK-negative NSCLC. We also identified miR-660-5p and miR-362-5p as potential predictors for response to crizotinib treatment.

Circulating microRNAs as novel biomarkers of ALK-positive non-small cell lung cancer and predictors of response to crizotinib therapy

PubMed Central

Fu, Han-Jiang; Zheng, Xiao-Fei; Tang, Chuan-Hao; Li, Xiao-Yan; Chen, Jian; Wang, Wei-Xia; Yang, Shao-Xing; Wang, Lin; Zhao, Guan-Hua; Lv, Pan-Pan; Zhang, Min; Lei, Yang-Yang; Qin, Hai-Feng; Wang, Hong; Gao, Hong-Jun; Liu, Xiao-Qing

2017-01-01

Circulating microRNAs are potential diagnostic and predictive biomarkers, but have not been investigated for patients with anaplastic lymphoma kinase (ALK)-positive lung cancer. In this exploratory study, we sought to identify potential plasma biomarkers for ALK-positive non-small cell lung cancer (NSCLC). A microRNA microarray was used to select ALK-related microRNAs in ALK-positive NSCLC (n = 3), ALK-negative NSCLC (n = 3), and healthy subjects (n = 3). Plasma levels of 21 microRNAs were differentially expressed for ALK-positive and ALK-negative NSCLC, including 14 down-regulated and 7 up-regulated microRNAs. We also identified 5s rRNA as the most stable endogenous control gene using geNorm and NormFinder algorithms. Candidate microRNAs in plasma from ALK-positive (n = 41) and ALK-negative NSCLC patients (n = 32) were quantified using real-time reverse transcriptase quantitative polymerase chain reaction. The expression levels of miR-28-5p, miR-362-5p, and miR-660-5p were all down-regulated in ALK-positive NSCLC, compared with ALK-negative NSCLC. The areas under the receiver operating characteristic curves of miR-28-5p, miR-362-5p, miR-660-5p, and 3-microRNAs panel were 0.873, 0.673, 0.760, and 0.876, respectively. The positive predictive values of miR-28-5p, miR-362-5p, and miR-660-5p were 96.43%, 80.77%, and 83.87%, respectively. Increased plasma levels of miR-660-5p after crizotinib treatment predicted good tumor response (p = 0.012). The pre-crizotinib levels of miR-362-5p were significantly associated with progression-free survival (p = 0.015). Thus, in this preliminary investigation, we identified a potential panel of 3 microRNAs for distinguishing between patients with ALK-positive and ALK-negative NSCLC. We also identified miR-660-5p and miR-362-5p as potential predictors for response to crizotinib treatment. PMID:28514730

Circulating milk fat globule-epidermal growth factor 8 levels are increased in pregnancy and gestational diabetes mellitus.

PubMed

Li, Yuanyuan; Ran, Wenzhuo; Zhang, Jiaqiang; Chen, Shi; Li, Yihang; Luo, Deng; Wang, Chen; Jia, Weiping

2017-07-01

Milk fat globule-epidermal growth factor 8 (MFG-E8) is the key mediator in anti-inflammatory responses that facilitate phagocytosis of apoptotic cells, and play an essential role in type 2 diabetes and pregnancy, both of which are under a low-grade inflammatory state. However, the action of MFG-E8 in gestational diabetes mellitus (GDM) is unclear. We measured plasma MFG-E8 levels in pregnancy and GDM for the first time, and elucidated possible relationships between its plasma levels and various metabolic parameters. Plasma MFG-E8 levels were quantified by enzyme-linked immunosorbent assay in 66 women with GDM, 70 with normal pregnancy (p-NGT) and 44 healthy non-pregnant controls (CON), who were matched for age and body mass index. Inflammatory factors tumor necrosis factor-α (TNF-α) and C-reactive protein levels were measured, oral glucose tolerance test was carried out and β-cell function was evaluated. Plasma MFG-E8 levels were remarkably higher in p-NGT than in CON (P = 0.024), and were further elevated in GDM vs p-NGT (P = 0.016). MFG-E8 concentrations correlated positively with hemoglobin A1c, glucose levels and insulin resistance (homeostasis model assessment for insulin resistance), and correlated inversely with TNF-α and insulin secretion evaluated by disposition indices in pregnancies. Fasting glucose levels, disposition index of first phase insulin secretion and TNF-α were independent predictors of MFG-E8 levels in pregnancies. Logistic regression analyses showed that women in the third tertile of MFG-E8 levels had a markedly elevated risk of GDM. Circulating MFG-E8 levels are dramatically elevated in pregnancy, and are significantly higher in GDM vs p-NGT. MFG-E8 concentrations are significantly associated with TNF-α, fasting glucose levels, homeostasis model assessment for insulin resistance and disposition indices. However, further studies are required to elucidate the regulation mechanism of MFG-E8 during pregnancy and GDM. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Brief report: Circulating markers of fibrosis are associated with immune reconstitution status in HIV-infected men.

PubMed

Tobolowsky, F A; Wada, N; Martinez-Maza, O; Magpantay, L; Koletar, S L; Palella, F J; Brown, T T; Lake, J E

2018-01-01

Lymphoid tissue fibrosis may contribute to incomplete immune reconstitution on antiretroviral therapy (ART) via local CD4+ T lymphocyte (CD4) depletion. Hyaluronic acid (HA) increases with fibrotic burden. CXCL4 concentrations increase in response to pro-fibrotic stimuli, but lower CXCL4 concentrations in HIV-infected individuals may reflect successful immune evasion by HIV. We investigated relationships between circulating HA and CXCL4 concentrations and immune reconstitution on ART in HIV-infected Multicenter AIDS Cohort Study participants. HIV-infected men on ART for >1 year with cryopreserved plasma samples and suppressed post-ART HIV-1 RNA were included. Men with post-ART CD4 <200 cells/mm3 were defined as immunologic non-responders (n = 25). Age-/race-matched men with post-ART CD4 >500 cells/mm3 served as controls (n = 49). HA and CXCL4 concentrations were measured via ELISA. Median pre-ART CD4 was 297 cells/mm3 for non-responders vs 386 cells/mm3 for controls. Median post-ART CD4 was 141 cells/mm3 for non-responders and 815 cells/mm3 for controls. HIV infection duration was 23 years, with median time on ART 13 years for non-responders vs 11 years for controls. Pre-ART HA and CXCL4 concentrations did not vary by eventual immune reconstitution status. Post-ART HA concentrations tended to be higher (85 vs 36 ng/mL, p = 0.07) and CXCL4 concentrations were lower (563 vs 1459 ng/mL, p = 0.01) among non-responders. Among men with paired pre-/post-ART samples, non-responders had greater HA increases and CXCL4 decreases than controls (HA: 50 vs 12 ng/mL, p = 0.04; CXCL4: -1258 vs -405 ng/mL, p = 0.01). Higher circulating concentrations of HA and lower concentrations of CXCL4 are associated with failure of immune reconstitution on ART.

Brief report: Circulating markers of fibrosis are associated with immune reconstitution status in HIV-infected men

PubMed Central

Wada, N.; Martinez-Maza, O.; Magpantay, L.; Koletar, S. L.; Palella, F. J.; Brown, T. T.; Lake, J. E.

2018-01-01

Introduction Lymphoid tissue fibrosis may contribute to incomplete immune reconstitution on antiretroviral therapy (ART) via local CD4+ T lymphocyte (CD4) depletion. Hyaluronic acid (HA) increases with fibrotic burden. CXCL4 concentrations increase in response to pro-fibrotic stimuli, but lower CXCL4 concentrations in HIV-infected individuals may reflect successful immune evasion by HIV. We investigated relationships between circulating HA and CXCL4 concentrations and immune reconstitution on ART in HIV-infected Multicenter AIDS Cohort Study participants. Methods HIV-infected men on ART for >1 year with cryopreserved plasma samples and suppressed post-ART HIV-1 RNA were included. Men with post-ART CD4 <200 cells/mm3 were defined as immunologic non-responders (n = 25). Age-/race-matched men with post-ART CD4 >500 cells/mm3 served as controls (n = 49). HA and CXCL4 concentrations were measured via ELISA. Results Median pre-ART CD4 was 297 cells/mm3 for non-responders vs 386 cells/mm3 for controls. Median post-ART CD4 was 141 cells/mm3 for non-responders and 815 cells/mm3 for controls. HIV infection duration was 23 years, with median time on ART 13 years for non-responders vs 11 years for controls. Pre-ART HA and CXCL4 concentrations did not vary by eventual immune reconstitution status. Post-ART HA concentrations tended to be higher (85 vs 36 ng/mL, p = 0.07) and CXCL4 concentrations were lower (563 vs 1459 ng/mL, p = 0.01) among non-responders. Among men with paired pre-/post-ART samples, non-responders had greater HA increases and CXCL4 decreases than controls (HA: 50 vs 12 ng/mL, p = 0.04; CXCL4: -1258 vs -405 ng/mL, p = 0.01). Conclusions Higher circulating concentrations of HA and lower concentrations of CXCL4 are associated with failure of immune reconstitution on ART. PMID:29381717

Different effect of hydrogelation on anti-fouling and circulation properties of dextran–iron oxide nanoparticles

PubMed Central

Karmali, Priya Prakash; Chao, Ying; Park, Ji-Ho (Joe); Sailor, Michael J.; Ruoslahti, Erkki; Esener, Sadik C.; Simberg, Dmitri

2012-01-01

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by crosslinking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that: (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles. PMID:22243419

Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

PubMed

Lundell, Anna-Carin; Hesselmar, Bill; Nordström, Inger; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

2015-10-01

A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Emergence of MET hyper-amplification at progression to MET and BRAF inhibition in colorectal cancer.

PubMed

Oddo, Daniele; Siravegna, Giulia; Gloghini, Annunziata; Vernieri, Claudio; Mussolin, Benedetta; Morano, Federica; Crisafulli, Giovanni; Berenato, Rosa; Corti, Giorgio; Volpi, Chiara Costanza; Buscarino, Michela; Niger, Monica; Dunne, Philip D; Rospo, Giuseppe; Valtorta, Emanuele; Bartolini, Alice; Fucà, Giovanni; Lamba, Simona; Martinetti, Antonia; Di Bartolomeo, Maria; de Braud, Filippo; Bardelli, Alberto; Pietrantonio, Filippo; Di Nicolantonio, Federica

2017-07-25

Combined MET and BRAF inhibition showed clinical benefit in a patient with rectal cancer carrying BRAF V600E and MET amplification. However after 4 months, acquired resistance emerged and the patient deceased shortly after disease progression. The mechanism of resistance to this drug combination is unknown. We analysed plasma circulating tumour DNA obtained at progression by exome sequencing and digital PCR. MET gene and mRNA in situ hybridisation analyses in two bioptic specimens obtained at progression were used to confirm the plasma data. We identified in plasma MET gene hyper-amplification as a potential mechanism underlying therapy resistance. Increased MET gene copy and transcript levels were detected in liver and lymph node metastatic biopsies. Finally, transduction of MET in BRAF mutant colorectal cancer cells conferred refractoriness to BRAF and MET inhibition. We identified in a rectal cancer patient MET gene hyper-amplification as mechanism of resistance to dual BRAF and MET inhibition.

Regulating intestinal function to reduce atherogenic lipoproteins.

PubMed

Hussain, M Mahmood; Leung, Tung Ming; Zhou, Liye; Abu-Merhi, Sarah

2013-08-01

Significant knowledge regarding different molecules involved in the transport of dietary fat into the circulation has been garnered. Studies point to the possibility that accumulation of intestine-derived lipoproteins in the plasma could contribute to atherosclerosis. This article provides a brief overview of dietary lipid metabolism and studies in mice supporting the hypothesis that intestinal lipoproteins contribute to atherosclerosis. Deficiencies in lipoprotein lipase and Gpihbp1, and overexpression of heparanse in mice, are associated with increases in atherosclerosis, suggesting that defects in catabolism of larger lipoproteins in the plasma contribute to atherosclerosis. Furthermore, inositol-requiring enzyme 1β-deficient mice that produce more intestinal lipoproteins also develop more atherosclerosis. Thus, increases in plasma intestinal lipoproteins due to either overproduction or reduced catabolism result in augmented atherosclerosis. Intestinal lipoproteins tend to adhere strongly to subendothelial proteoglycans, elicit an inflammatory response by endothelial cells and activate macrophages, contributing to the initiation and progression of the disease. Thus, molecules that reduce intestinal lipid absorption can be useful in lowering atherosclerosis.

Epstein-Barr Virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies.

PubMed

Ryan, Julie L; Fan, Hongxin; Swinnen, Lode J; Schichman, Steven A; Raab-Traub, Nancy; Covington, Mary; Elmore, Sandra; Gulley, Margaret L

2004-06-01

Epstein-Barr Virus (EBV), a ubiquitous gamma herpes virus, infects more than 95% of the human population before adulthood. Life-long persistence, usually without adverse health consequences, relies on a balance between viral latency, viral replication, and host immune response. Patients with EBV-related disease often have high levels of EBV DNA in their plasma. This study addresses whether this circulating, cell-free EBV DNA is encapsidated in virions or exists as naked genomes. First, an assay was developed, combining DNase I and quantitative real-time PCR, to discriminate encapsidated from naked EBV DNA. EBV DNA was almost always naked in the plasma of AIDS-related lymphoma patients (n = 11) and immunosuppressed/posttransplantation patients (n = 8). In contrast, infectious mononucleosis patients (n = 30) often had a mixture of encapsidated and naked EBV DNA. These findings may be important in understanding how viral load relates to disease status and in predicting response to nucleoside analogs and other antiviral therapies.

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients

PubMed Central

Fucikova, Jitka; Truxova, Iva; Hensler, Michal; Becht, Etienne; Kasikova, Lenka; Moserova, Irena; Vosahlikova, Sarka; Klouckova, Jana; Church, Sarah E.; Cremer, Isabelle; Kepp, Oliver; Kroemer, Guido; Galluzzi, Lorenzo; Salek, Cyril

2016-01-01

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response. PMID:27802968

Plasma wake field XUV radiation source

DOEpatents

Prono, Daniel S.; Jones, Michael E.

1997-01-01

A XUV radiation source uses an interaction of electron beam pulses with a gas to create a plasma radiator. A flowing gas system (10) defines a circulation loop (12) with a device (14), such as a high pressure pump or the like, for circulating the gas. A nozzle or jet (16) produces a sonic atmospheric pressure flow and increases the density of the gas for interacting with an electron beam. An electron beam is formed by a conventional radio frequency (rf) accelerator (26) and electron pulses are conventionally formed by a beam buncher (28). The rf energy is thus converted to electron beam energy, the beam energy is used to create and then thermalize an atmospheric density flowing gas to a fully ionized plasma by interaction of beam pulses with the plasma wake field, and the energetic plasma then loses energy by line radiation at XUV wavelengths Collection and focusing optics (18) are used to collect XUV radiation emitted as line radiation when the high energy density plasma loses energy that was transferred from the electron beam pulses to the plasma.

EJE PRIZE 2018: A gut feeling about glucagon.

PubMed

Knop, Filip K

2018-06-01

Hyperglucagonaemia (in the fasting as well as in the postprandial state) is considered a core pathophysiological component of diabetes and is found to contribute substantially to the hyperglycaemic state of diabetes. Hyperglucagonaemia is usually viewed upon as a consequence of pancreatic alpha cell insensitivity to the glucagon-suppressive effects of glucose and insulin. Since we observed that the well-known hyperglucagonaemic response to oral glucose in patients with type 2 diabetes is exchanged by normal suppression of plasma glucagon levels following isoglycaemic intravenous glucose administration in these patients, we have been focusing on the gut and gut-derived factors as potential mediators of diabetic hyperglucagonaemia. In a series of clinical experiments, we have elucidated the role of gut-derived factors in diabetic hyperglucagonaemia and shown that glucose-dependent insulinotropic polypeptide promotes hyperglucagonaemia and that glucagon, hitherto considered a pancreas-specific hormone, may also be secreted from extrapancreatic tissues - most likely from proglucagon-producing enteroendocrine cells. Furthermore, our observation that fasting hyperglucagonaemia is unrelated to the diabetic state, but strongly correlates with obesity, liver fat content and circulating amino acids, has made us question the common 'pancreacentric' and 'glucocentric' understanding of hyperglucagonaemia and led to the hypothesis that steatosis-induced hepatic glucagon resistance (and reduced amino acid turnover) and compensatory glucagon secretion mediated by increased circulating amino acids constitute a complete endocrine feedback system: the liver-alpha cell axis. This article summarises the physiological regulation of glucagon secretion in humans and considers new findings suggesting that the liver and the gut play key roles in determining fasting and postabsorptive circulating glucagon levels. © 2018 European Society of Endocrinology.

CIRCULATING MICROPARTICLES IN PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES: CHARACTERIZATION AND ASSOCIATIONS

PubMed Central

Chaturvedi, Shruti; Cockrell, Erin; Espinola, Ricardo; Hsi, Linda; Fulton, Stacey; Khan, Mohammad; Li, Liang; Fonseca, Fabio; Kundu, Suman; McCrae, Keith R.

2014-01-01

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte, platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-β2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2 hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R = 0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with APS and their correlation with anti-β2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for β2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies. PMID:25467081

Effect of Immobilized Antithrombin III on the Thromboresistance of Polycarbonate Urethane.

PubMed

Lukas, Karin; Stadtherr, Karin; Gessner, Andre; Wehner, Daniel; Schmid, Thomas; Wendel, Hans Peter; Schmid, Christof; Lehle, Karla

2017-03-24

The surface of foils and vascular grafts made from a thermoplastic polycarbonate urethanes (PCU) (Chronoflex AR) were chemically modified using gas plasma treatment, binding of hydrogels-(1) polyethylene glycol bisdiamine and carboxymethyl dextran (PEG-DEX) and (2) polyethyleneimine (PEI)-and immobilization of human antithrombin III (AT). Their biological impact was tested in vitro under static and dynamic conditions. Static test methods showed a significantly reduced adhesion of endothelial cells, platelets, and bacteria, compared to untreated PCU. Modified PCU grafts were circulated in a Chandler-Loop model for 90 min at 37 °C with human blood. Before and after circulation, parameters of the hemostatic system (coagulation, platelets, complement, and leukocyte activation) were analyzed. PEI-AT significantly inhibited the activation of both coagulation and platelets and prevented the activation of leukocytes and complement. In conclusion, both modifications significantly reduce coagulation activation, but only PEI-AT creates anti-bacterial and anti-thrombogenic functionality.

Dietary fatty acids modulate associations between genetic variants and circulating fatty acids in plasma and erythrocyte membranes: meta-analysis of nine studies in the CHARGE consortium

USDA-ARS?s Scientific Manuscript database

Scope: Tissue concentrations of omega-3 fatty acids may reduce cardiovascular disease risk, and genetic variants are associated with circulating fatty acids concentrations. Whether dietary fatty acids interact with genetic variants to modify circulating omega-3 fatty acids is unclear. We evaluated i...

Potentialities of aberrantly methylated circulating DNA for diagnostics and post-treatment follow-up of lung cancer patients.

PubMed

Ponomaryova, Anastasia A; Rykova, Elena Yu; Cherdyntseva, Nadezda V; Skvortsova, Tatiana E; Dobrodeev, Alexey Yu; Zav'yalov, Alexander A; Bryzgalov, Leonid O; Tuzikov, Sergey A; Vlassov, Valentin V; Laktionov, Pavel P

2013-09-01

To date, aberrant DNA methylation has been shown to be one of the most common and early causes of malignant cell transformation and tumors of different localizations, including lung cancer. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA circulating in the blood (cirDNA) is a convenient tumor-associated DNA marker that can be used as a minimally invasive diagnostic test. In the current study, we investigated the methylation status in blood samples of 32 healthy donors and 60 lung cancer patients before and after treatment with neoadjuvant chemotherapy followed by total tumor resection. Using quantitative methylation-specific PCR, we found that the index of methylation (IM), calculated as IM = 100 × [copy number of methylated/(copy number of methylated + unmethylated gene)], for the RASSF1A and RARB2 genes in the cirDNA isolated from blood plasma and cell-surface-bound cirDNA was elevated 2- to 3-fold in lung cancer patients compared with healthy donors. Random forest classification tree model based on these variables combined (RARB2 and RASSF1A IM in both plasma and cell-surface-bound cirDNA) lead to NSCLC patients' and healthy subjects' differentiation with 87% sensitivity and 75% specificity. An association of increased IM values with an advanced stage of non-small-cell lung cancer was found for RARB2 but not for RASSF1A. Chemotherapy and total tumor resection resulted in a significant decrease in the IM for RARB2 and RASSF1A, in both cirDNA fractions, comparable to the IM level of healthy subjects. Importantly, a rise in the IM for RARB2 was detected in patients within the follow-up period, which manifested in disease relapse at 9 months, confirmed with instrumental and pathologic methods. Our data indicate that quantitative analysis of the methylation status of the RARB2 and RASSF1A tumor suppressor genes in both cirDNA fractions is a useful tool for lung cancer diagnostics, evaluation of cancer treatment efficiency and post-treatment monitoring. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Circulating microRNAs as biomarkers of disease and typification of the atherothrombotic status in antiphospholipid syndrome

PubMed Central

Pérez-Sánchez, Carlos; Arias-de la Rosa, Iván; Aguirre, María Ángeles; Luque-Tévar, María; Ruiz-Limón, Patricia; Barbarroja, Nuria; Jiménez-Gómez, Yolanda; Ábalos-Aguilera, María Carmen; Collantes-Estévez, Eduardo; Segui, Pedro; Velasco, Francisco; Herranz, María Teresa; Lozano-Herrero, Jesús; Hernandez-Vidal, María Julia; Martínez, Constantino; González-Conejero, Rocío; Radin, Massimo; Sciascia, Savino; Cecchi, Irene; Cuadrado, María José; López-Pedrera, Chary

2018-01-01

We aimed to identify the plasma miRNA profile of antiphospholipid syndrome (APS) patients and to investigate the potential role of specific circulating miRNAs as non-invasive disease biomarkers. Ninety APS patients and 42 healthy donors were recruited. Profiling of miRNAs by PCR-array in plasma of APS patients identified a set of miRNAs differentially expressed and collectively involved in clinical features. Logistic regression and ROC analysis identified a signature of 10 miRNA ratios as biomarkers of disease. In addition, miRNA signature was related to fetal loss, atherosclerosis, and type of thrombosis, and correlated with parameters linked to inflammation, thrombosis, and autoimmunity. Hard clustering analysis differentiated 3 clusters representing different thrombotic risk profile groups. Significant differences between groups for several miRNA ratios were found. Moreover, miRNA signature remained stable over time, demonstrated by their analysis three months after the first sample collection. Parallel analysis in two additional cohorts of patients, including thrombosis without autoimmune disease, and systemic lupus erythematosus without antiphospholipid antibodies, each displayed specific miRNA profiles that were distinct from those of APS patients. In vitro, antiphospholipid antibodies of IgG isotype promoted deregulation in selected miRNAs and their potential atherothrombotic protein targets in monocytes and endothelial cells. Taken together, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients’ atherothrombotic status, thus constituting a useful tool in the management of the disease. PMID:29545345

Plasma thyroid hormone pattern in king penguin chicks: a semi-altricial bird with an extended posthatching developmental period.

PubMed

Cherel, Yves; Durant, Joël M; Lacroix, André

2004-05-01

Plasma concentrations of thyroid hormones (TH) were investigated during the extended posthatching developmental period (approximately 11 months) of a semi-altricial bird species, the king penguin (Aptenodytes patagonicus). The first period of growth in summer was marked by a progressive rise in plasma T4 concentration that paralleled rapid increases in body mass and in structural and down growth. By contrast, plasma T3 concentration had already reached adult levels in newly hatched chicks and did not change thereafter. Circulating TH of king penguin chicks thus follow an original pattern when comparing to altricial and precocial species. During the austral winter, the long period of undernutrition of king penguin chicks was characterized by a decrease in circulating TH that can be related to a seasonal stop in growth and energy saving mechanisms. Plasma TH concentrations increased again during the second growth phase in spring, and they reached their highest levels at the end of the fledging period, slightly before juveniles initiated their first foraging trip at sea. As expected, plasma T4 levels were elevated when chicks moulted, developing a true-adult type waterproof plumage. The data also suggest that T4 plays a major role in skeletal development and pectoral muscle maturation in anticipation of marine life. Plasma T3 was at its highest during the period when juveniles improved resistance to cold waters by going back and forth to the sea, suggesting a role for circulating T3 in cold acclimatization occurring at that time.

Elevated Plasma IL-6 Associates with Increased Risk of Advanced Fibrosis and Cholangiocarcinoma in Individuals Infected by Opisthorchis viverrini

PubMed Central

Sripa, Banchob; Thinkhamrop, Bandit; Mairiang, Eimorn; Laha, Thewarach; Kaewkes, Sasithorn; Sithithaworn, Paiboon; Periago, Maria Victoria; Bhudhisawasdi, Vajarabhongsa; Yonglitthipagon, Ponlapat; Mulvenna, Jason; Brindley, Paul J.; Loukas, Alex; Bethony, Jeffrey M.

2012-01-01

Opisthorchis viverrini is considered among the most important of the food-borne trematodes due to its strong association with advanced periductal fibrosis and bile duct cancer (cholangiocarcinoma). We investigated the relationship between plasma levels of Interleukin (IL)-6 and the risk of developing advanced fibrosis and bile duct cancer from chronic Opisthorchis infection. We show that IL-6 circulates in plasma at concentrations 58 times higher in individuals with advanced fibrosis than age, sex, and nearest-neighbor matched controls and 221 times higher in individuals with bile duct cancer than controls. We also observed a dose-response relationship between increasing levels of plasma IL-6 and increasing risk of advanced fibrosis and bile duct cancer; for example, in age and sex adjusted analyses, individuals with the highest quartiles of plasma IL-6 had a 19 times greater risk of developing advanced periductal fibrosis and a 150 times greater risk of developing of bile duct cancer than individuals with no detectable level of plasma IL-6. Finally, we show that a single plasma IL-6 measurement has excellent positive predictive value for the detection of both advanced bile duct fibrosis and bile duct cancer in regions with high O. viverrini transmission. These data support our hypothesis that common mechanisms drive bile duct fibrosis and bile duct tumorogenesis from chronic O. viverrini infection. Our study also adds a unique aspect to the literature on circulating levels of IL-6 as an immune marker of hepatobiliary pathology by showing that high levels of circulating IL-6 in plasma are not related to infection with O. viverrini, but to the development of the advanced and often lethal pathologies resulting from chronic O. viverrini infection. PMID:22629477

Acute Effects of Lysergic Acid Diethylamide on Circulating Steroid Levels in Healthy Subjects.

PubMed

Strajhar, P; Schmid, Y; Liakoni, E; Dolder, P C; Rentsch, K M; Kratschmar, D V; Odermatt, A; Liechti, M E

2016-03-01

Lysergic acid diethylamide (LSD) is a serotonin 5-hydroxytryptamine-2A (5-HT2A ) receptor agonist that is used recreationally worldwide. Interest in LSD research in humans waned after the 1970s, although the use of LSD in psychiatric research and practice has recently gained increasing attention. LSD produces pronounced acute psychedelic effects, although its influence on plasma steroid levels over time has not yet been characterised in humans. The effects of LSD (200 μg) or placebo on plasma steroid levels were investigated in 16 healthy subjects using a randomised, double-blind, placebo-controlled, cross-over study design. Plasma concentration-time profiles were determined for 15 steroids using liquid-chromatography tandem mass-spectrometry. LSD increased plasma concentrations of the glucocorticoids cortisol, cortisone, corticosterone and 11-dehydrocorticosterone compared to placebo. The mean maximum concentration of LSD was reached at 1.7 h. Mean peak psychedelic effects were reached at 2.4 h, with significant alterations in mental state from 0.5 h to > 10 h. Mean maximal concentrations of cortisol and corticosterone were reached at 2.5 h and 1.9 h, and significant elevations were observed 1.5-6 h and 1-3 h after drug administration, respectively. LSD also significantly increased plasma concentrations of the androgen dehydroepiandrosterone but not other androgens, progestogens or mineralocorticoids compared to placebo. A close relationship was found between plasma LSD concentrations and changes in plasma cortisol and corticosterone and the psychotropic response to LSD, and no clockwise hysteresis was observed. In conclusion, LSD produces significant acute effects on circulating steroids, especially glucocorticoids. LSD-induced changes in circulating glucocorticoids were associated with plasma LSD concentrations over time and showed no acute pharmacological tolerance. © 2016 British Society for Neuroendocrinology.

Interactions between dietary oil treatments and genetic variants modulate fatty acid ethanolamides in plasma and body weight composition.

PubMed

Pu, Shuaihua; Eck, Peter; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Jones, Peter J H

2016-03-28

Fatty acid ethanolamides (FAE), a group of lipid mediators derived from long-chain fatty acids (FA), mediate biological activities including activation of cannabinoid receptors, stimulation of fat oxidation and regulation of satiety. However, how circulating FAE levels are influenced by FA intake in humans remains unclear. The objective of the present study was to investigate the response of six major circulating FAE to various dietary oil treatments in a five-period, cross-over, randomised, double-blind, clinical study in volunteers with abdominal obesity. The treatment oils (60 g/12 552 kJ per d (60 g/3000 kcal per d)) provided for 30 d were as follows: conventional canola oil, high oleic canola oil, high oleic canola oil enriched with DHA, flax/safflower oil blend and corn/safflower oil blend. Two SNP associated with FAE degradation and synthesis were studied. Post-treatment results showed overall that plasma FAE levels were modulated by dietary FA and were positively correlated with corresponding plasma FA levels; minor allele (A) carriers of SNP rs324420 in gene fatty acid amide hydrolase produced higher circulating oleoylethanolamide (OEA) (P=0·0209) and docosahexaenoylethanolamide (DHEA) levels (P=0·0002). In addition, elevated plasma DHEA levels in response to DHA intake tended to be associated with lower plasma OEA levels and an increased gynoid fat mass. In summary, data suggest that the metabolic and physiological responses to dietary FA may be influenced via circulating FAE. Genetic analysis of rs324420 might help identify a sub-population that appears to benefit from increased consumption of DHA and oleic acid.

The disposition index does not reflect β-cell function in IGT subjects treated with pioglitazone.

PubMed

DeFronzo, Ralph A; Tripathy, Devjit; Abdul-Ghani, Muhammad; Musi, Nicolas; Gastaldelli, Amalia

2014-10-01

The insulin secretion/insulin resistance (IR) (disposition) index (ΔI/ΔG ÷ IR, where Δ is change from baseline, I is insulin, and G is glucose) is commonly used as a measure of β-cell function. This relationship is curvilinear and becomes linear when log transformed. ΔI is determined by 2 variables: insulin secretion rate (ISR) and metabolic clearance of insulin. We postulated that the characteristic curvilinear relationship would be lost if Δ plasma C-peptide (ΔCP) (instead of Δ plasma insulin) was plotted against insulin sensitivity. A total of 441 individuals with impaired glucose tolerance (IGT) from ACT NOW received an oral glucose tolerance test and were randomized to pioglitazone or placebo for 2.4 years. Pioglitazone reduced IGT conversion to diabetes by 72% (P < .0001). ΔI/ΔG vs the Matsuda index of insulin sensitivity showed the characteristic curvilinear relationship. However, when ΔCP/ΔG or ΔISR/ΔG was plotted against the Matsuda index, the curvilinear relationship was completely lost. This discordance was explained by 2 distinct physiologic effects that altered plasma insulin response in opposite directions: 1) increased ISR and 2) augmented metabolic clearance of insulin. The net result was a decline in the plasma insulin response to hyperglycemia during the oral glucose tolerance test. These findings demonstrate a physiologic control mechanism wherein the increase in ISR ensures adequate insulin delivery into the portal circulation to suppress hepatic glucose production while delivering a reduced but sufficient amount of insulin to peripheral tissues to maintain the pioglitazone-mediated improvement in insulin sensitivity without excessive hyperinsulinemia. These results demonstrate the validity of the disposition index when relating the plasma insulin response to insulin sensitivity but underscore the pitfall of this index when drawing conclusions about β-cell function, because insulin secretion declined despite an increase in the plasma insulin response.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients.

PubMed

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-08-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell‑free DNA (cfDNA) extracted from the plasma of advanced non‑small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) ‑sensitive (19DEL, L858R) and TKI‑resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR‑TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra‑sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR‑TKI decision‑making for advanced NSCLC patients.

Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules

PubMed Central

2011-01-01

Background Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer. Methods Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined. Results We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis. Conclusion Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs. PMID:22151235

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.

PubMed

Dickinson, Brent A; Semus, Hillary M; Montgomery, Rusty L; Stack, Christianna; Latimer, Paul A; Lewton, Steven M; Lynch, Joshua M; Hullinger, Thomas G; Seto, Anita G; van Rooij, Eva

2013-06-01

Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease. In order to define circulating miRNAs that change during hypertension-induced heart failure and that respond to therapeutic treatment, we performed miRNA arrays on plasma RNA from hypertensive rats that show signs of heart failure. Array analysis indicated that approximately one-third of the miRNAs on the array are detectable in plasma. Quantitative real-time polymerase chain reaction (PCR) analysis for a selected panel of miRNAs indicated that circulating levels of miR-16, miR-20b, miR-93, miR-106b, miR-223, and miR-423-5p were significantly increased in response to hypertension-induced heart failure, while this effect was blunted in response to treatment with antimiR-208a as well as an ACE inhibitor. Moreover, treatment with antimiR-208a resulted in a dramatic increase in one miRNA, miR-19b. A time course study indicated that several of these miRNA changes track with disease progression. Circulating levels of miRNAs are responsive to therapeutic interventions and change during the progression of hypertension-induced heart disease.

Different effects of fenofibrate on metabolic and cardiovascular risk factors in mixed dyslipidemic women with normal thyroid function and subclinical hypothyroidism.

PubMed

Krysiak, Robert; Gilowski, Wojciech; Szkrobka, Witold; Okopien, Bogusław

2014-12-01

Subclinical hypothyroidism is suggested to increase cardiovascular risk. No previous study compared the effect of any fibrate on plasma levels of lipids and other cardiovascular risk factors in patients with different thyroid function status. The study included three age-, weight- and lipid-matched groups of women with mixed dyslipidemia in different thyroid function status: patients with untreated subclinical hypothyroidism (group 1, n = 18), women with treated hypothyroidism (group 2, n = 15), and subjects without thyroid disorders (group 3, n = 19). Plasma lipids, glucose homeostasis markers, as well as plasma levels of uric acid, high-sensitivity C-reactive protein (hsCRP), homocysteine, and fibrinogen were determined before and after 12 weeks of fenofibrate therapy. Despite similar plasma lipid levels, mixed dyslipidemic patients with untreated hypothyroidism had decreased insulin sensitivity, as well as higher circulating levels of uric acid, hsCRP, homocysteine, and fibrinogen in comparison with the other groups. The effect of fenofibrate on plasma lipids and, with the exception of homocysteine, on circulating levels of all investigated risk factors was stronger in patients from groups 2 and 3 than in patients from group 1. The obtained results indicate that the effect of fenofibrate on plasma lipids and circulating levels of cardiovascular risk factors is partially related to thyroid function. They also suggest that to improve the strength of fibrate action in dyslipidemic patients with subclinical hypothyroidism, they should be administered together with L-thyroxine. © 2014 John Wiley & Sons Ltd.

Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells.

PubMed

Khan, Mahfuz B; Lang, Michelle J; Huang, Ming-Bo; Raymond, Andrea; Bond, Vincent C; Shiramizu, Bruce; Powell, Michael D

2016-04-01

In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

Evidence for circulating cancer stem-like cells and epithelial-mesenchymal transition phenotype in the pleurospheres derived from lung adenocarcinoma using liquid biopsy.

PubMed

Mirza, Sheefa; Jain, Nayan; Rawal, Rakesh

2017-03-01

Lung cancer stem cells are supposed to be the main drivers of tumor initiation, maintenance, drug resistance, and relapse of the disease. Hence, identification of the cellular and molecular aspects of these cells is a prerequisite for targeted therapy of lung cancer. Currently, analysis of circulating tumor cells has the potential to become the main diagnostic technique to monitor disease progression or therapeutic response as it is non-invasive. However, accurate detection of circulating tumor cells has remained a challenge, as epithelial cell markers used so far are not always trustworthy for detecting circulating tumor cells, especially during epithelial-mesenchymal transition. As cancer stem cells are the only culprit to initiate metastatic tumors, our aim was to isolate and characterize circulating tumor stem cells rather than circulating tumor cells from the peripheral blood of NSCLC adenocarcinoma as limited data are available addressing the gene expression profiling of lung cancer stem cells. Here, we reveal that CD44(+)/CD24(-) population in circulation not only exhibit stem cell-related genes but also possess epithelial-mesenchymal transition characteristics. In conclusion, the use of one or more cancer stem cell markers along with epithelial, mesenchymal and epithelial mesenchymal transition markers will prospectively provide the most precise assessment of the threat for recurrence and metastatic disease and has a great potential for forthcoming applications in harvesting circulating tumor stem cells and their downstream applications. Our results will aid in developing diagnostic and prognostic modalities and personalized treatment regimens like dendritic cell-based immunotherapy that can be utilized for targeting and eliminating circulating tumor stem cells, to significantly reduce the possibility of relapse and improve clinical outcomes.

Soy-based infant formula supplemented with DHA and ARA supports growth and increases circulating levels of these fatty acids in infants.

PubMed

Hoffman, Dennis; Ziegler, Ekhard; Mitmesser, Susan H; Harris, Cheryl L; Diersen-Schade, Deborah A

2008-01-01

Healthy term infants (n = 244) were randomized to receive: (1) control, soy-based formula without supplementation or (2) docosahexaenoic acid-arachidonic acid (DHA + ARA), soy-based formula supplemented with at least 17 mg DHA/100 kcal (from algal oil) and 34 mg ARA/100 kcal (from fungal oil) in a double-blind, parallel group trial to evaluate safety, benefits, and growth from 14 to 120 days of age. Anthropometric measurements were taken at 14, 30, 60, 90, and 120 days of age and 24-h dietary and tolerance recall were recorded at 30, 60, 90, and 120 days of age. Adverse events were recorded throughout the study. Blood samples were drawn from subsets of 25 infants in each group. Capillary column gas chromatography was used to analyze the percentages of fatty acids in red blood cell (RBC) lipids and plasma phospholipids. Compared with the control group, percentages of fatty acids such as DHA and ARA in total RBC and plasma phospholipids were significantly higher in infants in the DHA + ARA group at 120 days of age (P < 0.001). Growth rates did not differ significantly between feeding groups at any assessed time point. Supplementation did not affect the tolerance of formula or the incidence of adverse events. Feeding healthy term infants soy-based formula supplemented with DHA and ARA from single cell oil sources at concentrations similar to human milk significantly increased circulating levels of DHA and ARA when compared with the control group. Both formulas supported normal growth and were well tolerated.

Short-term high-fat diet alters postprandial glucose metabolism and circulating vascular cell adhesion molecule-1 in healthy males.

PubMed

Numao, Shigeharu; Kawano, Hiroshi; Endo, Naoya; Yamada, Yuka; Takahashi, Masaki; Konishi, Masayuki; Sakamoto, Shizuo

2016-08-01

Short-term intake of a high-fat diet aggravates postprandial glucose metabolism; however, the dose-response relationship has not been investigated. We hypothesized that short-term intake of a eucaloric low-carbohydrate/high-fat diet (LCHF) would aggravate postprandial glucose metabolism and circulating adhesion molecules in healthy males. Seven healthy young males (mean ± SE; age: 26 ± 1 years) consumed either a eucaloric control diet (C, approximately 25% fats), a eucaloric intermediate-carbohydrate/intermediate-fat diet (ICIF, approximately 50% fats), or an LCHF (approximately 70% fats) for 3 days. An oral meal tolerance test (MTT) was performed after the 3-day dietary intervention. The concentrations of plasma glucose, insulin, glucagon-like peptide-1 (GLP-1), intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 (VCAM-1) were determined at rest and during MTT. The incremental area under the curve (iAUC) of plasma glucose concentration during MTT was significantly higher in LCHF than in C (P = 0.009). The first-phase insulin secretion indexes were significantly lower in LCHF than in C (P = 0.04). Moreover, the iAUC of GLP-1 and VCAM-1 concentrations was significantly higher in LCHF than in C (P = 0.014 and P = 0.04, respectively). The metabolites from ICIF and C were not significantly different. In conclusion, short-term intake of eucaloric diet containing a high percentage of fats in healthy males excessively increased postprandial glucose and VCAM-1 concentrations and attenuated first-phase insulin release.

Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease.

PubMed

Anguiano, Lidia; Riera, Marta; Pascual, Julio; Valdivielso, José Manuel; Barrios, Clara; Betriu, Angels; Mojal, Sergi; Fernández, Elvira; Soler, María José

2015-07-01

Patients with cardiovascular (CV) disease have an increased circulating angiotensin-converting enzyme 2 (ACE2) activity, but there is little information about changes in ACE2 in chronic kidney disease (CKD) patients without history of CV disease. We examined circulating ACE2 activity in CKD patients at stages 3-5 (CKD3-5) and in dialysis (CKD5D) without any history of CV disease. Circulating ACE2 activity was measured in human ethylenediamine-tetraacetic acid (EDTA)-plasma samples from the NEFRONA study (n = 2572): control group (CONT) (n = 568), CKD3-5 (n = 1458) and CKD5D (n = 546). Different clinical and analytical variables such as gender; age; history of diabetes mellitus (DM), dyslipidemia and hypertension; glycaemic, renal, lipid and anaemia profiles; vitamin D analogues treatment and antihypertensive treatments (angiotensin-converting enzyme inhibitor and angiotensin receptor blockade) were analysed. Circulating ACE2 and ACE activities were measured using modified fluorimetric assay for EDTA-plasma samples, where zinc chloride was added to recover enzymatic activity. In CKD3-5 and CKD5D, significant decrease in circulating ACE2 activity was observed when compared with CONT, but no differences were found between CKD3-5 and CKD5 when performing paired case-control studies. By multivariate linear regression analysis, male gender and advanced age were identified as independent predictors of ACE2 activity in all groups. Diabetes was identified as independent predictor of ACE2 activity in CKD3-5. Significant increase in the activity of circulating ACE was found in CKD3-5 and CKD5D when compared with CONT and in CKD5D when compared with CKD3-5. By multiple regression analysis, female gender and younger age were identified as independent predictors of ACE activity in CONT and CKD3-5. Diabetes was also identified as an independent predictor of ACE activity in CKD3-5 patients. Circulating ACE2 and ACE activities can be measured in human EDTA-plasma samples with zinc added to recover enzymatic activity. In a CKD population without previous history of CV disease, ACE2 activity from human EDTA-plasma samples directly correlated with the classical CV risk factors namely older age, diabetes and male gender. Our data suggest that circulating ACE2 is altered in CKD patients at risk for CV event. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, C.; Keppens, R.

Solar prominences are long-lived cool and dense plasma curtains in the hot and rarefied outer solar atmosphere or corona. The physical mechanism responsible for their formation and especially for their internal plasma circulation has been uncertain for decades. The observed ubiquitous downflows in quiescent prominences are difficult to interpret because plasma with high conductivity seems to move across horizontal magnetic field lines. Here we present three-dimensional numerical simulations of prominence formation and evolution in an elongated magnetic flux rope as a result of in situ plasma condensations fueled by continuous plasma evaporation from the solar chromosphere. The prominence is bornmore » and maintained in a fragmented, highly dynamic state with continuous reappearance of multiple blobs and thread structures that move mainly downward, dragging along mass-loaded field lines. The circulation of prominence plasma is characterized by the dynamic balance between the drainage of prominence plasma back to the chromosphere and the formation of prominence plasma via continuous condensation. Plasma evaporates from the chromosphere, condenses into the prominence in the corona, and drains back to the chromosphere, establishing a stable chromosphere–corona plasma cycle. Synthetic images of the modeled prominence with the Solar Dynamics Observatory Atmospheric Imaging Assembly closely resemble actual observations, with many dynamical threads underlying an elliptical coronal cavity.« less

Angiotensin 1-7 Is a Negative Modulator of Aldosterone Secretion In Vitro and In Vivo.

PubMed

Shefer, Gabi; Marcus, Yonit; Knoll, Esther; Dolkart, Oleg; Foichtwanger, Shulamit; Nevo, Nava; Limor, Rona; Stern, Naftali

2016-08-01

Angiotensin (1-7) [Ang 1-7] is a 7 amino acid peptide generated predominantly from Ang II by the action of Ang-converting enzyme 2. We previously showed that Ang 1-7 reduced plasma aldosterone and plasma renin activity in high fructose-fed rats, and that the reduction in circulating aldosterone seemed to accord a parallel reduction in plasma renin activity. Here, we tested the possibility that Ang 1-7 affects aldosterone secretion acting directly in glomerulosa cells. First, as detected by immunofluorescence, the receptor for Ang 1-7, Mas1 is localized predominantly at the rat adrenal subcapsular region. Second, in isolated rat glomerulosa cells incubates, Ang 1-7 attenuated the aldosterone response to Ang II, with the strongest effect seen on Ang II (10(-9) M) (control 22±2.5 pg/10(5) cells; Ang II [10(-9) M] 189±11 pg/10(5) cells; Ang II [10(-9) M]+Ang 1-7 [10(-6) M] 33±3.6 pg/10(5) cells; P<0.001) and the largest effect on adrenocorticotropic hormone (10(-8) M) (control 30±3.4 pg/10(5) cells; ACTH [10(-8) M] 409±32.5 pg/10(5) cells; ACTH [10(-8) M]+Ang 1-7 [10(-6) M] 280±12.5 pg/10(5) cells; P<0.001). In contrast, Ang 1-7 did not affect the aldosterone response to potassium (K(+)). In rats subjected to a low-salt diet for 7 days, continuous infusion of Ang 1-7 (576 μg/kg per day) resulted in a lesser rise in aldosterone (salt deplete+Ang 1-7, 16.4±4.8 ng/dL) compared with rats receiving vehicle (salt deplete+vehicle, 27.6±5.3 ng/dL; P<0.01) but did not modify plasma renin activity. Taken together, these results indicate that Ang 1-7 can act as a negative modulator of aldosterone secretion in vitro and in vivo. © 2016 American Heart Association, Inc.

Elevated circulating soluble thrombomodulin activity, tissue factor activity and circulating procoagulant phospholipids: new and useful markers for pre-eclampsia?

PubMed

Rousseau, Aurélie; Favier, Rémi; Van Dreden, Patrick

2009-09-01

One of the most frequently proposed mechanisms for pre-eclampsia refers to uteroplacental thrombosis. However, the contribution of classical thrombotic risk factors remains questionable. The aims of this study were to investigate the activities of thrombomodulin, tissue factor and procoagulant phospholipids to assess endothelial cell injury in pregnant women with pre-eclampsia and to compare them with other classical markers of vascular injury and thrombotic risk. Using three new functional assays we studied the plasma levels of these new markers in 35 healthy women, 30 healthy pregnant women, and 35 women with pre-eclampsia. We found that plasma levels of thrombomodulin activity, tissue factor activity and procoagulant phospholipids were significantly elevated in women with pre-eclampsia versus normal pregnant and non-pregnant women. It is thus suggested that elevated levels of these parameters in pre-eclampsia may reflect vascular endothelium damage, and may be a more valuable biomarker than antigen for the assessment of endothelial damage in pre-eclampsia. The high increased levels of procoagulant phospholipids and tissue factor activities in pre-eclampsia could suggest that the procoagulant potential may be implicated in this complication and makes these markers very promising for the understanding, follow-up and therapeutic handling of complicated pregnancy.

Exacerbation of Acute Traumatic Brain Injury by Circulating Extracellular Vesicles.

PubMed

Hazelton, Isla; Yates, Abi; Dale, Ashley; Roodselaar, Jay; Akbar, Naveed; Ruitenberg, Marc J; Anthony, Daniel C; Couch, Yvonne

2018-02-15

Inflammatory lesions in the brain activate a systemic acute-phase response (APR), which is dependent on the release of extracellular vesicles (EVs) into the circulation. The resulting APR is responsible for regulating leukocyte mobilization and subsequent recruitment to the brain. Factors that either exacerbate or inhibit the APR will also exacerbate or inhibit central nervous system (CNS) inflammation as a consequence and have the potential to influence ongoing secondary damage. Here, we were interested to discover how the circulating EV population changes after traumatic brain injury (TBI) and how manipulation of the circulating EV pool impacts on the outcome of TBI. We found the number of circulating EVs increased rapidly post-TBI, and this was accompanied by an increase in CNS and hepatic leukocyte recruitment. In an adoptive transfer study, we then evaluated the outcomes of TBI after administering EVs derived from either in vitro macrophage or endothelial cell lines stimulated with lipopolysaccharide (LPS), or from murine plasma from an LPS challenge using the air-pouch model. By manipulating the circulating EV population, we were able to demonstrate that each population of transferred EVs increased the APR. However, the characteristics of the response were dependent on the nature of the EVs; specifically, it was significantly increased when animals were challenged with macrophage-derived EVs, suggesting that the cellular origins of EVs may determine their function. Selectively targeting EVs from macrophage/monocyte populations is likely to be of value in reducing the impact of the systemic inflammatory response on the outcome of traumatic CNS injury.

Cell-free plasma DNA as a novel marker of aseptic inflammation severity related to exercise overtraining.

PubMed

Fatouros, Ioannis G; Destouni, Aspasia; Margonis, Konstantinos; Jamurtas, Athanasios Z; Vrettou, Christina; Kouretas, Dimitrios; Mastorakos, George; Mitrakou, Asimina; Taxildaris, Kiriakos; Kanavakis, Emmanouel; Papassotiriou, Ioannis

2006-09-01

Circulating free plasma DNA is implicated in conditions associated with tissue injury, including exercise-induced inflammation, and thus is a potential marker for athletic overtraining. We measured free plasma DNA along with C-reactive protein (CRP), creatine kinase (CK), and uric acid (UA) in 17 recreationally trained men participating in a 12-week resistance training regimen (8 resistance multi-joint exercises selected to stress the entire musculature: bench press, squat, leg press, snatch, hang clean, dead lifts, barbell arm curls, and rowing), consisting of 4 training periods (t1, t2, t3, and t4). Plasma DNA concentrations increased markedly after t1, t2, and t3 and returned to baseline after t4. There were substantial differences between t2 and t1 and between t3 and t2 plasma DNA concentrations. CRP increased by 300% after t2 and by 400% after t3 (there was no difference between t2 and t3 CRP values) compared with baseline (t0). CK increased only after t3. UA increased after t2 and t3, with a greater increase after t3. This study demonstrates that, after chronic excessive resistance exercise, plasma DNA concentrations increase in proportion to training load, suggesting that plasma DNA may be a sensitive marker for overtraining-induced inflammation.

Prevalence of polyreactive innate clones among graft--infiltrating B cells in human cardiac allograft vasculopathy.

PubMed

Chatterjee, Debanjana; Moore, Carolina; Gao, Baoshan; Clerkin, Kevin J; See, Sarah B; Shaked, David; Rogers, Kortney; Nunez, Sarah; Veras, Yokarla; Addonizio, Linda; Givertz, Michael M; Naka, Yoshifumi; Mancini, Donna; Vasilescu, Rodica; Marboe, Charles; Restaino, Susan; Madsen, Joren C; Zorn, Emmanuel

2018-03-01

Cardiac allograft vasculopathy (CAV) has been associated with graft-infiltrating B cells, although their characteristics are still unclear. In this study we examined the frequency, localization and reactivity profile of graft-infiltrating B cells to determine their contribution to the pathophysiology of CAV. B cells, plasma cells and macrophages were examined by immunohistochemistry in 56 allografts with CAV, 49 native failed hearts and 25 autopsy specimens. A total of 102 B-cell clones were immortalized directly from the infiltrates of 3 fresh cardiac samples with CAV. Their secreted antibodies were assessed using enzyme-linked immunoassay and flow cytometry. B-cell infiltration was observed around coronary arteries in 93% of allograft explants with CAV. Comparatively, intragraft B cells were less frequent and less dense in the intraventricular myocardium from where routine biopsies are obtained. Plasma cells and macrophages were also detected in 85% and 95% of explants, respectively. Remarkably, B-cell infiltrates were not associated with circulating donor-specific antibodies (DSA) or prior episodes of antibody-mediated rejection (AMR). Among all B-cell clones generated from 3 explants with CAV, a majority secreted natural antibodies reactive to multiple autoantigens and apoptotic cells, a characteristic of innate B cells. Our study reveals a high frequency of infiltrating B cells around the coronary arteries of allografts with CAV, independent of DSA or AMR. These cells are enriched for innate B cells with a polyreactive profile. The findings shift the focus from conventional DSA-producing B cells to the potentially pathogenic polyreactive B cells in the development of clinical CAV. Copyright © 2018 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Paricalcitol modulates ACE2 shedding and renal ADAM17 in NOD mice beyond proteinuria.

PubMed

Riera, Marta; Anguiano, Lidia; Clotet, Sergi; Roca-Ho, Heleia; Rebull, Marta; Pascual, Julio; Soler, Maria Jose

2016-03-15

Circulating and renal activity of angiotensin-converting enzyme 2 (ACE2) is increased in non-obese diabetic (NOD) mice. Because paricalcitol has been reported to protect against diabetic nephropathy, we investigated the role of paricalcitol in modulating ACE2 in these mice. In addition, renal ADAM17, a metalloprotease implied in ACE2 shedding, was assessed. NOD female and non-diabetic control mice were studied for 21 days after diabetes onset and divided into various treatment groups. Diabetic animals received either vehicle; 0.4 or 0.8 μg/kg paricalcitol, aliskiren, or a combination of paricalcitol and aliskiren. We then studied the effect of paricalcitol on ACE2 expression in proximal tubular epithelial cells. Paricalcitol alone or in combination with aliskiren resulted in significantly reduced circulating ACE2 activity in NOD mice but there were no changes in urinary albumin excretion. Serum renin activity was significantly decreased in mice that received aliskiren but no effect was found when paricalcitol was used alone. Renal content of ADAM17 was significantly decreased in animals that received a high dose of paricalcitol. Renal and circulating oxidative stress (quantified by plasma H2O2 levels and immunolocalization of nitrotyrosine) were reduced in high-dose paricalcitol-treated mice compared with non-treated diabetic mice. In culture, paricalcitol incubation resulted in a significant increase in ACE2 expression compared with nontreated cells. In NOD mice with type 1 diabetes, paricalcitol modulates ACE2 activity, ADAM17, and oxidative stress renal content independently from the glycemic profile and urinary albumin excretion. In tubular cells, paricalcitol may modulate ACE2 by blocking its shedding. In the early stage of diabetic nephropathy, paricalcitol treatment counterbalances the effect of diabetes on circulating ACE2 activity. Our results suggest that additional use of paricalcitol may be beneficial in treating patients with diabetes under standard therapeutic strategies. Copyright © 2016 the American Physiological Society.

Complement Activation on Platelets Correlates with a Decrease in Circulating Immature Platelets in Patients with Immune Thrombocytopenic Purpura

PubMed Central

Peerschke, Ellinor I.B.; Andemariam, Biree; Yin, Wei; Bussel, James B.

2010-01-01

The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non-immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non-immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A-IPF) (<15 × 109/L) (p = 0.027) and thrombocytopenia (platelet count less than 100K/μl) (p= 0.024). The positive predictive value of an enhanced CAC for a low A-IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacologic therapies, an enhanced response to splenectomy was noted (p <0.063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes. PMID:19925495

In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

NASA Astrophysics Data System (ADS)

He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

2016-12-01

Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing.

PubMed

Kinnings, Sarah L; Geis, Jennifer A; Almasri, Eyad; Wang, Huiquan; Guan, Xiaojun; McCullough, Ron M; Bombard, Allan T; Saldivar, Juan-Sebastian; Oeth, Paul; Deciu, Cosmin

2015-08-01

Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction. The MaterniT21™ PLUS test uses massively parallel sequencing to analyze cell-free fetal DNA in maternal plasma and detect chromosomal abnormalities. We assess the impact of a variety of factors, both maternal and fetal, on the fetal fraction across a large number of samples processed by Sequenom Laboratories. The rate of increase in fetal fraction with increasing gestational age varies across the duration of the testing period and is also influenced by fetal aneuploidy status. Maternal weight trends inversely with fetal fraction, and we find no added benefit from analyzing body mass index or blood volume instead of weight. Strong correlations exist between fetal fractions from aliquots taken from the same patient at the same blood draw and also at different blood draws. While a number of factors trend with fetal fraction across the cohort as a whole, they are not the sole determinants of fetal fraction. In this study, the variability for any one patient does not appear large enough to justify postponing testing to a later gestational age. © 2015 John Wiley & Sons, Ltd.

Noninvasive in vivo plasma volume and hematocrit in humans: observing long-term baseline behavior to establish homeostasis for intravascular volume and composition

NASA Astrophysics Data System (ADS)

Dent, Paul; Deng, Bin; Goodisman, Jerry; Peterson, Charles M.; Narsipur, Sriram; Chaiken, J.

2016-04-01

A new device incorporating a new algorithm and measurement process allows simultaneous noninvasive in vivo monitoring of intravascular plasma volume and red blood cell volume. The purely optical technique involves probing fingertip skin with near infrared laser light and collecting the wavelength shifted light, that is, the inelastic emission (IE) which includes the unresolved Raman and fluorescence, and the un-shifted emission, that is, the elastic emission (EE) which includes both the Rayleigh and Mie scattered light. Our excitation and detection geometry is designed so that from these two simultaneous measurements we can calculate two parameters within the single scattering regime using radiation transfer theory, the intravascular plasma volume fraction and the red blood cell volume fraction. Previously calibrated against a gold standard FDA approved device, 2 hour monitoring sessions on three separate occasions over a three week span for a specific, motionless, and mostly sleeping individual produced 3 records containing a total of 5706 paired measurements of hematocrit and plasma volume. The average over the three runs, relative to the initial plasma volume taken as 100%, of the plasma volume±1σ was 97.56+/-0.55 or 0.56%.For the same three runs, the average relative hematocrit (Hct), referenced to an assumed initial value of 28.35 was 29.37+/-0.12 or stable to +/-0.4%.We observe local deterministic circulation effects apparently associated with the pressure applied by the finger probe as well as longer timescale behavior due to normal ebb and flow of internal fluids due to posture changes and tilt table induced gravity gradients.

Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

PubMed

Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

2015-04-10

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytokine-Mediated Loss of Blood Dendritic Cells During Epstein-Barr Virus-Associated Acute Infectious Mononucleosis: Implication for Immune Dysregulation.

PubMed

Panikkar, Archana; Smith, Corey; Hislop, Andrew; Tellam, Nick; Dasari, Vijayendra; Hogquist, Kristin A; Wykes, Michelle; Moss, Denis J; Rickinson, Alan; Balfour, Henry H; Khanna, Rajiv

2015-12-15

Acute infectious mononucleosis (IM) is associated with altered expression of inflammatory cytokines and disturbed T-cell homeostasis, however, the precise mechanism of this immune dysregulation remains unresolved. In the current study we demonstrated a significant loss of circulating myeloid and plasmacytoid dendritic cells (DCs) during acute IM, a loss correlated with the severity of clinical symptoms. In vitro exposure of blood DCs to acute IM plasma resulted in loss of plasmacytoid DCs, and further studies with individual cytokines showed that exposure to interleukin 10 could replicate this effect. Our data provide important mechanistic insight into dysregulated immune homeostasis during acute IM. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Aging, Atherosclerosis, and IGF-1

PubMed Central

Higashi, Yusuke; Sukhanov, Sergiy; Anwar, Asif; Shai, Shaw-Yung

2012-01-01

Insulin-like growth factor 1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation, and tissue repair. Recent evidence indicates that IGF-1 reduces atherosclerosis burden and improves features of atherosclerotic plaque stability in animal models. Potential mechanisms for this atheroprotective effect include IGF-1–induced reduction in oxidative stress, cell apoptosis, proinflammatory signaling, and endothelial dysfunction. Aging is associated with increased vascular oxidative stress and vascular disease, suggesting that IGF-1 may exert salutary effects on vascular aging processes. In this review, we will provide a comprehensive update on IGF-1's ability to modulate vascular oxidative stress and to limit atherogenesis and the vascular complications of aging. PMID:22491965

The effect of anticoagulants on the distribution of chromium VI in blood fractions.

PubMed

Afolaranmi, Grace A; Tettey, Justice N A; Murray, Helen M; Meek, R M Dominic; Grant, M Helen

2010-01-01

Metal-on-metal resurfacing arthroplasty is associated with elevated circulating levels of cobalt and chromium ions. To establish the long-term safety of metal-on-metal resurfacing arthroplasty, it has been recommended that during clinical follow-up of these patients, the levels of these metal ions in blood be monitored. In this article, we provide information on the distribution of chromium VI ions (the predominant form of chromium released by cobalt-chrome alloys in vivo and in vitro) in blood fractions. Chromium VI is predominantly partitioned into red blood cells compared with plasma (analysis of variance, P < .05). The extent of accumulation in red blood cells is influenced by the anticoagulant used to collect the blood, with EDTA giving a lower partitioning into red cells compared with sodium citrate and sodium heparin. 2010 Elsevier Inc. All rights reserved.

The number and function of circulating CD34+CD133+ progenitor cells decreased in stable coronary artery disease but not in acute myocardial infarction

PubMed Central

Kondo, Takahisa; Shintani, Satoshi; Maeda, Kengo; Hayashi, Mutsuharu; Inden, Yasuya; Numaguchi, Yasushi; Sugiura, Kaichiro; Morita, Yasuhiro; Kitamura, Tomoya; Kamiya, Haruo; Sone, Takahito; Ohno, Miyoshi; Murohara, Toyoaki

2010-01-01

Objective Circulating CD34+CD133+ cells are one of the main sources of circulating endothelial progenitor cells (EPCs). Age is inversely related to the number and function of CD34+CD133+ progenitor cells in stable coronary artery disease (CAD), but the relationship remains unclear in acute myocardial infarction (AMI). The authors aimed to clarify how ageing affects the number and function of mobilised CD34+CD133+ progenitor cells in AMI. Design and results Circulating CD34+CD133+ progenitor cells were measured by flow cytometry. Measurements were made at admission for CAD, or on day 7 after the onset of AMI. In stable CAD (n=131), circulating CD34+CD133+ cells decreased with age (r=−0.344, p<0.0001). In AMI, circulating CD34+CD133+ cells did not correlate with age (n=50), and multivariate analysis revealed that the decreased number of circulating CD34+CD133+ cells was associated with male sex and higher peak creatinine kinase. The ability to give rise to functional EPCs, which show good migratory and tube-forming capabilities, deteriorated among stable CAD subjects (n=10) compared with AMI subjects (N=6). Conclusions In stable CAD, the number and function of circulating CD34+CD133+ progenitor cells decreased with age, whereas those mobilised and circulating in AMI did not. PMID:27325937

TLR-adjuvanted nanoparticle vaccines differentially influence the quality and longevity of responses to malaria antigen Pfs25.

PubMed

Thompson, Elizabeth A; Ols, Sebastian; Miura, Kazutoyo; Rausch, Kelly; Narum, David L; Spångberg, Mats; Juraska, Michal; Wille-Reece, Ulrike; Weiner, Amy; Howard, Randall F; Long, Carole A; Duffy, Patrick E; Johnston, Lloyd; O'Neil, Conlin P; Loré, Karin

2018-05-17

Transmission-blocking vaccines (TBVs) are considered an integral element of malaria eradication efforts. Despite promising evaluations of Plasmodium falciparum Pfs25-based TBVs in mice, clinical trials have failed to induce robust and long-lived Ab titers, in part due to the poorly immunogenic nature of Pfs25. Using nonhuman primates, we demonstrate that multiple aspects of Pfs25 immunity were enhanced by antigen encapsulation in poly(lactic-co-glycolic acid)-based [(PLGA)-based] synthetic vaccine particles (SVP[Pfs25]) and potent TLR-based adjuvants. SVP[Pfs25] increased Ab titers, Pfs25-specific plasmablasts, circulating memory B cells, and plasma cells in the bone marrow when benchmarked against the clinically tested multimeric form Pfs25-EPA given with GLA-LSQ. SVP[Pfs25] also induced the first reported Pfs25-specific circulating Th1 and Tfh cells to our knowledge. Multivariate correlative analysis indicated several mechanisms for the improved Ab responses. While Pfs25-specific B cells were responsible for increasing Ab titers, T cell responses stimulated increased Ab avidity. The innate immune activation differentially stimulated by the adjuvants revealed a strong correlation between type I IFN polarization, induced by R848 and CpG, and increased Ab half-life and longevity. Collectively, the data identify ways to improve vaccine-induced immunity to poorly immunogenic proteins, both by the choice of antigen and adjuvant formulation, and highlight underlying immunological mechanisms.

Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

2015-03-01

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

PLGA/liposome hybrid nanoparticles for short-chain ceramide delivery.

PubMed

Zou, Peng; Stern, Stephan T; Sun, Duxin

2014-03-01

Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA). BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor). FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h. The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.

Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma.

PubMed

Lim, Ji Hyae; Kim, Mee Jin; Kim, Shin Young; Kim, Hye Ok; Song, Mee Jin; Kim, Min Hyoung; Park, So Yeon; Yang, Jae Hyug; Ryu, Hyun Mee

2011-02-01

To perform a reliable non-invasive detection of the fetal achondroplasia using maternal plasma. We developed a quantitative fluorescent-polymerase chain reaction (QF-PCR) method suitable for detection of the FGFR3 mutation (G1138A) causing achondroplasia. This method was applied in a non-invasive detection of the fetal achondroplasia using circulating fetal-DNA (cf-DNA) in maternal plasma. Maternal plasmas were obtained at 27 weeks of gestational age from women carrying an achondroplasia fetus or a normal fetus. Two percent or less achondroplasia DNA was reliably detected by QF-PCR. In a woman carrying a normal fetus, analysis of cf-DNA showed only one peak of the wild-type G allele. In a woman expected an achondroplasia fetus, analysis of cf-DNA showed the two peaks of wild-type G allele and mutant-type A allele and accurately detected the fetal achondroplasia. The non-invasive method using maternal plasma and QF-PCR may be useful for diagnosis of the fetal achondroplasia.

Melanin-concentrating hormone in peripheral circulation in the human.

PubMed

Naufahu, J; Alzaid, F; Fiuza Brito, M; Doslikova, B; Valencia, T; Cunliffe, A; Murray, J F

2017-03-01

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide with a well-characterised role in energy homeostasis and emergent roles in diverse physiologic functions such as arousal, mood and reproduction. Work to date has predominantly focused on its hypothalamic functions using animal models; however, little attention has been paid to its role in circulation in humans. The aims of this study were to (a) develop a radioimmunoassay for the detection of MCH in human plasma; (b) establish reference ranges for circulating MCH and (c) characterise the pattern of expression of circulating MCH in humans. A sensitive and specific RIA was developed and cross-validated by RP-HPLC and MS. The effective range was 19.5-1248 pg MCH/mL. Blood samples from 231 subjects were taken to establish a reference range of 19.5-55.4 pg/mL for fasting MCH concentrations. There were no significant differences between male and female fasting MCH concentrations; however, there were correlations between MCH concentrations and BMI in males and females with excess fat (P < 0.001 and P = 0.020) and between MCH concentrations and fat mass in females with excess fat (P = 0.038). Plasma MCH concentrations rose significantly after feeding in a group of older individuals (n = 50, males P = 0.006, females P = 0.023). There were no robust significant correlations between fasting or post-prandial MCH and resting metabolic rate, plasma glucose, insulin or leptin concentrations although there were correlations between circulating MCH and leptin concentrations in older individuals (P = 0.029). These results indicate that the role of circulating MCH may not be reflective of its regulatory hypothalamic role. © 2017 Society for Endocrinology.

Autologous circulating angiogenic cells treated with osteopontin and delivered via a collagen scaffold enhance wound healing in the alloxan-induced diabetic rabbit ear ulcer model.

PubMed

O'Loughlin, Aonghus; Kulkarni, Mangesh; Vaughan, Erin E; Creane, Michael; Liew, Aaron; Dockery, Peter; Pandit, Abhay; O'Brien, Timothy

2013-01-01

Diabetic foot ulceration is the leading cause of amputation in people with diabetes mellitus. Peripheral vascular disease is present in the majority of patients with diabetic foot ulcers. Despite standard treatments there exists a high amputation rate. Circulating angiogenic cells previously known as early endothelial progenitor cells are derived from peripheral blood and support angiogenesis and vasculogenesis, providing a potential topical treatment for non-healing diabetic foot ulcers. A scaffold fabricated from Type 1 collagen facilitates topical cell delivery to a diabetic wound. Osteopontin is a matricellular protein involved in wound healing and increases the angiogenic potential of circulating angiogenic cells. A collagen scaffold seeded with circulating angiogenic cells was developed. Subsequently the effect of autologous circulating angiogenic cells that were seeded in a collagen scaffold and topically delivered to a hyperglycemic cutaneous wound was assessed. The alloxan-induced diabetic rabbit ear ulcer model was used to determine healing in response to the following treatments: collagen seeded with autologous circulating angiogenic cells exposed to osteopontin, collagen seeded with autologous circulating angiogenic cells, collagen alone and untreated wound. Stereology was used to assess angiogenesis in wounds. The cells exposed to osteopontin and seeded on collagen increased percentage wound closure as compared to other groups. Increased angiogenesis was observed with the treatment of collagen and collagen seeded with circulating angiogenic cells. These results demonstrate that topical treatment of full thickness cutaneous ulcers with autologous circulating angiogenic cells increases wound healing. Cells exposed to the matricellular protein osteopontin result in superior wound healing. The wound healing benefit is associated with a more efficient vascular network. This topical therapy provides a potential novel therapy for the treatment of non-healing diabetic foot ulcers in humans.

A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G>A (IVS25+1G>A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor

PubMed Central

Persu, Alexandre; Lambert, Michel; Deinum, Jaap; Cossu, Marta; de Visscher, Nathalie; Irenge, Leonid; Ambroise, Jerôme; Minon, Jean-Marc; Nesterovitch, Andrew B.; Churbanov, Alexander; Popova, Isolda A.; Danilov, Sergei M.; Danser, A. H. Jan; Gala, Jean-Luc

2013-01-01

Background Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. Methods and Results Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. Conclusions We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. PMID:23560051

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

PubMed

Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

2011-04-01

Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study.

PubMed

Hierso, Régine; Lemonne, Nathalie; Villaescusa, Rinaldo; Lalanne-Mistrih, Marie-Laure; Charlot, Keyne; Etienne-Julan, Maryse; Tressières, Benoit; Lamarre, Yann; Tarer, Vanessa; Garnier, Yohann; Hernandez, Ada Arce; Ferracci, Serge; Connes, Philippe; Romana, Marc; Hardy-Dessources, Marie-Dominique

2017-03-01

Painful vaso-occlusive crisis, a hallmark of sickle cell anaemia, results from complex, incompletely understood mechanisms. Red blood cell (RBC) damage caused by continuous endogenous and exogenous oxidative stress may precipitate the occurrence of vaso-occlusive crises. In order to gain insight into the relevance of oxidative stress in vaso-occlusive crisis occurrence, we prospectively compared the expression levels of various oxidative markers in 32 adults with sickle cell anaemia during vaso-occlusive crisis and steady-state conditions. Compared to steady-state condition, plasma levels of free haem, advanced oxidation protein products and myeloperoxidase, RBC caspase-3 activity, as well as the concentrations of total, neutrophil- and RBC-derived microparticles were increased during vaso-occlusive crises, whereas the reduced glutathione content was decreased in RBCs. In addition, natural anti-band 3 autoantibodies levels decreased during crisis and were negatively correlated with the rise in plasma advanced oxidation protein products and RBC caspase-3 activity. These data showed an exacerbation of the oxidative stress during vaso-occlusive crises in sickle cell anaemia patients and strongly suggest that the higher concentration of harmful circulating RBC-derived microparticles and the reduced anti-band 3 autoantibodies levels may be both related to the recruitment of oxidized band 3 into membrane aggregates. © 2016 John Wiley & Sons Ltd.

Circulating YKL-40‡ levels during human endotoxaemia

PubMed Central

Johansen, J S; Krabbe, K S; Møller, K; Pedersen, B K

2005-01-01

YKL-40 is secreted by macrophages and neutrophils and patients with bacterial infections have elevated circulating YKL-40. The aim was to evaluate changes in plasma YKL-40 (determined by enzyme-linked immunosorbent assay (ELISA) at 0, 2, 4, 8, 24 and 32 h) in eight healthy volunteers after injection with Esherichia coli endotoxin or saline. Plasma YKL-40 increased after endotoxin injection from 31 µg/l (range 19–39 µg/l) to a maximum of 159 µg/l (61–552 µg/l, P < 0·01) at 24 h. The finding that plasma YKL-40 increased after endotoxin injection compared with saline (P < 0·001) suggests that YKL-40 has a functional role in infections. PMID:15807860

Method of acetaldehyde measurement with minimal artifactual formation in red blood cells and plasma of actively drinking subjects with alcoholism.

PubMed

Hernandez-Munoz, R; Ma, X L; Baraona, E; Lieber, C S

1992-07-01

After alcohol consumption, a substantial amount of acetaldehyde that is reversibly bound to protein and nonprotein components of the red blood cells circulates in the blood and could cause extrahepatic toxicity. However, acetaldehyde measurement in human red blood cells is hampered by considerable ex vivo artifactual formation as a result of nonenzymatic oxidation of ethanol during protein precipitation. To eliminate this source of artifactual formation, free and reversibly bound acetaldehyde were trapped with semicarbazide from red blood cell hemolysates, and both the stroma and the hemoglobin were sequentially removed by centrifugation and ion-exchange chromatography in carboxymethyl Sephadex, respectively. The eluted semi-carbazone was dissociated with perchloric acid, and the acetaldehyde that was released in the protein-free supernatants was measured by head-space gas chromatography. Maximal retention of hemoglobin by carboxymethyl Sephadex and complete recovery of acetaldehyde and ethanol were achieved at a pH of 5.3. The artifactual formation decreased from 2.62 +/- 0.32 mumol of acetaldehyde per millimole of ethanol in the initial hemolysates to 1.38 +/- 0.20 mumol after removal of the stroma and to a level that is comparable to measurements in plasma (0.09 +/- 0.02 mumol) after removal of both the stroma and the hemoglobin. In 12 actively drinking subjects with alcoholism, with blood ethanol levels that ranged between 9 and 81 mmol/L, the concentrations of acetaldehyde in red blood cells (11.50 +/- 1.46 mumol/L; range: 7.5 to 22 mumol/L) were minimally affected by blood ethanol levels and were three times as high as those in the plasma (3.74 +/- 1.49 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a monocarboxylate transporter in reticular cells of the mouse lymph node and its involvement in the uptake of exogenous particles.

PubMed

Zheng, Miao; Ishiguro-Oonuma, Toshina; Iwanaga, Toshihiko

2014-01-01

The monocarboxylate transporter (MCT)-1 plays an important role in the transfer of monocarboxylate metabolites such as lactate, ketone bodies, and acetic acid. The present study revealed the selective localization of MCT1 in reticular cells of the murine lymph node. An intense MCT1 immunoreactivity was found in the reticular cells forming a cellular network together with sinus-lining cells in the medullary sinuses and in cells covering the inside of subcapsular sinuses.Electron-microscopically, MCT1 was localized along the plasma membrane of the reticular cells.The medullary reticular cells vigorously ingested carboxylate-modified latex particles, but any reticular cells within the cortical lymphoid follicles and medullary cords neither expressed MCT1 nor incorporated latex particles. MCT1-immunoreactive reticular cells also expressed LYVE-1,which is a hyaluronan receptor abundant in both the lymphatic endothelium and hepatic sinusoidal epithelium. The selective localization of MCT1 and LYVE-1 suggests a high level of activity for lymphoid reticular cells in the uptake of carboxylate-modified and hyaluronate waste substances circulating in the body.

Circulating miR-200c is up-regulated in paediatric patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication of a ZEB1-dependent mechanism.

PubMed

D'Agostino, Marco; Martino, Francesco; Sileno, Sara; Barillà, Francesco; Beji, Sara; Marchetti, Lorenza; Gangi, Fabio Maria; Persico, Luca; Picozza, Mario; Montali, Anna; Martino, Eliana; Zanoni, Cristina; Avitabile, Daniele; Parrotto, Sandro; Capogrossi, Maurizio Colognesi; Magenta, Alessandra

2017-09-15

Hypercholesterolaemia provokes reactive oxygen species (ROS) increase and is a major risk factor for cardiovascular disease (CVD) development. We previously showed that circulating miR-33a/b expression levels were up-regulated in children with familial hypercholesterolaemia (FH). miR-33a/b control cholesterol homoeostasis and recently miR-33b has been demonstrated to directly target the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1). The latter acts in a negative feedback loop with the miR-200 family. Our previous studies showed that the ROS-dependent miR-200c up-regulation induces endothelial dysfunction and provokes a ZEB1-dependent apoptosis and senescence. In the present study, we aimed to verify whether circulating miR-200c was induced in FH children, and whether a correlation existed with miR-33a/b Total RNA was extracted from plasma of 28 FH children and 25 age-matched healthy subjects (HS) and miR-200c levels were measured. We found that miR-200c was up-regulated in FH compared with HS (4.00 ± 0.48-fold increase, P <0.05) and exhibited a positive correlation with miR-33a/b. miR-200c did not correlate with plasma lipids, but correlated with C-reactive protein (CRP) plasma levels and glycaemia (GLI). Ordinary least squares (OLS) regression analysis revealed that miR-200c was significantly affected by GLI and by miR-33a ( P <0.01; P <0.001 respectively). Moreover, we found that miR-33 overexpression, in different cell lines, decreased ZEB1 expression and up-regulated both the intracellular and the extracellular miR-200c expression levels. In conclusion, circulating miR-200c is up-regulated in FH, probably due to oxidative stress and inflammation and via a miR-33a/b -ZEB1-dependent mechanism. The present study could provide the first evidence to point to the use of miR-33a/b and miR-200c , as early biomarkers of CVD, in paediatric FH. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

PubMed Central

Sofrenovic, Tanja; McEwan, Kimberly; Crowe, Suzanne; Marier, Jenelle; Davies, Robbie; Suuronen, Erik J.; Kuraitis, Drew

2012-01-01

Background Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs). Methods PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential. Results The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34+VEGFR2+CD133+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle. Conclusion Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34+VEGFR2+CD133+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation. PMID:23133548

Depression of the Lecithin-Cholesterol Acyltransferase Reaction in Vitamin E-Deficient Monkeys,

DTIC Science & Technology

Vitamin E deficiency in two species of monkeys reduced the esterification of cholesterol by the plasma lecithin -cholesterol acyltransferase reaction...depression in the concentration of circulating polyunsaturated fatty acid cholesteryl esters. Since the plasma lecithin -cholesterol acyltransferase...cholesterol by plasma from vitamin E-deficient monkeys is due to alteration of these sulfhydryl sites. A similar reduction in the plasma lecithin -cholesterol

Control of magnetohydrodynamic stability by phase space engineering of energetic ions in tokamak plasmas.

PubMed

Graves, J P; Chapman, I T; Coda, S; Lennholm, M; Albergante, M; Jucker, M

2012-01-10

Virtually collisionless magnetic mirror-trapped energetic ion populations often partially stabilize internally driven magnetohydrodynamic disturbances in the magnetosphere and in toroidal laboratory plasma devices such as the tokamak. This results in less frequent but dangerously enlarged plasma reorganization. Unique to the toroidal magnetic configuration are confined 'circulating' energetic particles that are not mirror trapped. Here we show that a newly discovered effect from hybrid kinetic-magnetohydrodynamic theory has been exploited in sophisticated phase space engineering techniques for controlling stability in the tokamak. These theoretical predictions have been confirmed, and the technique successfully applied in the Joint European Torus. Manipulation of auxiliary ion heating systems can create an asymmetry in the distribution of energetic circulating ions in the velocity orientated along magnetic field lines. We show the first experiments in which large sawtooth collapses have been controlled by this technique, and neoclassical tearing modes avoided, in high-performance reactor-relevant plasmas.

Plasma and cellular fibronectin: distinct and independent functions during tissue repair

PubMed Central

2011-01-01

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes. PMID:21923916

Clinical CVVH model removes endothelium-derived microparticles from circulation

PubMed Central

Abdelhafeez, Abdelhafeez H.; Jeziorczak, Paul M.; Schaid, Terry R.; Hoefs, Susan L.; Kaul, Sushma; Nanchal, Rahul; Jacobs, Elizabeth R.; Densmore, John C.

2014-01-01

Background Endothelium-derived microparticles (EMPs) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury, apoptosis or activation. We have previously demonstrated EMP-induced acute lung injury (ALI) in animal models and endothelial barrier dysfunction in vitro. Current treatment options for ALI are limited and consist of supportive therapies. We hypothesize that standard clinical continuous venovenous hemofiltration (CVVH) reduces serum EMP levels and may be adapted as a potential therapeutic intervention. Materials and methods EMPs were generated from plasminogen activation inhibitor-1 (PAI-1)-stimulated human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis was used to characterize EMPs as CD31- and annexin V-positive events in a submicron size gate. Enumeration was completed against a known concentration of latex beads. Ultimately, a concentration of ~650,000 EMP/mL perfusate fluid (total 470 mL) was circulated through a standard CVVH filter (pore size 200 μm, flow rate 250 mL/hr) for a period of 70 minutes. 0.5 mL aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis. EMP concentration in the dialysate was measured at the end of 4 hours to better understand the fate of EMPs. Results A progressive decrease in circulating EMP concentration was noted using standard CVVH at 250 mL/hr (a clinical standard rate) from a 470 mL volume modelling a patient's circulation. A 50% reduction was noted within the first 30 minutes. EMPs entering the dialysate after 4 hours were 5.7% of the EMP original concentration. Conclusion These data demonstrate that standard CVVH can remove EMPs from circulation in a circuit modelling a patient. An animal model of hemofiltration with induction of EMP release is required to test the therapeutic potential of this finding and potential of application in early treatment of ALI. PMID:24596654

Preferential Elimination of Older Erythrocytes in Circulation and Depressed Bone Marrow Erythropoietic Activity Contribute to Cadmium Induced Anemia in Mice

PubMed Central

Chatterjee, Sreoshi; Saxena, Rajiv K.

2015-01-01

Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid population indicating a stress response. In short cadmium exposure causes preferential clearance of older erythrocytes from circulation along with a depressed erythropoietic activity at higher doses. PMID:26161863

Digital PCR analysis of circulating nucleic acids.

PubMed

Hudecova, Irena

2015-10-01

Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.

PubMed

Caixeta, Luciano S; Giesy, Sarah L; Krumm, Christopher S; Perfield, James W; Butterfield, Anthony; Schoenberg, Katie M; Beitz, Donald C; Boisclair, Yves R

2017-11-01

Modern dairy cows meet the energy demand of early lactation by calling on hormonally driven mechanisms to increase the use of lipid reserves. In this context, we recently reported that fibroblast growth factor-21 (FGF21), a hormone required for efficient use of lipid reserves in rodents, is upregulated in periparturient dairy cows. Increased plasma FGF21 in early lactation coincides with elevated circulating concentrations of glucagon (GCG) and nonesterified fatty acids (NEFA). To assess the relative contribution of these factors in regulating FGF21, two experiments were performed in energy-sufficient, nonpregnant, nonlactating dairy cows. In the first study, cows were injected with saline or GCG every 8 h over a 72-h period. GCG increased hepatic FGF21 mRNA by an average of fivefold over matched controls but had no effect on plasma FGF21. In the second study, cows were infused and injected with saline, infused with Intralipid and injected with saline, or infused with Intralipid and injected with GCG. Infusions and injections were administered intravenously over 16 h and subcutaneously every 8 h, respectively. Intralipid infusion increased plasma NEFA from 92 to 550 µM within 3 h and increased plasma FGF21 from 1.3 to >11 ng/ml 6 h later; FGF21 mRNA increased by 34-fold in liver but remained invariant in adipose tissue. GCG injections during the Intralipid infusion had no additional effects on plasma NEFA, liver FGF21 mRNA, or plasma FGF21. These data implicate plasma NEFA as a key factor triggering hepatic production and increased circulating concentrations of FGF21 in early lactation. Copyright © 2017 the American Physiological Society.

Dissociation of a ferric maltol complex and its subsequent metabolism during absorption across the small intestine of the rat.

PubMed Central

Barrand, M. A.; Callingham, B. A.; Dobbin, P.; Hider, R. C.

1991-01-01

1. The fate and disposition of [59Fe]-ferric [3H]-maltol after intravenous administration were investigated in anaesthetized rats. Immediate dissociation of ferric iron from maltol took place in the circulation even with high doses of ferric maltol (containing 1 mg elemental iron). In plasma samples withdrawn within 1 min of injection and subjected to gel filtration, 59Fe eluted with the high molecular weight proteins whilst the tritium was associated with low molecular weight material. 2. The rates of elimination of 59Fe and of tritium from the plasma and their ultimate fate were very different. The half life for 59Fe in the plasma was around 70 min and 59Fe appeared mainly in the bone marrow and liver. There was an initial rapid exit of tritium from the plasma with a half life of around 12 min. This was followed either by a plateau or by a rise in tritium levels, involving entry of maltol metabolites into the circulation. These metabolites could be recovered in the urine. 3. Entry of 59Fe and of tritium into the blood plasma after intraduodenal administration of [59Fe]-ferric [3H]-maltol was also very different. At low doses of ferric maltol (containing 100 micrograms elemental iron), the tritium appeared in the plasma in highest amounts within seconds and then decreased whilst there was a slow rise in 59Fe levels. At higher doses of ferric maltol (containing 7 mg elemental iron), levels of 59Fe in the plasma were highest at 5 min and then fell whereas tritium levels rose steadily. Mucosal processing of 59Fe prevented further entry of iron at high dose into the circulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1364845